DEVELOPMENT OF CARBOHYDRATE BASED CONJUGATE VACCINES USING Qβ VIRUS LIKE PARTICLES WITH ANTI-BACTERIAL OR ANTI-CANCER PROPERTIES By Zahra Rashidijahanabad A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Chemistry—Doctor of Philosophy 2021 ABSTRACT DEVELOPMENT OF CARBOHYDRATE BASED CONJUGATE VACCINES USING Qβ VIRUS LIKE PARTICLES WITH ANTI-BACTERIAL OR ANTI-CANCER PROPERTIES By Zahra Rashidijahanabad Chimeric antigen receptor (CAR) T cells and bispecific antibodies (BsAbs) are exciting directions to harness the power of the immune system to fight cancer. Chapter 1 is focused on GD2 ganglioside and the mucin-1 (MUC1) protein, two important tumor associated carbohydrate antigens, and latest advances in CAR T cells and bispecific antibodies targeting these two antigens are presented. The roles of co-stimulatory molecules, structures of the sequences for antigen binding, methods for CAR and antibody construction, as well as strategies to enhance solid tumor penetration and reduce T cell exhaustion and death are discussed. Furthermore, approaches to reduce “on target, off tumor” side effects are introduced. Besides CAR T cells and bispecific antibodies, carbohydrate-based vaccines hold great promise for a number of diseases, which will be the focus of the rest of this dissertation. Several challenges are associated with carbohydrate antigens in regard to inducing specific and protective antibodies as they are poorly immunogenic and the resulting antibodies induced by immunizing with carbohydrates only, typically have low affinity. Currently, developing carbohydrate-based vaccines requires covalent conjugation of the carbohydrate antigen with a protein carrier for optimal antibody response. Thus, generated antibodies have higher affinity against glycan structures. In chapter 2, a potential conjugate vaccine was developed by linking O-specific polysaccharide (OSP) antigen purified from Vibrio cholerae Inaba with Qβ virus like particles (VLPs) efficiently via squarate chemistry as one of the first examples of polysaccharide conjugation to VLPs. The Qβ-OSP conjugate was characterized with mass photometry on the whole particle level. Pertinent immunologic display of OSP was confirmed by immunoreactivity of the conjugate with convalescent phase samples from humans with cholera. Mouse immunization with the Qβ-OSP conjugate showed that the construct generated prominent and long-lasting IgG antibody responses against OSP, and the resulting antibodies could recognize the native lipopolysaccharide from Vibrio cholerae Inaba. This was the first time that Qβ was conjugated with a bacterial polysaccharide for vaccine development, broadening the scope of this powerful carrier. Tumor associated carbohydrate antigens (TACAs) are another class of attractive carbohydrate antigens for the development of anti-cancer immunotherapy with respect to monoclonal antibodies and vaccines. Tetrasaccharide sialyl-Lewisa is an attractive therapeutic target for cancer therapy since it is widely expressed on epithelial tumors of the gastrointestinal tract. The overexpression of sLea appears to be a key event in invasion and metastasis of many tumors and results in susceptibility to antibody-mediated lysis. In chapter 3, sialyl-Lewisa conjugate vaccine with Qβ was developed. The resulting construct, Qβ-sLea, induced antibody production in vivo and the resulting antibodies showed high selectivity for sLea antigen in in vitro studies and effectively reduced tumor growth in mice. To my beloved mother and father iv ACKNOWLEDGEMENTS These projects would not have been possible without the support of many people. I am extremely grateful to my supervisor, Prof. Xuefei Huang for his invaluable advice, continuous support, and patience during my PhD study. I have benefited greatly from his wealth of knowledge and meticulous editing. I am extremely grateful that he took me on as a student and continued to have faith in me over the years. I would also like to thank all my guidance committee members, Dr. Daniel Woldring, Dr. Jetze Tepe and Dr. Kevin Walker for reviewing my thesis, their assistance, good suggestions, and kindness. I am grateful to Dr. Babak Borhan, Dr. Gary Blanchard and other graduate research committee at chemistry department for giving me the opportunity to study at Michigan State University. I would like to thank all instrumental specialists who assisted me to run all experiments involving sophisticated analytical instruments. My special thanks are to Dr. Daniel Holmes who kindly trained me to set up and run all kinds of NMR experiments; Tony Schilmiller, who trained me to run mass spectrometers and assisted me to process all data; Dr. Louis King, Dr. Daniel Vocelle and Dr. Matthew Bernard for the flow cytometry training and their support with data analysis. I am indebted to all of my friends and colleagues from Huang group, especially Mehdi Hossaini Nasr and Suttipun Sungsuwan, who helped me to develop my research knowledge and skills. I appreciate Dr. Sherif Ramadan for our collaboration projects and his unlimited assistance during these years. Besides, I thank Dr. Herbert Kavunja for his invaluable assistance in my research. I also thank Xuanjun Wu, Tianlu Li, Shuyao Lang, Zibin Tan, Hunter McFall-Boegeman and Shivangi Chugh for their assistance in various aspect of my research. And all other members v in Huang lab: Weizhun Yang, Vincent Shaw, Jicheng Zhang, Changxin Huo, Peng Wang, Kedar Baryal, Mengxia Sun, Kunli Liu, Chia-wei Yang, Po-han Lin, Cameron Talbot, Setare Nick and Ida Shafieichaharberoud. Finally, I would like to express my deepest gratitude to my parents and siblings- Ali, Yaser and Sima- for their love, support, and encouragement through this hard time of separation. I also wish to thank my grandfather, Haddad, for the love and support he gave to me. He will be missed forever. vi TABLE OF CONTENTS LIST OF TABLES ......................................................................................................................... ix LIST OF FIGURES .........................................................................................................................x LIST OF SCHEMES......................................................................................................................xv KEY TO ABBREVIATIONS ...................................................................................................... xvi CHAPTER 1: Recent advances in tumor associated carbohydrate antigen based chimeric antigen receptor T cells and bispecific antibodies for anti-cancer immunotherapy .............1 1.1 Introduction ..........................................................................................................................1 1.2 GD2 CAR T cells: going beyond the anti-GD2 monoclonal antibodies .............................2 1.2.1 Building co-stimulatory signals into GD2 CAR T cells ................................................4 1.2.2 Enhancing homing and penetration of solid tumor by GD2 CAR T cells .....................5 1.2.3 Combination of GD2 CAR T therapy with checkpoint blockade and chemotherapy ...7 1.2.4 Reengineering of the GD2 CAR ....................................................................................8 1.3 MUC1 CAR T cells ...........................................................................................................11 1.3.1 MUC1 epitope structures for CAR T cell targeting .....................................................12 1.3.2 Building costimulatory signals into MUC1 based CAR T cells and dual targeting of tumor cells .............................................................................................................................15 1.4 Bispecific antibodies targeting GD2 and MUC1 ...............................................................17 1.4.1 GD2 targeting BsAbs ...................................................................................................18 1.4.2 MUC1 based BsAbs .....................................................................................................22 1.4.3 CAR T cells vs BsAbs .................................................................................................22 1.5 Conclusions and perspectives ............................................................................................23 REFERENCES ..............................................................................................................................27 CHAPTER 2: Virus like particle display of Vibrio cholerae O-specific polysaccharide as a potential vaccine against cholera ..........................................................................36 2.1 Introduction ........................................................................................................................36 2.2 Results ................................................................................................................................38 2.2.1 Conjugation of the OSP core antigen to Qβ ................................................................38 2.2.2 Immunogenicity of the Qβ-OSP conjugate ..................................................................45 2.3 Discussion ..........................................................................................................................50 2.4 Conclusions ........................................................................................................................52 2.5 Materials and methods ......................................................................................................54 2.5.1 General experimental procedures and methods for synthesis ......................................54 2.5.2 Qβ conjugation to lactoside 3 and purification ............................................................54 2.5.3 Qβ conjugation to OSP and purification ......................................................................55 2.5.4 MP procedure ...............................................................................................................55 2.5.5 Immunization ...............................................................................................................56 2.5.6 Evaluation of antibody titers by ELISA.......................................................................56 vii 2.5.7 Evaluation of Qβ-OSP conjugates using human serum ...............................................57 2.5.8 Serum vibriocidal responses ........................................................................................58 APPENDIX ....................................................................................................................................59 REFERENCES ..............................................................................................................................62 CHAPTER 3: Development of Sialyl-Lewis a conjugate vaccine for targeted cancer immunotherapy. .................................................................................................68 3.1 Introduction ........................................................................................................................68 3.2 Results ................................................................................................................................70 3.2.1 Synthesis of Qβ-sLea conjugate vaccine and mouse immunization ............................70 3.2.2 Qβ-sLea conjugate elicited high titers of IgG antibodies titers and longer lasting anti- sLea IgG antibodies in mice compared with the KLH-sLea conjugate as well as the admixture of Qβ and sLea.......................................................................................................................72 3.2.3 Qβ-sLea conjugate elicited antibodies capable of binding with sLea expressing tumor cells .......................................................................................................................................73 3.2.4 Antibodies induced by the Qβ-sLea conjugate were highly selective toward human pancreatic ductal adenocarcinoma tissues ............................................................................75 3.2.5 Antibodies induced by the Qβ-sLea conjugate were highly selective toward sLea binding based on glycan microarray analysis .......................................................................77 3.2.6 Vaccine activity in animal model for metastasis .........................................................77 3.2.7 Ongoing experiments ...................................................................................................81 3.3 Discussion ..........................................................................................................................82 3.4 Materials and methods .......................................................................................................84 3.4.1 General experimental procedures and methods for synthesis ......................................84 3.4.2 Synthesis of Qβ-sLea conjugate ...................................................................................86 3.4.3 KLH-sLea conjugation .................................................................................................86 3.4.4 Synthesis of BSA-sLea conjugate ................................................................................87 3.4.5 Mouse immunization ...................................................................................................87 3.4.6 Rabbit immunization ....................................................................................................88 3.4.7 Tumor challenge and antibody treatments ...................................................................88 3.4.8 Evaluation of antibody titers by ELISA.......................................................................88 3.4.9 Detection of antibody binding to cells by FACS .........................................................89 3.4.10 Complement dependent cytotoxicity .........................................................................90 3.4.11 Immunochemistry staining of cancer tissue microarrays...........................................90 3.4.12. Active tumor protection model .................................................................................91 3.4.13 Passive tumor protection model .................................................................................91 APPENDIX ....................................................................................................................................92 REFERENCES ............................................................................................................................121 viii LIST OF TABLES Table 3. 1 Human pancreatic ductal adenocarcinoma tissue microarray (XPAN024-01) specification which contains 24 cores and 24 cases. 1 core per case. Each core has 1.5 mm diameter with 5μm thickness and fixed with formalin. ............................................................................... 95 Table 3. 2 Human pancreatic cancer tissue microarray (XPAN048-01) specification which contains 48 cores and 48 cases. 1 core per case. It contains 3 cases of normal tissue, 2 cases of metastatic cancer, 2 cases of squamous cell carcinoma, 2 cases of adenosquamous carcinoma, and 31 cases of pancreatic adenocarcinoma. ....................................................................................... 96 Table 3. 3 Glycopeptide microarray screening results of antisera induced by Qβ-sLea or Qβ and sLea mixture. ............................................................................................................................... 104 ix LIST OF FIGURES Figure 1. 1 Structure of the GD2 ganglioside. ............................................................................... 2 Figure 1. 2 Schematic demonstration of various GD2 CAR constructs. a) The CAR includes the hinge region as well as the Fc domain (CH2 and CH3); b) CAR without the Fc domain; c) CAR with the hinge attached to the stalk of CD8a; and d) CAR with CD8a stalk only without the hinge region. (Image adapted from [38]) .................................................................................................. 9 Figure 1. 3 Construction of UniCAR and TM. A) UniCAR cells do not recognize tumor cells in the absence of TM due to the lack of receptor on T cells towards tumor antigens. B) Upon addition of the TM comprised of the conjugate of anti-GD2 scFv and E5B9, the UniCAR can bind with the TM through E5B9, thus gaining the abilities to recognize GD2+ tumor cells. ............................. 11 Figure 1. 4 Schematic demonstration of MUC1 glycoprotein structure. MUC1 is composed of a heterodimer of MUC1-N linked non-covalently with the transmembrane MUC1-C. MUC1-N contains a variable number of 20 amino acid VNTRs that are heavily glycosylated on serine or threonine residues of each VNTR in normal cells shielding the protein backbone for immune recognition. However, tumor associated MUC1 are hypoglycosylated exposing the protein backbone. ...................................................................................................................................... 12 Figure 1. 5 Schematic representation of various BsAb formats. a) IgG-like BsAbs: i) and ii) IgG- scFv, iii) triomab, iv) quadroma, and v) half molecule exchange format. And b) non-IgG-like BsAb: i) tandem scFv, ii) dual-affinity re-targeting antibody, iii) bi-nanobody, iv) scFv-human serum albumin-scFv. ..................................................................................................................... 18 Figure 1. 6 Schematic demonstration of the Hu3F8-BsAb structure. .......................................... 20 Figure 1. 7 scFv-based bispecific antibody format of Hu3F8-scBA (VH is the heavy chain of the variable region, and the VL is the light chain of the variable region). .......................................... 20 Figure 2. 1 Structure of O-specific polysaccharide (OSP) 1 of Vibrio cholerae O1 ................... 37 Figure 2. 2 SELDI-TOF MS result of the conjugation of Qβ triple mutant A38K/A40C/D102C and lactose squarate 3. a) Qβ before conjugation, b) 14 eq of 3 was added to Qβ and the reaction mixture was incubated for 20h, c) An additional 14eq of 3 was added to reaction and incubated for 72h. The mass difference between the peaks corresponds to the addition of a lactose squarate with MW of 577 Da. The average loading was calculated based on the ratio of the sum of respective antigen number of each peak multiplied by their intensity to the total intensity of all peaks. ..... 41 Figure 2. 3 The SDS-PAGE of different samples at non-reducing (Lanes 1-5) and reducing (Lanes 8-12) conditions. Lanes 1, 12: Molecular weight ladder; lanes 2, 11: unconjugated Qβ; lanes 3, 10: Qβ-lactose conjugate; lanes 4, 9: Qβ-OSP conjugate; lanes 5, 8: BSA. The Qβ monomer and dimer appeared at 14KDa and 28KDa under the reducing condition. The band corresponding to the Qβ- lactose 5 monomer shifted to about 19 kDa after conjugation, corresponding to the addition of x about 8 lactoses per monomer. Qβ-OSP conjugate showed up as a smear at higher MW on the gel. ....................................................................................................................................................... 42 Figure 2. 4 The MP result of Qβ triple mutant A38K/A40C/D102C. The same Qβ sample which was measured several times over time of this study showed a decrease of the MW. The right peak shifted from 2696 KDa in (a) to 2544KDa (b) and 2387KDa in (c)............................................. 44 Figure 2. 5 The MP result of Qβ without RNA. The measurement of Qβ sample without RNA was repeated after three months. The MW of right peak was 2388 KDa in (d) and 2443 KDa in (e) respectively. .................................................................................................................................. 44 Figure 2. 6 The MP result of wild-type Qβ without RNA f) before and g) after conjugation with lactoside 3. The right peak shifted from 2,443 KDa to 3,249 KDa, which suggests the conjugation of about 7 lactosides per Qβ monomer on average. ...................................................................... 45 Figure 2. 7 MP results of a) Qβ and b) Qβ-OSP conjugate. The right peak shifted from 2,544 KDa to 2,953 KDa, which suggests the conjugation of an average 68 OSP per full Qβ capsid calculated based on the mass of the intact particle. ....................................................................................... 45 Figure 2. 8 Immunoreactivities of human plasma toward Qβ and Qβ-OSP were measured by acute phase plasma (day 2 sample) versus convalescent phase plasma (day 7 sample) of patients with cholera versus typhoid fever in Dhaka, Bangladesh. .................................................................... 46 Figure 2. 9 Evaluation of Qβ-OSP immunogenicity. a) Immunization and blood collection schedule. Each group received 3 immunization three weeks apart with blood collected at day 0 and on days 56, 86, 118, 170, 265 and 272 respectively. b) OSP-specific IgG titer of pooled sera from Qβ and Qβ-OSP groups up to day 272 post-immunization. The red arrow indicates a booster injection at day 265. c) individual mouse serum OSP-specific IgG titer of Qβ and Qβ-OSP groups at day 56. The statistical significance was determined through a two tailed t-test using GraphPad Prism. ** p < 0.0001. .................................................................................................................... 48 Figure 2. 10 ELISA analysis showed significant IgG binding to BSA–OSP by post-immune sera at d49 and d56 (p=0.0014 and 0.0065 respectively), compared to the control sera from mice immunized with Qβ only. Each bar represents data for 5 mice at 20,000 fold of serum dilution. 48 Figure 2. 11 Binding of mouse serum immunized with Qβ-OSP and Qβ to LPS from Inaba vs E. coli. Serum binding against Inaba LPS was observed in the Qβ-OSP immunized group while sera from the Qβ group had lower binding. The binding to E. coli LPS was lower by sera from both Qβ-OSP and Qβ immunized mice. The statistical significance was determined through an unpaired two tailed t-test using GraphPad Prism. * p < 0.05. ..................................................................... 49 Figure 2. 12 Vibriocidal responses in vaccine cohorts. We defined responders as having an increase in vibriocidal titer by 4-fold at day 56 than day 0. ......................................................... 50 Figure 2. 13 SELDI-TOF MS result of conjugation of a) wild type Qβ or b) mutant Qβ to squarate- OSP. The broad weak peak could be observed at ~20 KDa which correlates to conjugate 6 with loading 1 OSP. .............................................................................................................................. 60 xi Figure 2. 14 Mass spectrum of wild-type Qβ without RNA. ....................................................... 60 Figure 2. 15 Mass spectrum of wild-type Qβ without RNA after conjugation to lactoside. The average loading of lactoside is 6 per Qβ monomer....................................................................... 61 Figure 2. 16 SELDI-TOF MS result of a) wild type Qβ without RNA or b) wild type Qβ without RNA conjugated to compound 3. The average loading of lactoside is about 6 per Qβ monomer. ....................................................................................................................................................... 61 Figure 3. 1 Structure of sLea-isothiocyanate (sLea-NCS) ............................................................ 71 Figure 3. 2 Immunization and blood collection schedule. Groups of five C57BL/6 female mice received 3 immunizations of Qβ-sLea conjugate or sLea and Qβ mixture two weeks apart with blood collected at days 0, 35, 65, 95, 125, 185, 277, and 356. At day 365, both groups were inoculated i.v. with 5 × 105 B16-FUT3 tumor cells and lungs were collected 14 days later........ 72 Figure 3. 3 a) Titers of anti-sLea IgG antibodies from mice immunized with the Qβ-sLea conjugate against BSA-sLea (each symbol represents one mouse, n=5 mice for Qβ-sLea or sLea and Qβ mixture group and n=3 for KLH-sLea group). Pooled sera from 5 mice were used for Qβ and day 0. The statistical significance was determined through an unpaired two tailed t-test using GraphPad Prism. **** p<0.0001. b) Changes of the titers of anti-sLea IgG antibodies from Qβ-sLea immunized mice over time. The IgG titers were determined with pooled sera. ........................... 73 Figure 3. 4 Recognition of cell surface expression of sLea with FACS (a) and CDC (b) of 1199FB cells in presence of Qβ-sLea antisera. a) 3x105 cells were incubated with mouse sera dilution (1:20), or 10 μg/ml of anti-sLea 121SLE mAb for 0.5h at 4°C and washed with FACS buffer. The sera binding to cells were assessed using PE conjugated anti-mouse IgG or IgM (121SLE) secondary Antibodies. b) 3x104 1199FB cells were incubated with mouse sera dilution (1:20), or 10 μg/ml of anti-sLea 121SLE mAb for 1h at 4°C. Then cells were washed, and rabbit sera complement at 1:10 dilution was added and further incubated at 37°C for 3h. The cell viability was tested with MTS assay. **** p<0.0001. The p values were determined through a two-tailed unpaired t test using GraphPad Prism. ........................................................................................................................... 75 Figure 3. 5 Individual XPAN024 microarray slides stained with pooled serum of 5 mice immunized with Qβ-sLea conjugate(a), sLea and Qβ mixture (b) at day 35 at 1:1000 serum dilution or with 5B1 recombinant antibody at 1.08 μg/ml final concentration (c). Similar cores have been shown here to compare the intensity of staining. Characterization of each core has been provided in appendix, table 3.1 based on their map ID and the pictures of all the cores in appendix, figure 3.15 and 3.16. The brown/red color was due to antibody binding to tissues. The lack of brown/red staining indicates little binding of antibodies to the tissues. Scale bar is 100μm. ........................ 76 Figure 3. 6 XPAN048 microarray stained with pooled serum of 5 mice immunized with Qβ-sLea conjugate at day 35 at 1:1000 serum dilution. Selected cores: N2 and N3 are normal pancreatic tissue, N4 metastatic pancreatic malignant islet cell tumor, N11 Neuroendocrine tumor, N14 Squamous cell carcinoma, N15 Adenosquamous carcinoma, N23 and N32 Adenocarcinoma. Characterization of each core has been provided in appendix, table 3.2 based on their core number and the pictures of all the cores in appendix, figure 3.17. The brown/red color was due to antibody xii binding to tissues. The lack of brown/red staining indicates little binding of antibodies to the tissues. Scale bar is 100μm. .......................................................................................................... 76 Figure 3. 7 Expression of sLea confirmed through FACS experiment by using a) 121SLE mAb and chimeric recombinant antibody 5B1 at 10ug/ml concentration in EL4-FUT3 cells. Pooled serum from group immunized with Qβ-sLea conjugate showed significantly higher binding toward EL4-FUT3 cell line. The absence of non-specific binding was confirmed by using EL4 parent cell line. b) 121SLE mAb at different concentration and pooled sera from Qβ-sLea conjugate in B16- FUT3 cells which showed binding to sLea expressed on cell surface. PE-anti mouse IgG or IgM was used as secondary antibody. .................................................................................................. 78 Figure 3. 8 a) WT C57BL/6 mice were inoculated i.v. with 5 × 105 B16-FUT3 (n=3) or EL4-FUT3 (n=6) tumor cells. 100μg of anti-sLea 5B1 Ab was administered i.p. on days 1, 4, 7, and 11 and survival was assessed daily for EL4-FUT3 group (b), p= 0.0448 and for B16-FUT3 group, mice were euthanized, lungs were excised and fixed fourteen days after cell inoculation (c). ............. 79 Figure 3. 9 The binding of pooled mice sera from day 35, 95, 185 and 356 against sLea antigen at 1:10 dilution with B16-FUT3 cell line were analyzed with flow cytometry with two biological replicates. 356 days after immunization the sera from Qβ-sLea conjugate vaccinated group showed binding toward cell surface expression of sLea antigen. ............................................................... 80 Figure 3. 10 Total tumor area ratio to lung tissue after analyzing the pictures with ImageJ. The tumor area in Qβ-sLea conjugate vaccinated group had lower trending compared with group vaccinated with sLea and Qβ mixture. .......................................................................................... 80 Figure 3. 11 Two New Zealand rabbits were injected subcutaneously on day 0 with 0.1 mL Qβ- sLea constructs (at 8µg glycan) as emulsions in Complete Freund’s Adjuvant according to manufacturer’s instructions. Boosters were given subcutaneously on days 14, 28 and 42 (at 4µg glycan) mixed with Incomplete Freund’s Adjuvant. Serum samples were collected on days 0 (before immunization), 35, 49 and 63. .......................................................................................... 81 Figure 3. 12 ESI-TOF HRMS spectrum for Qβ-sLea conjugate. Mass spectrometry analysis of the Qβ-sLea conjugate showed average loading of 300 tetrasaccharides on viral capsid. Each peaks shows the addition of sLea with MW of 918 Da. The average loading was calculated based on ratio of sum of peaks loading multiplied by their intensity to total intensity of all peaks. ................... 93 Figure 3. 13 MALDI-TOF characterization of BSA-sLea. The molecular weight of BSA shifted from 66,417 Da to 70,146 Da after conjugation. The difference of MW before and after conjugation is divided to the sLea MW (918) to obtain the average loading of four sLea sugar per BSA molecule. ....................................................................................................................................................... 93 Figure 3. 14 Sialyl Lewis a expression in 1242 and 1245 FB cell line versus neo cells was analyzed with flow cytometry. Sera from mice immunized with Qβ-sLea conjugate bind to FB cells very strongly, while binding to Neo cells was similar to the cells without addition of serum in presence or absence of secondary Ab which served as negative control cells. The sLea and Qβ mixture did not show binding to FB or neo cell lines. Anti-Sialyl Lewis a mAb, 121 SLE, served as positive control. ***P = 0.0004, ****P < 0.0001 (unpaired two tailed t test). (The experiments for 1242 xiii and 1245 were done separately with different instrument setting which resulted in different background level).......................................................................................................................... 94 Figure 3. 15 XPAN024 microarray stained with pooled serum of 5 mice immunized with Qβ-sLea conjugate at day 35 at 1:1000 serum dilution. Characterization of each core has been provided in table 1 based on their map ID. The brown/red color was due to antibody binding to tissues. The lack of brown/red staining indicates little binding of antibodies to the tissues. Scale bar is 100μm. ....................................................................................................................................................... 97 Figure 3. 16 XPAN024 microarray stained with pooled serum of 5 mice immunized with sLea and Qβ mixture at day35 at 1:1000 serum dilution. Characterization of each core has been provided in table 1 based on their map ID. The brown/red color was due to antibody binding to tissues. The lack of brown/red staining indicates little binding of antibodies to the tissues. Scale bar is 100μm. ....................................................................................................................................................... 98 Figure 3. 17 XPAN048 microarray stained with pooled serum of 5 mice immunized with Qβ-sLea conjugate at day 35 at 1:1000 serum dilution. Characterization of each core has been provided in table 2 based on their number. The brown/red color was due to antibody binding to tissues. The lack of brown/red staining indicates little binding of antibodies to the tissues. Scale bar is 100μm. ....................................................................................................................................................... 99 Figure 3. 18 XPAN024 microarray hematoxylin and eosin (H&E) staining. Pictures were obtained to determine the inflammatory stage of tissues. Areas with dense nucleus population (dark blue spots) suggests the presence of macrophages. Characterization of each core has been provided in table 1 based on their map ID. Scale bar is 100μm. ................................................................... 101 Figure 3. 19 XPAN024 microarray stained with 5B1 recombinant antibody at 1.08 µg/ml final concentration. Characterization of each core has been provided in table 1 based on their map ID. The brown/red color was due to antibody binding to tissues. The lack of brown/red staining indicates little binding of antibodies to the tissues. Scale bar is 100μm..................................... 102 Figure 3. 20 Images of lung tissues obtained from the mice immunized with a) sLea and Qβ mixture, and b) Qβ-sLea conjugate which challenged with B16-FUT3 cell line 356 days post immunization. ............................................................................................................................. 103 Figure 3. 21 ESI-TOF HRMS spectrum for the 2nd batch of Qβ-sLea conjugate that used for rabbit study. Mass spectrometry analysis of the Qβ-sLea conjugate showed that the number of tetrasaccharides on viral capsid were 455 on average. ............................................................... 103 xiv LIST OF SCHEMES Scheme 2. 1 Qβ conjugation to lactose squarate 3. a) 0.5M pH 7.0 phosphate buffer, r.t., 3h, 76%; b) 0.5M borate buffer, pH 9.0, r.t., 90h......................................................................................... 39 Scheme 2. 2 Conjugation of OSP 1 to Qβ VLP. OSP 1 was activated with dimethyl squarate 2, and subsequently added to a Qβ solution in 0.5M borate buffer, pH=9.0. After 120h, the reaction was worked up by ultrafiltration against pH 7.2 (1x) PBS buffer. ............................................... 42 Scheme 3. 1 synthesis of a) Qβ-sLea conjugate. To a solution of Qβ (1 mg, 10 mg/ml concentration, 0.4 nmol subunit, 0.36 μmol reactive amine) in 0.1 M potassium phosphate (KPB) buffer pH 8.5, was added sLea-NCS (2.5 equivalent to reactive amine) and the reaction was incubated at 37 ℃ overnight and worked up by ultrafiltering the reaction mixture against 0.1 M KPB buffer (pH 7.0, 0.1 M). b) KLH-sLea conjugate was synthesized under a similar condition. ............................... 71 xv KEY TO ABBREVIATIONS ADCC Antibody dependent cell-mediated cytotoxicity APC Antigen presenting cell BCR B-cell receptor BMDC Bone marrow dendritic cells BSA Bovine serum albumin BsAbs Bispecific antibodies CAR Chimeric antigen receptor CDC Complement-dependent cytotoxicity CFA Complete Freund's adjuvant CTL Cytotoxic T cell DC Dendritic cell DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethyl sulfoxide ELISA Enzyme-linked immuno-sorbent assay FACS Fluorescence activated cell sorting FBS Fetal bovine serum FDA Food and Drug Administration FITC Fluorescein isothiocyanate GD2 Disialoganglioside HRP Horseradish peroxidase IFA Incomplete Freund's adjuvant xvi IgG Immunoglobulin G IgM Immunoglobulin M KLH Keyhole limpet hemocyanin LCMS Liquid chromatography–mass spectrometry LPS Lipopolysaccharide mAb Monoclonal antibody MALDI-TOF Matrix assisted laser desorption ionization-time of flight MFI Mean fluorescence intensities MHC I Major histocompatibility complex class I MHC II Major histocompatibility complex class II MPLA Monophosphoryl lipid A mQβ Mutant Qβ MS Mass spectrometry MUC1 Glycoprotein Mucin 1 MW Molecular weight MWCO Molecular weight cut-off OD Optical density OSP O-specific polysaccharide PBS Phosphate-buffered saline PBST Phosphate-buffered saline tween PD-1 Programmed cell death protein 1 PD-L1 programmed death-ligand 1 PE Phycoerythrin xvii Qβ Bacteriophage Qbeta (Qubevirus durum) SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEC Size-exclusion chromatography SELDI-TOF Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry sLea sialyl Lewisa scFv Single-chain variable fragment ST Sialylated Thomsen-Friedenreich antigen STn Sialylated Thomsen-nouveau antigen T antigen Thomsen-Friedenreich antigen TACA Tumor-associated carbohydrate antigen TCR T cell receptor TEM Transmission electron microscopy TFA Trifluoroacetic acid Th Helper T cell TLR Toll-like receptor Tn antigen Thomsen-nouveau antigen TT Tetanus toxoid VLP Virus-like particle wtQβ Wild-type Qβ xviii CHAPTER 1: Recent advances in tumor associated carbohydrate antigen based chimeric antigen receptor T cells and bispecific antibodies for anti-cancer immunotherapy 1.1 Introduction Cancer is a leading cause of death worldwide. The global cancer burden, which has been estimated as 18.1 million new cases and 9.6 million deaths in 2018, is predicted to rise to 29.5 million new incidences in 2040 [1,2]. The alarming increase in cancer burden accentuates the urgent need to develop innovative approaches for more effective and less toxic cancer treatments complementing conventional therapeutics such as chemotherapy, radiotherapy, surgery and palliative care. Cancer immunotherapy holds great promises in cancer treatment, which was named as the Breakthrough of the Year in 2013 by Science [3]. Tumor associated carbohydrate antigens (TACAs) are a class of attractive antigens for anti- cancer immunotherapy development [4,5]. Multiple TACAs are over-expressed on the surface of cancer cells compared to those on normal cells [6]. In addition, TACAs can be found at high levels on many types of cancer, rendering them intriguing targets for potential broad spectrum anti-cancer immunotherapy [7]. Tremendous efforts have been devoted to the development of anti-cancer vaccines targeting TACAs with innovative designs of the antigen structures, the carrier moieties to deliver the antigens to the immune system, as well as the incorporation of immune activation elements including adjuvants and cytotoxic T cell epitopes into the vaccine constructs [8–11]. Besides vaccines, chimeric antigen receptor (CAR) T cells and bispecific antibodies (BsAbs) have emerged as appealing immunotherapeutic strategies to combat cancer, as evident from multiple products approved by the FDA [12]. Recently, great progresses have been achieved in the development of CAR T cells and BsAbs targeting two TACAs, i.e., gangliosides GD2 and glycoprotein mucin-1 (MUC1). In this review, we will discuss the advances and challenges in these areas to stimulate further development. 1 1.2 GD2 CAR T cells: going beyond the anti-GD2 monoclonal antibodies Disialoganglioside GD2 is an N-acetyl neuraminic acid containing glycolipid antigen composed of five monosaccharides anchored to the lipid bilayer of plasma membrane through a ceramide lipid (Figure 1.1). Normal tissues have low expression levels of GD2 [13], but in various types of cancers such as neuroblastoma, small-cell lung cancer, melanoma, glioma, and sarcomas, the expression of this weakly immunogenic antigen can reach 107 molecules per cell [8,14,15]. Furthermore, GD2 has been discovered as a potential biomarker for the purported breast cancer stem cells [16]. GD2 can induce tyrosine phosphorylation leading to the activation of a variety of kinase pathways, resulting in increased proliferation, cell migration and invasion of cancer cells. Knockdown of GD2 levels abrogated tumor formation in vivo [16]. With its high expression on tumor cells and importance in tumor development, GD2 was ranked as one of the top antigenic targets for cancer [14,17]. OH OH - O2C OH HO OH O HO O O O O AcHN AcHN HO -O C HO 2 OH OH HN OH O O O O O O AcHN HO HO OH OH OH N-acetyl neuraminic acid Figure 1. 1 Structure of the GD2 ganglioside. Monoclonal antibodies against GD2 have achieved clinical success in cancer treatment. Early generation of anti-GD2 cancer immunotherapy utilized murine monoclonal antibodies, 14G2a and 3F8, in patients with melanoma, neuroblastoma, and osteosarcoma [14]. However, due to the mouse origin of these antibodies, human anti-mouse antibodies were induced, limiting the dose that can be administered and reducing their anti-tumor efficacy. In addition, administration of these antibodies has been associated with side effects such as pain, fever, hypertension and 2 urticarial reactions, which limit their wide applications. To overcome these challenges, human- mouse chimeric anti-GD2 monoclonal antibodies have been generated [14,18]. ch14.18 consisting of the variable regions of murine 14G2a mAb fused with the constant regions of human IgG1, has higher antibody-dependent cell-mediated cytotoxicity and longer half-life compared to 14G2a. Further development of humanized anti GD2 antibody, hu3F8 and hu14.18K322A with a K322A mutation of the Fc region to prevent complement fixation, improved their half-lives and tolerable dose. However, in clinical trials, similar side effects as m3F8, including pain and hypertension, were observed. Adoptive transfer of CAR T cells is a promising immunotherapy strategy to treat cancer in an MHC-independent manner. CAR T cells are designed by linking the single-chain variable fragment (scFv) of a monoclonal antibody with the T cell receptor (TCR) ζ-chain transmembrane and cytoplasmic regions. Further development of CAR Ts by the addition of costimulatory signal (CD28) for full activation of these cells has led to 2nd generation of CAR Ts and the third generation includes additional signaling domain (CD27, 4-1BB or OX40) aimed at improving proliferation, survival and cytokine release from the cells. With the high anti-tumor efficacy in acute lymphoblastic leukemia, two anti-CD19 CAR T cell-based immunotherapies (KYMRIAH and CARTA) have been approved by FDA. However, applications of CAR T therapy in solid tumors have met with limited successes. Some of the main barriers of CAR T cell therapy include: limited ability of CAR T cells to proliferate, inefficient trafficking of CAR T cells to tumor tissues, limited T cell extravasation into solid tumor, and suppressive tumor microenvironment that dampens T cell proliferation and cytokine production[19]. With the importance and targetability of GD2 for solid tumors, anti GD2 CAR T cell immunotherapy has been investigated in various types of tumors. 3 1.2.1 Building co-stimulatory signals into GD2 CAR T cells One of the first CAR T cells products tested in children was an anti-GD2 CAR containing only the CD3ζ endodomain but no costimulatory domain [20]. In preclinical models, Rossig et al. demonstrated that GD2 was a viable CAR T cell target for neuroblastoma. However, the culture of these first generation CAR T cells could not be maintained for longer than 8 weeks upon stimulation with GD2+ tumor cells. In addition, these CAR T cells failed to proliferate when incubated with GD2+ tumor cells presumably due to a lack of co-stimulatory signals upon antigen binding. Further investigations of GD2 CAR T focused on CAR constructs incorporating endodomain CD28 and OX40 together with the anti-GD2 scFv [21]. The importance of costimulatory signals has been studied in a clinical trial in eleven individuals with neuroblastoma, who had Epstein-Barr virus (EBV)-associated malignancies [22]. EBV-specific cytotoxic T lymphocytes (CTLs) or bulk T cells activated with anti-CD3 antibodies (ATCs) were transduced with a CAR specific for GD2 antigen. Equal numbers of GD2 CAR-CTLs and CAR-ATCs were injected into patients. Persistence of the EBV specific GD2 CAR-CTLs was detectable beyond 6 weeks, which were almost two times longer compared to the GD2 CAR- ATCs. These results highlight the importance of costimulation of native antigen receptor on CAR- CTLs, when these cells engaged EBV antigens on professional antigen-presenting cells through their native receptors. Four of the eight patients (50%) with evaluable tumors had shown tumor necrosis or regressions. A subsequent study with 19 patients including the original 11 patients for up to 4 years showed that although both CAR-CTLs and CAR-ATCs were low or undetectable beyond 6 weeks, the continued presence of even low levels of CAR T cells was associated with a significantly longer time to disease progression. Three patients achieved complete remission out of the 11 patients with active disease at the time of infusion. The proportion of CD4+ helper cells 4 and central memory cells present in infused CAR T cells correlated with the long term persistence of CAR T cells [23]. To sustain the survival of GD2 CAR T cells, additional stimulatory molecule such as interleukin-15 (IL15) has been incorporated generating the GD2 CAR.15 T cells [24]. IL15 provided the survival signals to GD2 CAR.15 T cells while they were in circulation, and costimulation when the cells reached the tumor site. The GD2 CAR.15 T cells expressed lower amounts of programmed cell death protein-1 (PD-1), an immune checkpoint protein, and could proliferate and persist longer in vitro and in vivo even in the absence of exogenous cytokines or antigen support. GD2 CAR.15 T cells were more effective in controlling tumor growth with a lower number of total infused T cells, and in improving survival after rechallenging experiments. Further clinical trials (NCT03294954) for GD2 CAR.15 T cells are ongoing. Besides IL15, another CAR T design incorporated the CD27 costimulatory domains [25], which can augment survival and tumor killing activities of T cells [26]. These CAR T cells showed long term persistence with 30% of infused CAR T cells lasting up to 180 days. Two patients had markedly tumor regression and the 1-year overall survival rate reached 74%. 1.2.2 Enhancing homing and penetration of solid tumor by GD2 CAR T cells An important factor in dictating CAR T efficacy is the ability of these cells to reach solid tumor sites. To aid in trafficking and homing of CAR T cells, chemokine receptor can be incorporated [27]. While earlier finding confirmed the presence of C-C chemokine receptor type 2 (CCR2) on human activated T cells and T cell migration in response to C-C motif ligand 2 (CCL2) chemokine, CCR2 expression was deficient in CAR T cells due to culture conditions and the usage of anti-CD28 antibody, which reduced the levels of CCR2 following TCR activation. In this regard, modified GD2 CAR T cells expressing CCR2b was able to improve the homing and 5 expansion of T cells at tumor site in vivo by day 14. This increased the frequency of T cells at the tumor site correlated with reduced tumor growth. Hence, the CCL2 chemokine concentration at the tumor site can be exploited to attract effector cells to tumor environment. In addition to the incorporation of the chemokine receptor, modification of the tumor microenvironment and vascularity with anti-angiogenic agents such as anti-vascular endothelial growth factor antibody (Bevacizumab, BEV) can improve the trafficking of CAR T cells to tumor [28]. In a mouse neuroblastoma (NB) model, administration of anti-GD2 CAR T cells alone failed to lead to any increases in survival compared to the control group receiving non-transduced T cells. To address this, the combination of BEV and anti-GD2 CAR T was tested. While injection of BEV at a relatively low dose (2 mg/kg) did not significantly affect the number of micro-blood vessels in tumor tissues, addition of BEV to the anti-GD2 CAR T cell treatment regime led to enhanced migration and infiltration to the inner tumor core by these T cells. This was confirmed by double CD31/CD3 immunostaining. The T cells produced interferon-γ and reprogrammed the tumor microenvironment, with the mice receiving the combination therapy showing modest improvements in survival. Interestingly, NB cells were found to upregulate the levels of PD-L1. As GD2-CAR T cells in tumors were found to express programmed cell death protein-1 (PD-1), this study suggested the interactions of PD-1 with its ligand PD-L1 may limit the anti-tumor efficacy. Once reaching tumor tissues, the ability of CAR T cells to penetrate stroma-rich solid tumors is critical for antitumor effects. To facilitate tissue penetration, extracellular matrix (ECM), including the heparan sulfate proteoglycans (HSPGs) need to be degraded. The effect of in vitro culture of T lymphocytes on HSPG degrading heparanase enzyme (HPSE) has been investigated [29]. In a Matrigel - based cell invasion assay, briefly activated T cells showed 1.5 times higher 6 invasion of ECM compared to freshly isolated resting T cells. In contrast, long-term ex vivo– expanded T cells had 3 times lower invasion than the freshly isolated T cells. Mechanistic studies showed that the freshly isolated and briefly activated T cells retained the expression of active HPSE, while in long-term expanded T cells, the enzyme was not detected by either Western blotting or immunofluorescence. Building on this knowledge, CAR T cells were engineered to express HPSE, which exhibited improved capability of ECM degradation and deeper tumor penetration. While both HPSE modified and un-modified GD2-CAR T cells had similar efficiencies in lysing GD2+ human cell lines in vitro, HPSE expressing GD2-CAR T cells provided significantly improved protection to mice from GD2+ tumor cell induced death. 1.2.3 Combination of GD2 CAR T therapy with checkpoint blockade and chemotherapy Improvement of GD2 CAR T cell therapy was investigated in combination with anti-PD-1 blockade to reduce T cell exhaustion and improve survival. In vitro study showed that repeated antigen stimulation reduced the percentage of viable GD2 CAR T cells [30]. This activation induced cell death (AICD) of CAR T cells is dependent on the level of antigen expression since CAR T cell survival was higher in cell lines with lower GD2 expression. The AICD process could be potentially reversed with anti-PD-1 blockade, as treatment with the anti-PD-1 mAb was able to restore CAR T-cell survival similar to the level observed for CAR T cells cultured with GD2- cell lines. Interestingly, while siRNA knockdown of PD-1 enhanced the CAR T-cell viability, PD-L1 was found to have a more complex role. PD-L1 signaling via PD-1 caused inhibition or deletion of activated PD-1+ T cells [31,32], whereas signaling via CD80 stimulated naive T cells [33]. Furthermore, PD-L1 could mediate reverse signaling upon binding to PD-1 within PD-L1+ T cells and promote their survival. SiRNA knockdown of PD-L1 significantly reduced cell survival. Following the detailed in vitro study, the GD2 CAR T construct was administrated to four 7 metastatic melanoma patients. The GD2-CAR population declined in all patients beyond day 28 post-infusion and failed to persist in two patients. These observations suggested that CAR T cells were also being depleted in vivo. Based on the in vitro data, combination of PD-1 blockade with CAR T therapy may help to augment the efficacy and persistence of CAR T cells. GD2-CAR T cell treatment can be combined with chemotherapy to enhance efficacy. Heczey et. al [34] reported their clinical results in NB treatment, with three cohorts receiving GD2- CAR T, GD2-CAR T with cyclophosphamide and fludarabine (Cy/Flu) prior to cell infusions, and GD2-CAR T co-administered with Cy/Flu and PD-1 inhibitor respectively. GD2-CAR T and Cy/Flu treatment led to superior T cell expansion and higher levels of IL15 in the blood. Co- administering a PD-1 inhibitor did not make any differences in expansion, persistence of the cells, or circulating cytokine levels in this report. In contrast, other CAR T studies demonstrated beneficial clinical outcome when combined with PD-1 inhibitors [35–37], which may be due to variations in cancer types, antigen targets and timing/duration of PD-1 inhibitor treatment. Thus, careful optimization is necessary for each treatment protocol. 1.2.4 Reengineering of the GD2 CAR As many anti-GD2 mAbs originated from mice, administration of CAR T cells based on these antibodies can potentially generate anti-idiotype or anti-mouse antibodies recognizing the CAR and causing immune rejections. Thomas et.al [38] modified GD2-CAR T to incorporate humanized scFv of anti-GD2 mAb. This humanized CAR T had the same efficiency in proliferation and cytokine release as its murine counterpart. In addition, the effect of spacer between the ScFv and CD28 on CAR function was investigated. The inclusion of IgG Fc domain (Figure 1.2a) showed optimal efficiency compared to CAR T comprising of the hinge alone (Figure 1.2b), the hinge attached to the stalk of CD8a (Figure 1.2c) or the CD8a stalk alone 8 (Figure 1.2d). These results showed the flexibility and binding ability to target antigen can play an important role in CAR T activation and cytokine release. Figure 1. 2 Schematic demonstration of various GD2 CAR constructs. a) The CAR includes the hinge region as well as the Fc domain (CH2 and CH3); b) CAR without the Fc domain; c) CAR with the hinge attached to the stalk of CD8a; and d) CAR with CD8a stalk only without the hinge region. (Image adapted from [38]) Effective targeting of GD2 specific CAR T cells is a challenging task. Richman et.al.[39] showed by mutation of the aspartic acid residue 101 in the heavy chain of CAR scFv to lysine (E101HK), the affinity of GD2-CAR T cells could be significantly improved towards various GD2+ cell lines. In a mouse liver cancer model, histologic analysis of livers of mice treated with this construct showed less than 1% tumor cell foci compared to >95% in control and parent GD2 CAR T cell treated groups highlighting the efficacy of these engineered CAR T cells. However, all the mice treated developed severe neurotoxicity associated with T cell infiltration and proliferation into their brains presumably due to the expression of GD2 in brain tissues and the “on-target, off- tumor tissue” toxicity. Enhancing the safety profile of CAR T cell therapy while achieving high anti-cancer efficacy of GD2 CAR T cells requires further cellular engineering. One potential cause for toxicity is the recognition of Fc on CAR-T by cells with Fcγ receptors. Since IgG Fc is the natural ligand for high affinity Fcγ receptors (CD64), mutation of the Fc domain, which is commonly used as the spacer between the antigen recognition domain and the intracellular signaling domain in CAR 9 structure (Figure 1.2a), significantly reduced the off target toxicity of GD2-CAR T cells by preventing the engagement of CD64 expressing myeloid cells with CAR T cells [38]. An alternative approach to avoid the “on-target, off-tumor” effect is to build in inducible suicide genes. The inducible Caspase 9 (iCasp9) suicide gene was incorporated into the CAR of GD2 CAR T cells [38,40]. The iCasp9 was not toxic at basal expression levels. Administration of the small molecule dimerizer drug to the cells induced the expression of iCasp9, resulting in rapid onset of apoptosis in transduced cells. Activated cells expressing the CAR were preferentially killed, providing a safety switch. Similarly, the iCasp9 suicide gene has been applied to another type of GD2 CAR T, i.e., GD2 CAR.15 T cells, enhancing the safety profile of the construct [24]. Mitwasi et al. [41] designed a new type CAR T, termed universal CAR T (UniCAR), whose activation is dependent on a target module (TM), and can be potentially turned on and off via dosing of TM. The scFv binding domain of UniCAR recognized a short peptide epitope of 10 amino acid derived from the human nuclear autoantigen (5B9). The UniCAR cells were inactive after infusion since the target epitope did not exist on the cell surface. A TM was synthesized by fusing the 5B9 antigen to GD2 binding scFv (Figure 1.3). In the presence of the TM, UniCARs were able to specifically target GD2+ tumor in a TM-dependent manner. At effector to target cell ratio of 5:1, only less than 0.1 nM of GD2-TMs was needed for uniCAR T to mediate the lysis of tumor cells. No killings were observed in control cell lines or in the absence of TMs. In contrast to other safety switches such as modification with inducible suicide genes, the uniCAR T cells were unique that the activities could be modulated rapidly via dosing of a target module. 10 A) B) Figure 1. 3 Construction of UniCAR and TM. A) UniCAR cells do not recognize tumor cells in the absence of TM due to the lack of receptor on T cells towards tumor antigens. B) Upon addition of the TM comprised of the conjugate of anti-GD2 scFv and E5B9, the UniCAR can bind with the TM through E5B9, thus gaining the abilities to recognize GD2+ tumor cells. 1.3 MUC1 CAR T cells The human mucin 1 (MUC1) are high molecular weight glycoproteins expressed on the epithelial cell layers in the lung, breast, pancreas, kidney, ovary, colon, and other tissues to provide protection of these cells, which are exposed to external environments [42]. MUC1 consists of two subunits, MUC1-N and MUC1-C, which form a stable non-covalent complex at the cell surface. MUC1-N contains a variable number of 20 amino acid tandem repeats (VNTR) that are heavily glycosylated on serine or threonine residues of each VNTR in normal cells (Figure 1.4). However, on cancer cells, owing to altered glycosyltransferase expression, tumor-associated MUC1 is decorated by a preponderance of shorter glycans, including Thomsen-nouveau (Tn), sialyl Tn (STn), Thomsen-Friedenreich (Tf), and sialyl-T (STf) (Figure 1.4). Under-glycosylation of MUC1 unmasks cryptic epitopes within the extracellular domain, enabling tumor-selective binding by antibodies. 11 Figure 1. 4 Schematic demonstration of MUC1 glycoprotein structure. MUC1 is composed of a heterodimer of MUC1-N linked non-covalently with the transmembrane MUC1-C. MUC1-N contains a variable number of 20 amino acid VNTRs that are heavily glycosylated on serine or threonine residues of each VNTR in normal cells shielding the protein backbone for immune recognition. However, tumor associated MUC1 are hypoglycosylated exposing the protein backbone. 1.3.1 MUC1 epitope structures for CAR T cell targeting Multiple mAbs are available against the MUC1-N domain. A representative example is AS1402, which has been evaluated in phase 1 clinical trial for breast cancer patients [43]. However, the result showed that the addition of AS1402 to chemotherapy had the same efficacy compared to chemotherapy treatment alone [44]. As MUC1-N is known to be shed from cancer cell surface into circulation, the extracellular pool of MUC1-N in plasma is considered a major barrier for antibody-dependent cellular toxicity against MUC1-N. In addition to passive immunity using anti-MUC1 mAbs, vaccines targeting tumor associated MUC1 have been evaluated, 12 including L-BLP25 and PANVAC-V, which have been tested in late-stage clinical trials for the treatment of breast cancer [43,45–47]. However, no successful anti-MUC1 vaccines are available yet. As an alternative to mAbs and vaccines, anti-MUC1 CAR T cells have been investigated. One of the key considerations in designing anti-MUC1 CAR T cells is the epitope structure targeted by the CAR. As MUC1 is a large glycoprotein, there are many potential sequences with MUC1 that can be targeted. Early MUC1 CAR T design utilized scFvs derived from anti-MUC1 mAb SM3 [48]. While SM3 recognized well the unglycosylated PDTR peptide within the VNTR region of MUC1-N, it bound poorly with glycopeptides such as the STf antigen present on many types of cancer cells. As a result, the SM3 MUC1 CAR T cells did not respond strongly to MUC1 expressing tumor cells. Nevertheless, MUC1 CAR T cells expressing a mutated analog of SM3 to improve MUC1 binding, were evaluated in a first-in-human study by direct injection into tumor lesions [49]. Reduction of MUC1 expression due to apoptosis and necrosis of tumor cells were observed in solid tumors treated with these CAR T cells, providing new options for improved CAR T therapy. To overcome the low MUC1 affinity of SM3 mAb, another anti-MUC1 mAb, HMFG2 was utilized to construct the CAR [48]. HMFG2 has higher affinities to MUC1 than SM3 and can bind STf bearing MUC1 glycopeptides. In order to reduce the steric hindrance posted by immobilized MUC1, an IgD hinge was inserted into the CAR yielding a potent receptor containing a fused CD28/OX40/CD3ζ endodomain, so that the Fab regions can engage antigen in virtually any orientations. These HMFG2-CD28-OX40-CD3ζ CAR T cells proliferated upon encountering the MUC1 antigen, mediated production of proinflammatory cytokines, and killed MUC1+ tumor cells. When tested in a preclinical model, these CAR T cells led to a significant delay of tumor growth [48,50,51]. 13 A potential drawback of the HMFG2 antibody is that besides the glycosylated form of MUC1, HMFG2 also recognizes unglycosylated MUC1 peptide, which raises concerns of possible auto-immunity due to the binding with unglycosylated MUC1 in normal tissues. Anti-MUC1 mAb 5E5 is an antibody that has been shown to have high affinities for the Tn glycosylated MUC1 (MUC1-Tn) with little cross-reactivity with the non-glycosylated MUC1-60-mer peptide or normal human tissues, supporting the more cancer specific expression of Tn-glycopeptide epitopes [52]. With the knowledge of high tumor selectivity by 5E5, Posey et. al. developed MUC1-Tn CAR T cells using 5E5, which recognized multiple tumor cell lines expressing MUC1-Tn [53]. Increasing the expression of enzymes in tumor cells that converts Tn to other glycans reduced the levels of MUC1-Tn on tumor cells. As a result, the 5E5 CAR T cell induced cytotoxicity to these cells were significantly suppressed, which may be concerning for cancer treatment due to the heterogeneity of MUC1 glycosylation patterns on cancer cells. Interestingly, when evaluated in xenograft model of human pancreatic cancer studies, 5E5 CAR T cells provided complete protection to tumor bearing mice, suggesting that 5E5 driven CAR T cells are able to detect low levels of antigen targets, and antigenic diversity does not preclude tumor eradication [53]. Another example of CAR-T design against the exposed peptide backbone of aberrantly glycosylated MUC1 is to use scFv derived from anti-MUC1 mAb (TAB004) for recognition of STAPPVHNV peptide sequence within the VNTR region. The scFv was fused to CD28 and CD3ζ T cell intracellular signaling molecule to generate the CAR T cells, which were evaluated in triple- negative breast cancer (TNBC) [54]. MUC1 CAR T cell mediated tumor cell lysis correlated with MUC1 expression levels in vitro without significant lysis of normal breast epithelial tissues, suggesting their high tumor selectivities. Treatment of HCC70 tumor-bearing mice with this construct dramatically reduced the tumor growth from as early as 4-days post T cell injection, 14 which lasted up to 57 days. However, the tumor started to progress faster after about 60 days post treatment compared to the control group. Histological analysis showed that MUC1 expression levels were similar in both treatment groups, suggesting MUC1 tumor antigen down-regulation to avoid immune clearance by MUC1 CAR T cells is likely not a major contributing factor to tumor escape. Instead, an approximate two-fold increase of PD-1 expression on tumor cells was observed after CAR T cell treatment, which may be a major reason causing the exhaustion of CAR T cells. Increased lysis potency by MUC1 CAR T cells with pretreatment of an anti-PD1 antibody suggested the potential beneficial combination of CAR T cells with an anti-PD1 antibody for future evaluations. 1.3.2 Building costimulatory signals into MUC1 based CAR T cells and dual targeting of tumor cells To enhance the anti-tumor efficacy and reduce tumor escape from the immune pressure, an attractive strategy is to combine multiple types of CAR T cells. This combinational CAR T cell therapy has been investigated for the treatment of non-small-cell lung cancer [55]. Prostate stem cell antigen (PSCA) is frequently expressed in non-small-cell lung cancer along with MUC1 antigen. MUC1 and PSCA CAR T cells were constructed based on HMFG2 and humanized 1G8 antibodies respectively. When administered individually, MUC1 and PSCA CAR T cells significantly reduced tumor growth in a mouse non-small-cell lung cancer model compared to mock groups receiving non-tumor targeting CAR T cells. Combining the MUC1 and PSCA CAR T cells led to further reduction of tumor sizes, highlighting the power of this combinatorial approach. Costimulatory signals such as CD28 and CD3ζ are critical to sustain CAR T cell proliferation, enhance cytotoxicity, and resist AICD. However, as the CAR T cells become more 15 powerful, toxicities have emerged [56,57], which are often attributed to “on target, off tumor” effects due to the presence of tumor – associated antigens in healthy tissues off tumor sites. CAR T cells are commonly designed with both CD28 and CD3ζ on the same gene (also known as cis arrangement). Upon CAR engagement, CD28 and CD3ζ are both activated with CD3ζ signaling eliciting cytotoxicity and interferon-γ production, and CD28 creating signal 2 to promote T-cell proliferation and interleukin (IL)-2 production. One novel design to increase the specificity of CARs and improve their activity and safety is to physically separate the two stimulatory signals by incorporating them into 2 distinct CARs specific for 2 different antigens (trans signaling). In this regard, Wilkie and his coworkers engineered CAR T cells to simultaneously target ErbB2 and MUC1, two common breast cancer associated antigens [58]. The resulting CAR T, called ITH, coexpresses the ErbB2 specific CAR using scFv of anti ErbB2 mAb followed by CD3ζ signaling domain (Iz1) and MUC1 specific CARs based on scFv of anti MUC1 mAb, HMFG2, coupled to CD28 domain (HDF28). The two transgenes were co-expressed in equal amounts by separating the inserts with a Thosea asigna (T2A) peptide. The ITH construct could deliver complementary signals required for proliferation and lead to greater expansion of T cells compared to control CAR Ts with CD28 or CD3ζ alone. ITH CAR T cells showed similar cytolytic efficiency as anti-ErbB2 CAR T cells against multiple breast cancer cell lines with varying levels of ErbB2 and MUC1 expression. Deeper analysis of ITH CAR T cells was performed. It was notable that the production of IL-2 by ITH CARs was much lower compared to the CARs with cis fused CD28 + CD3ζ endodomain. Possible reasons included the conformational changes of scFv induced after binding of the first antigen to its corresponding scFv, which may hinder the optimal binding of the second scFv with its respective antigen, posing steric challenge for simultaneous targeting of both target 16 antigens [59]. Alternatively, co-stimulatory signaling from the ITH CAR T cells may be either less efficient or distinct from that provided by CARs containing a fused CD28 + CD3ζ endodomain. Besides the MUC1 and ErbB2 combination for ITH CAR T cells, other dual targeting CAR T cells with CAR pairs such as prostate antigens PSMA/PSCA and mesothelin/folate receptor have been evaluated in vitro and in vivo [60,61]. These studies showed that co-transduced trans- signaling CAR T cells could destroy tumors expressing both antigens, without affecting those with either antigen alone. Thus, a dual targeting, trans-signaling CAR approach can enhance the anti- cancer efficacy of CAR T cells while minimizing off tumor side effects against normal tissues bearing single antigen. 1.4 Bispecific antibodies targeting GD2 and MUC1 Bispecific antibodies (BsAbs) are engineered antibodies that can simultaneously engage two unique epitopes, such as ligands, receptors, and cytokines. While there are many different designs of BsAbs in development, they can be generally classified into two categories: immunoglobulin G (IgG)-like molecules and non-IgG-like molecules [62]. With two heavy and two light chains, the overall geometry of IgG like BsAbs resemble those of IgG (Figure 1.5a). However, unlike traditional IgGs, the two chains of BsAbs recognize two distinct epitopes. The IgG like BsAbs contain the Fc domain, which can mediate effector functions such as antibody- dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antibody- dependent cellular phagocytosis. In comparison, the non-IgG like BsAbs lack the entire Fc domain (Figure 1.5b). These BsAbs can include chemically linked Fabs, various types of bivalent and trivalent single-chain variable fragments (scFvs) with different epitope specificities. 17 Figure 1. 5 Schematic representation of various BsAb formats. a) IgG-like BsAbs: i) and ii) IgG- scFv, iii) triomab, iv) quadroma, and v) half molecule exchange format. And b) non-IgG-like BsAb: i) tandem scFv, ii) dual-affinity re-targeting antibody, iii) bi-nanobody, iv) scFv-human serum albumin-scFv. BsAbs may improve tumor targeting specificity by binding two tumor-associated antigens. Furthermore, BsAbs can be designed to engage immune system effector cells, usually through CD3 on cytotoxic T cells or CD16 on natural killer cells. This can bring the immune cells into close proximity with cancer cells and lead to the killing of cancer cells through perforin/granzyme- mediated non-MHC-restricted specific antitumor cytotoxicity [63–66]. 1.4.1 GD2 targeting BsAbs The most common TACA based BsAbs target GD2. One type of BsAbs was produced by chemically conjugating anti-CD3 (OKT3) and anti-GD2 (3F8) antibodies through a heterobifunctional linker [67]. This BsAb could coat expanded T cells in vitro, and help direct the activated T cells to neuroblastoma cells expressing GD2 antigens. The BsAb treatment could kill GD2 positive cells more effectively than T cells or 3F8 antibody treatment alone. Furthermore, it is highly promising that compared to intravenous injection of 3F8 mAb alone, the dose required for 3F8BsAb was 200 times lower for similar efficacy. This is a major advantage, as the lower 18 dose needed can potentially reduce the dose-limiting pain caused by 3F8 binding to peripheral nerve fibers. A second type anti-GD2 BsAb, Ektomab, was built in a quadroma-based format. One binding arm of Ektomab is from mouse IgG2a ME361, which can recognize ganglioside GD2 albeit with a lower affinity compared to 3F8. The other binding arm is from rat IgG2b recognizing CD3 on human T cells. Significant lytic activity of Ektomab could be observed down to 40 ng/ml. However, both murine and rat components could induce neutralizing antibodies in humans, which diminish their effects rendering the need for higher doses. To reduce anti-mouse/rat antibodies generated in humans, the first humanized IgG-scFv BsAb was developed for targeting GD2. In this work, the scFv of humanized mouse anti CD3 was attached to the carboxy end of anti GD2 light chain antibody (Figure 1.6) [68]. The BsAb has the same binding affinity for GD2 as parental IgG but CD3 binding was significantly reduced compared to parental anti-CD3 mAb. Using humanized anti GD2 and CD3 antibodies lowered the amounts of neutralizing antibodies generated when administered in humans. The Hu3f8-BsAb was highly potent against GD2+ tumor with cytotoxicity at femtomolar concentrations and greater than 105 fold selectivity over normal tissues. In addition, asparagine 297 of the Fc was mutated to alanine to remove the Fc glycosylation, which was proposed to reduce the risk of cytokine storm syndrome by preventing Fc receptor mediated binding. The Hu3F8-BsAb effectively reduced tumor growth in humanized mouse model with a high safety profile. Mechanistic studies showed that besides T cells, monocytes also play critical roles in sustaining T-cell infiltration of tumor stroma, survival, or proliferation, and contributing significantly to the exceptional antitumor effect of Hu3f8-BsAb. 19 hu3F8-BsAb Anti GD2 Anti GD2 Anti CD3 Anti CD3 N297A Figure 1. 6 Schematic demonstration of the Hu3F8-BsAb structure. A non-IgG like anti-GD2 BsAb was designed by a tandem fusion of single chain variable fragment of Hu3F8 to anti-CD3 antibody HuOKT3-scFv with a 15-residue linker (GGGGS)3 producing the hu3F8-scBA (Figure 1.7) [69]. This engineered BsAb contained the human anti GD2 (Hu3F8-scFv) rather than the murine anti-GD2 mAb 5F11 in earlier generation of the BsAb, resulting in a 13 fold higher affinity for GD2. This higher affinity led to stronger T cell activation and cytokine release in vitro. T cell cytotoxicity assays against GD2 expressing human cancer cells showed that Hu3F8-scFv was highly potent with EC50 values in the femtomolar range, which was up to 5,000-fold stronger than the 5F11 version of the BsAb. Hu3f8-scBA showed significant suppression of tumor growth in DKO mice with human neuroblastoma/melanoma xenografts. One drawback of Hu3F8-scFv is that this antibody has lower thermal stability, which needs to be improved for future clinical applications. Despite the decrease in thermal stability, this study highlighted that even relatively modest increases in antigen affinity could lead to substantial enhancement of the functional properties of the BsAb. hu3F8-scBA VL VH Anti-GD2 scFv VL VH Anti-huCD3 scFv Figure 1. 7 scFv-based bispecific antibody format of Hu3F8-scBA (VH is the heavy chain of the variable region, and the VL is the light chain of the variable region). 20 BsAbs can be applied in combinatorial therapy to enhance the effectiveness of the therapy and address immune suppression encountered. Deppisch et. al. constructed a BsAb antibody termed Surek, which targets both GD2 and the CD3 receptor on murine T cells with the Fc region consisting of mouse IgG2a and rat IgG2b isotypes recognizable by Fc receptors [70]. The combination of mouse IgG2a and rat IgG2b isotypes can facilitate the purification of quadroma bispecific antibodies by affinity chromatography using protein A taking advantage of the differential affinities of various IgG isotypes with protein A. The Surek was able to successfully recruit T cells to tumor tissues. However, detailed analysis showed that cytotoxic T-lymphocyte- associated protein 4 (CTLA-4), another immune checkpoint protein, was upregulated in these redirected T cells, which led to the addition of an anti-CTLA-4 mAb for treatment. The combined Surek and anti-CTLA-4 mAb regime increased the overall survival of mice challenged with B78- D14 melanoma to 90% compared to the Surek alone (60%), while only 20% of the mice in the group receiving anti-CTLA-4 mAb alone survived the tumor. However, the survival advantage was more modest in another melanoma model B16-EpCAM. Besides the direct effect of Surek on tumor cells, Surek could induce tumor-specific humoral immune responses [16], enhancing tumor protection in vivo. Combined administration of Surek with CTLA-4 blockade improved humoral immunity against cancer, which correlated with increased serum titers of melanoma-reactive IgG2a antibodies. The combinatorial vaccination markedly increased the number of memory CD4+ T cells [70]. Survival rate of mice challenged with tumor three weeks after immunization receiving both Surek and anti-CTLA-4 antibodies was 40% better than the group immunized with Surek only. 21 1.4.2 MUC1 based BsAbs Compared to GD2 based BsAbs, there are far fewer studies on MUC1 based BsAbs. Two BsAbs, i.e., MUC1 x CD3 and MUC1 x CD28 were constructed [71]. These two BsAbs reacted well with MUC1+ tumor cells, and in the presence of interleukin-12 activated killer cells, were able to exhibit significant cytotoxicity to tumor cells. The cytotoxicity by MUC1 x CD3 BsAb alone was similar to those when the MUC1 x CD3 and MUC1 x CD28 BsAbs were combined. When evaluated in a mouse tumor model, MUC1 x CD3 BsAb was able to significantly slow down the growth of TFK-1, a type of human bile duct carcinoma. In addition to T cells, natural killer cells (NK cells) are another type of effector cells capable of killing infected or malignant cells. NK cell mediated antibody-dependent cellular cytotoxicity plays a major role in antibody mediated targeted cancer therapy, including anti-MUC1 and anti-GD2 monoclonal antibody treatment [72–74]. As an alternative to T cell recruiting BsAbs, bispecific antibody capable of binding with NK cells has been investigated for TACAs. A new non-IgG like BsAb was designed by linking single domain anti-MUC1 and anti-CD16 antibodies through a short dipeptide linker. This BsAb could be efficiently expressed in E. coli, and recruit NK cells to bestow potent killing of MUC1 expressing tumor cells. In mouse xenograft tumor models, the BsAb provided significant protection from human colon cancer LS174 growth [75]. 1.4.3 CAR T cells vs BsAbs A direct comparison study of GD2 targeting BsAb (Hu3F8 x anti CD3) vs CAR T cells was reported by Cheung and coworkers [76]. When incubated with GD2 expressing tumor cells in vitro, a majority of CAR T cells with high density receptors were depleted while T cells stimulated with BsAb survived. Upregulations of PD1 and LAG3 marker expression were seen in both groups after stimulation with target cells suggesting that over-expression of PD1 was not the exhaustion 22 signal in this study and blocking of PD1 did not improve the CAR T cell survival. CAR T cell exhaustion was observed in all generated CAR T cells with different affinities, indicating that the density of CAR on the cell surface rather than the affinity of the CAR itself may be the important determinant of the GD2 CAR T cell depletion. BsAb in conjunction with untransduced T cells showed superior antitumor activity in vivo in human melanoma xenograft model. While both CAR T cells and BsAb recruited T cells penetrated into the tumor sites, the number of tumor infiltrating lymphocytes (TIL) was greater with untransduced T cells plus BsAb treatment compared to the CAR T cell group, which can be the result of higher proliferation of T cells in presence of BsAb. The TIL showed equal percentage of CD4+ and CD8+ T cell population for BsAb group versus CAR T group with almost all the CAR T cells expressing CD8+. This absence of CD4+ T cell may have compromised the therapeutic response of CAR T cells [76]. 1.5 Conclusions and perspectives With the overexpression on a wide range of cancer cells, TACAs are attractive targets for immunotherapy development. Anti-TACA CAR T cells and BsAbs are exciting directions for TACA based immunotherapy. While the concept of CAR T cells can be relatively straightforward, multiple parameters need to be established for effective CAR T therapy. Early studies of GD2 CAR T cells demonstrated that it is important to build in costimulatory domains such as CD28 and OX40 into the T cells in order to maintain the abilities of cells to proliferate, as well as to reduce T cell exhaustion and AICD. Other stimulatory molecules including IL15 and CD27 have been incorporated into GD2 CAR T cells, which enabled long term persistence of the cells in human patients and led to improved clinical outcome. Co-administration of immune checkpoint blockade 23 through anti-PD-1 antibody may help further augment the efficacy and persistence of GD2 CAR T cells. To overcome low penetration of CAR T cells into solid tumor tissues, GD2 CAR T therapy has been combined with anti-angiogenic agents as well as heparanase for digestion of extracellular matrix. This led to significantly improved protection of mice from GD2+ tumor induced death. As GD2 CAR T cells become more potent, the “on target, off tumor” binding of T cells to low level of GD2 expressed in normal tissues can cause serious side effects including neurotoxicity. To address this challenge, CAR engineering can be performed to reduce Fc receptor binding to CAR. Furthermore, innovative research has been established to introduce inducible suicide gene or design target modules to turn on and turn off the CAR T responses when necessary. Similar to GD2 CAR T cells, the design of effective MUC1 CAR T cells required built-in co-stimulatory signals to sustain CAR T cell proliferation, enhance cytotoxicity, and resist AICD. Another major consideration for MUC1 CAR T cells is the MUC1 epitope to be targeted as MUC1 glycoprotein contains many potential antigenic sites. Early generations of the cells were based on antibodies recognizing the unglycosylated MUC1 peptides in the VNTR region of MUC1-N. While in preclinical models, these CAR T cells delayed tumor growth, there were concerns regarding potential auto-immunity. To address this issue, MUC1 CAR T cells were developed targeting the more tumor specific MUC1-Tn glycopeptides, which recognized multiple tumor cell lines. When evaluated in xenograft model of human pancreatic cancer studies, these CAR T cells provided complete protection to tumor bearing mice. As an alternative to CAR T cells, bispecific antibodies have shown great promises in anti- cancer therapy. The most common type of TACA based BsAbs targets GD2 and CD3 on T cells. Engineering of the BsAbs include humanization of the non-human origin scFv to reduce the level 24 of neutralization antibodies, and Fc mutation to reduce the risk of cytokine storm syndrome. These BsAbs can direct activated T cells to GD2 expressing tumor tissues, leading to significant tumor lysis even with femtomolar concentrations of the BsAbs. A direct comparison study has been performed to compare GD2 BsAbs and CAR T cells. Administration of BsAbs in conjunction with untransduced T cells were found to lead to longer survival of activated T cells. Furthermore, BsAbs-T cells provided more effective tumor protection in tumor models. The superiority of BsAbs-T cells could be partly attributed to the presence of CD4+ helper T cells in BsAbs-T cells, while the infused CAR T cells were almost exclusively CD8+ T cells. Building on the results from the reported CAR T cell and BsAb studies, a promising direction to explore is to test novel combination therapies. As cancer cells may mutate to downregulate the levels of targeted antigen under immune pressure, constructs that can potentially bind with multiple types of antigens may help reduce the chances of tumor escape and enhance treatment efficacy. Other combinations can include the addition of checkpoint inhibition such as anti-PD1 and anti-CTLA4 mAbs, agents that can enhance the access and penetration of immune cells to solid tumor, as well as chemotherapeutic agents. In order to guide such studies, it is critical to gain a more thorough understanding of the tumor microenvironment and factors in suppressing the immune responses. These approaches may overcome the inhibitory signals in tumor microenvironment, high interstitial fluid pressure, compressed vasculature and dense fibrotic tissue surrounding solid tumors and increase the delivery of immunotherapeutic agents to solid tumors. A critical factor in the success of immunotherapy is in minimizing potential toxicity and side effects, especially considering many tumor antigens can also be found in normal tissues, albeit 25 at low levels. To reduce the “on target, off tumor” side effects, further investigation for characterization of TACA structures in solid tumor tissues can lead to the discovery of unique TACA derivatives. For example, the expression of the 9-O-acetylated GD2 gangliosides has been reported to be restricted to tumor tissues, while they are not found in normal tissues [77]. Furthermore, compared to normal cells, tumors tend to contain more N-glycolyl neuraminic acid [78,79]. These rarer structures may provide more specific targets for anti-cancer immunotherapy to enhance the safety of the treatment. 26 REFERENCES 27 REFERENCES [1] World Health Organisation, Latest global cancer data, Int. Agency Res. Cancer. 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Oncol. 4 MAR (2014) 33. doi:10.3389/fonc.2014.00033. [79] S. Inoue, C. Sato, K. Kitajima, Extensive enrichment of N-glycolylneuraminic acid in extracellular sialoglycoproteins abundantly synthesized and secreted by human cancer cells, Glycobiology 20 (2010) 752–762. doi:10.1093/glycob/cwq030. 35 CHAPTER 2: Virus like particle display of Vibrio cholerae O-specific polysaccharide as a potential vaccine against cholera 2.1 Introduction Cholera is an acute, secretory diarrheal disease caused by the highly transmissible bacterium Vibrio cholerae. V (V. cholerae). cholerae are gram-negative and highly motile bacteria with a single polar flagellum. There are more than 200 serogroups of V. cholerae based on the O antigen of surface lipopolysaccharide (LPS) structures, but only serogroups O1 and O139 are capable of causing epidemic cholera. V. cholerae O1 has two serotypes, i.e., Ogawa and Inaba, based on the presence or absence of a methyl group on the non-reducing terminal perosamine moiety of the surface O-specific polysaccharide (OSP, O-antigen) [1]. There are 2-3 million cases of cholera each year, resulting in tens of thousands of deaths annually [2]. Current cholera vaccines include oral killed whole cell vaccine with or without cholera toxin B subunit (CtxB), and attenuated oral cholera vaccine [3]. Inclusion of the cholera vaccine into global cholera control strategies has been transformative, but current oral vaccines have the lowest level and duration of protection in young children [4–8], who bear a large share of global cholera burden, especially in cholera-endemic countries [2,3,9–11]. As such, there is a need to develop new cholera vaccines that can provide high level and long-term immunity. Immunity protective against cholera infection targets the OSP of V. cholerae [12,13]. However, as O-antigens are T cell independent B cells antigens, direct administration of the O- antigens often only leads to low titers of low℃affinity IgM antibodies with limited duration of antibody responses and a lack of induction of immunological memory, rendering O-antigen-based vaccination suboptimal [14–16]. Covalent linkage of carbohydrate to a carrier protein provides a T cell-dependent immune response by activating CD4+ T cells and enables memory B cell proliferation for long lasting antibody protection. Recently, we have demonstrated that self℃ 36 assembled virus℃like particles (VLPs) such as bacteriophage Qβ could be used to conjugate with carbohydrate antigens such as the Thomsen-Nouveau (Tn) antigen and ganglioside GM2 and GD2 as potential vaccines [17,18]. The resulting glycoconjugates were able to induce strong glycan specific IgG antibody responses. However, to date, only low molecular weight (MW generally below 2,000 Da) glycans have been investigated for Qβ based anti-carbohydrate vaccine studies. It is not known whether bacterial polysaccharide antigens could be conjugated with Qβ and whether such conjugates could induce strong IgG antibody responses to such polysaccharides. Herein, we report that the native O-specific polysaccharide (OSP) 1 of Vibrio cholerae O1, Inaba serotype was successfully conjugated with Qβ through squarate chemistry, which is the first time that a bacterial polysaccharide antigen is covalently linked with Qβ as a potential vaccine. This approach provides direct conjugation without prior introduction of a linker to the protein carrier. High levels of anti-polysaccharide IgG antibodies were induced by the conjugate in mice, and the antibodies were effective in killing the bacteria. Figure 2. 1 Structure of O-specific polysaccharide (OSP) 1 of Vibrio cholerae O1 37 2.2 Results 2.2.1 Conjugation of the OSP core antigen to Qβ To efficiently link the OSP antigen to Qβ, we built upon previous work using squarate based conjugation of polysaccharide (the conjugation and characterization part of the work were performed by Dr. Peng Xu) [19–21]. 3,4-Dimethoxy-3-cyclobutene-1,2-dione (dimethyl squarate) 2 is a unique bifunctional linker, both of whose methoxy groups are reactive with amines but under different pH conditions. The first methoxy group can be substituted by a primary amine in neutral pH and the second one is active toward amines in a basic solution. The squaramide moiety itself is stable to hydrolysis under the aqueous reaction conditions [22]. To optimize the conjugation reaction with the squarate ester chemistry, Qβ was conjugated with a squarate functionalized lactose 3[23] as a model reaction. Lactoside 3 was prepared by derivatizing lactose 4[23] with dimethyl squarate 2 in 76% yield (Scheme 2.1a). In parallel, the coat protein of Qβ triple mutant A38K/A40C/D102C was expressed in E. coli, which self-assembled into nanoparticles with average diameters of 28 nm consisting of 180 copies of the monomer [24]. The lactoside 3 was then incubated with Qβ (14eq per monomer) at 22°C for 20 hours leading to the Qβ-lactose conjugate 5 (Scheme 2.1b). In order to quantify the degree of modification, surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry analysis was performed (Figure 2.1). Based on the intensities of the mass spectrometry (MS) peaks for Qβ coat protein monomer conjugated with lactosides, it was estimated there was an average loading of 4.5 haptens per Qβ monomer, corresponding to 810 copies per Qβ capsid. Increasing the amount of 3 to 28 eq led to an average of 8 lactosides conjugated per Qβ monomer unit (1,440 copies per Qβ capsid). As each monomer of the Qβ triple mutant A38K/A40C/D102C has 9 total free amines (8 lysines plus the free N-terminus), the ability to nearly fully functionalize Qβ suggests the squarate 38 chemistry is highly efficient in promoting glycan conjugation with Qβ. To further characterize the conjugate, gel electrophoresis analysis was carried out (Figure 2.2). In the presence of reducing agents, the Qβ capsid disassembled to its subunits showing bands at 14KDa and 28KDa corresponding to the monomer and dimer of the coat protein respectively (Figure 2.2, lane 11). After conjugation, the monomer band of the Qβ coat protein shifted to about 19 KDa, correlating well with the addition of ~8 lactose units per Qβ monomer on average (lane 10). Scheme 2. 1 Qβ conjugation to lactose squarate 3. a) 0.5M pH 7.0 phosphate buffer, r.t., 3h, 76%; b) 0.5M borate buffer, pH 9.0, r.t., 90h. We next explored the conjugation of Vibrio cholerae O1 Inaba OSP from PIC018[21] with Qβ using the squarate chemistry. V. cholerae O1 Inaba OSP 1 was treated with dimethyl squarate 2 first, which was then incubated with Qβ. To preserve the valuable material, the amount of OSP- squarate was reduced to 8 equivalents per Qβ monomer (Scheme 2.2). After 120 h, the conjugation reaction was analyzed by SELDI-TOF, which only showed the peak for Qβ monomer at 14.1 KDa with very weak signals from the potential OSP adduct (Appendix, Figure 2.12). The SDS-PAGE of the Qβ-OSP conjugate (Figure 2.2, lane 9) showed a very faint band close to the Qβ monomer MW under the reducing condition with the majority of the protein sample appearing smeared at the high MW region of the gel. The incomplete disassembly of Qβ-OSP under the reducing 39 condition as compared to Qβ-lactose may be due to the large size of the OSP (~6 kDa), which may sterically hinder the reduction of disulfides thus holding multiple coat protein monomers together. As there was little Qβ monomer observed on the gel, the low signal from the OSP conjugate observed in SELDI-TOF (Appendix, Figure 2.12) was most likely due to the difficulty in ionizing the Qβ-OSP conjugate by MS. Thus, we needed to employ additional techniques beyond SELDI- TOF and SDS-PAGE to provide more quantitative information on the degree of OSP functionalization on Qβ. 40 Figure 2. 2 SELDI-TOF MS result of the conjugation of Qβ triple mutant A38K/A40C/D102C and lactose squarate 3. a) Qβ before conjugation, b) 14 eq of 3 was added to Qβ and the reaction mixture was incubated for 20h, c) An additional 14eq of 3 was added to reaction and incubated for 72h. The mass difference between the peaks corresponds to the addition of a lactose squarate with MW of 577 Da. The average loading was calculated based on the ratio of the sum of respective antigen number of each peak multiplied by their intensity to the total intensity of all peaks. 41 Figure 2. 3 The SDS-PAGE of different samples at non-reducing (Lanes 1-5) and reducing (Lanes 8-12) conditions. Lanes 1, 12: Molecular weight ladder; lanes 2, 11: unconjugated Qβ; lanes 3, 10: Qβ-lactose conjugate; lanes 4, 9: Qβ-OSP conjugate; lanes 5, 8: BSA. The Qβ monomer and dimer appeared at 14KDa and 28KDa under the reducing condition. The band corresponding to the Qβ- lactose 5 monomer shifted to about 19 kDa after conjugation, corresponding to the addition of about 8 lactoses per monomer. Qβ-OSP conjugate showed up as a smear at higher MW on the gel. Scheme 2. 2 Conjugation of OSP 1 to Qβ VLP. OSP 1 was activated with dimethyl squarate 2, and subsequently added to a Qβ solution in 0.5M borate buffer, pH=9.0. After 120h, the reaction was worked up by ultrafiltration against pH 7.2 (1x) PBS buffer. 42 We next tested Mass Photometry (MP)[25] to quantify OSP functionalization of Qβ. While the aforementioned SELDI-TOF and SDS-PAGE methods for VLP analysis rely on disassembly of the particles and assessing the conjugation at the individual monomeric coat protein levels, MP measures light scattering from the intact particle, which is proportional to the mass of the scattering particles. By analyzing hundreds to thousands of particles, the mass distribution of the sample is generated. To the best of our knowledge, MP has only been applied twice to study VLPs to date [26,27]. When we measured the Qβ sample, there were two populations and the MW was decreasing over time (Figure 2.3). This could be resulting from the instability and degradation of RNA encapsulated inside the Qβ. To test this possibility, we prepared a Qβ sample without RNA by cleaving the RNA chemically with Lead acetate and measured the empty particles with MP. Although we still observed two populations in the sample, the MW remained stable in two different measurements of the same sample over a 3-month interval (Figure 2.4). This supported that the result is reproducible and that MW reduction over time observed with full Qβ was likely due to RNA degradation. The population of Qβ particles with smaller molecular weight that was observed in the MP spectrum may be the result of partially disassembled VLPs. In order to calibrate the mass shifts in MP, we conjugated the Qβ capsid without RNA with lacoside 3 as a control sample. Based on the MW of this conjugate obtained from MP, the lactose loading was about 7 per monomer (Figure 2.5), which was close to the average loading of 6 obtained from the same sample on QTOF-ESI (Appendix, Figure 2.14) and SELDI-TOF (Appendix, Figure 2.15). With this result, we confirmed that the mass shift in Qβ sample after conjugation is due to the loading of the carbohydrate and hence the difference in mass can be used to calculate the loading. 43 We analyzed the Qβ-OSP conjugate with MP next. Upon OSP conjugation, the mean mass shifted about 400 kDa based on the MW of intact particles (Figure 2.6). With the average MW of OSP at ~6,000 Da, it was calculated that on average there were 68 OSP molecules per Qβ capsid. The OSP conjugation and MP protocols were reproducible, giving 66-68 OSP units per Qβ on two independent batches of Qβ-OSP conjugates. Figure 2. 4 The MP result of Qβ triple mutant A38K/A40C/D102C. The same Qβ sample which was measured several times over time of this study showed a decrease of the MW. The right peak shifted from 2696 KDa in (a) to 2544KDa (b) and 2387KDa in (c). Figure 2. 5 The MP result of Qβ without RNA. The measurement of Qβ sample without RNA was repeated after three months. The MW of right peak was 2388 KDa in (d) and 2443 KDa in (e) respectively. 44 Figure 2. 6 The MP result of wild-type Qβ without RNA f) before and g) after conjugation with lactoside 3. The right peak shifted from 2,443 KDa to 3,249 KDa, which suggests the conjugation of about 7 lactosides per Qβ monomer on average. a) b) Figure 2. 7 MP results of a) Qβ and b) Qβ-OSP conjugate. The right peak shifted from 2,544 KDa to 2,953 KDa, which suggests the conjugation of an average 68 OSP per full Qβ capsid calculated based on the mass of the intact particle. 2.2.2 Immunogenicity of the Qβ-OSP conjugate With the Qβ-OSP conjugate in hand, to analyze whether the Qβ-OSP was displaying OSP in an immunologically relevant manner, we assessed the ability of convalescent plasma from humans recovering from cholera to recognize Qβ-OSP. Plasma was collected and analyzed as previously described [21]. As shown in figure 2.7, Qβ-OSP was recognized by convalescent phase 45 plasma of humans recovering from cholera (day 7 sample), but not by acute phase plasma (day 2 sample). This is most likely due to the production of protective antibodies in patients recovering from the infection. In comparison to Qβ-OSP, there was little binding of convalescent sera to Qβ by itself. Furthermore, there was no increased immune-recognition of Qβ-OSP by plasma from S. Typhi infected patients suggesting the binding of Qβ-OSP was a result from cholera infection. 150 β -OSP Qβ mOD/min Cholera 01 Plasma 100 Typhi Plasma β Qβ 50 0 d2 d7 d2 d7 d2 d7 d2 d7 Figure 2. 8 Immunoreactivities of human plasma toward Qβ and Qβ-OSP were measured by acute phase plasma (day 2 sample) versus convalescent phase plasma (day 7 sample) of patients with cholera versus typhoid fever in Dhaka, Bangladesh. Next, we evaluated the ability of Qβ-OSP to generate anti-OSP antibodies in animals. A group of five female Swiss-Webster (3–5 week old) mice was injected intramuscularly on days 0, 21 and 42 with the Qβ-OSP construct (10 µg OSP per mouse) in the absence of any exogenous adjuvants (Figure 2.8a). Blood was collected from the immunized mice on days 0, 7, 28, 49, 56 and during the study as shown in figure 2.8a. A control group of Swiss-Webster mice received Qβ only following the same protocol. To analyze the levels of anti-OSP antibodies in the sera by enzyme-linked immunosorbent assay (ELISA), a bovine serum albumin (BSA) conjugate of OSP was prepared to avoid the interference of anti-Qβ antibodies. The anti-OSP IgG titer, the highest 46 dilution above background that gives optical density (OD) = 0.1, was determined in pooled sera at different time points by ELISA. While there were weak IgG responses one week after the first immunization, after the second immunization, significantly higher levels of IgG were observed on day 28, 49 and 56 (Figures 2.8b and 2.9). The average IgG titers reached the maximum value of 226,504 on day 56 (Figure 2.8c). The IgG titer from the Qβ-OSP group remained at high levels over time with IgG titers still detectable at day 265. In contrast, there was no detectable anti-OSP IgG responses in the control group at any time point suggesting Qβ-OSP potently induced antibody responses against OSP. No anti-OSP IgM antibodies were detected. The high anti-OSP IgG levels suggested that the Qβ-OSP constructs successfully induced the activation of helper T cells, which could promote the isotype switching to IgG. In order to assess whether memory responses were generated, on day 265 post initial immunization, mice received an additional vaccination. One week (day 272) after the booster, the average anti-OSP IgG antibody levels of the mice increased over 35 times compared to those on day 265 and reached the similar level of IgG titer as day 56. These results suggest that Qβ-OSP vaccination induced memory B cell responses and the anti-OSP humoral immunity could be boosted (Figure 2.8b). 47 a) Figure 2. 9 Evaluation of Qβ-OSP immunogenicity. a) Immunization and blood collection schedule. Each group received 3 immunization three weeks apart with blood collected at day 0 and on days 56, 86, 118, 170, 265 and 272 respectively. b) OSP-specific IgG titer of pooled sera from Qβ and Qβ-OSP groups up to day 272 post-immunization. The red arrow indicates a booster injection at day 265. c) individual mouse serum OSP-specific IgG titer of Qβ and Qβ-OSP groups at day 56. The statistical significance was determined through a two tailed t-test using GraphPad Prism. ** p < 0.0001. 3 ** ** Qβ-OSP Qβ OD 450 nm 2 1 0 d0 d28 d49 d56 d0 d28 d49 d56 Figure 2. 10 ELISA analysis showed significant IgG binding to BSA–OSP by post-immune sera at d49 and d56 (p=0.0014 and 0.0065 respectively), compared to the control sera from mice immunized with Qβ only. Each bar represents data for 5 mice at 20,000 fold of serum dilution. 48 In order to determine whether immunization with Qβ-OSP would elicit antibodies recognizing the native LPS containing OSP from V. cholerae, ELISA analysis was also performed using V. cholerae O1 Inaba LPS PIC018 as the coating antigen. Sera from mice immunized with Qβ-OSP had significantly higher levels of anti-LPS IgG antibodies as compared to those from mice receiving Qβ alone (Figure 2.10). Furthermore, serum binding to Inaba LPS was significantly higher compared to binding to LPS from E. coli suggesting the antibodies induced by Qβ-OSP were selective toward Inaba (Figure 2.10). * P= 0.020 2.0 * P= 0.022 * P= 0.037 1.5 LPS-Inaba OD 450 nm LPS-E.Coli 1.0 0.5 0.0 d0 d7 d28 d49 d56 d0 d7 d28 d49 d56 d0 d7 d28 d49 d56 d0 d7 d28 d49 d56 Qβ-OSP Qβ Qβ-OSP Qβ days Figure 2. 11 Binding of mouse serum immunized with Qβ-OSP and Qβ to LPS from Inaba vs E. coli. Serum binding against Inaba LPS was observed in the Qβ-OSP immunized group while sera from the Qβ group had lower binding. The binding to E. coli LPS was lower by sera from both Qβ-OSP and Qβ immunized mice. The statistical significance was determined through an unpaired two tailed t-test using GraphPad Prism. * p < 0.05. With the ability to selectively bind native V. cholerae O1 Inaba LPS and OSP by the Qβ- OSP induced antibodies established, we next measured the vibriocidal activities of the post- immune sera [21]. In the presence of an exogenous source of complement, V. cholerae cells are 49 incubated with serial serum dilutions. Anti-V. cholerae antibodies present in the serum sample(s) in combination with complement can lyse the live bacteria. While none of the mice from the Qβ immunized group showed any vibriocidal activities, sera from 2 of the 5 Qβ-OSP immunized mice were able to kill the bacteria at dilutions higher than those in mice immunized with Qβ alone (Figure 2.11). Figure 2. 12 Vibriocidal responses in vaccine cohorts. We defined responders as having an increase in vibriocidal titer by 4-fold at day 56 than day 0. 2.3 Discussion OSP of V. cholerae has been used as an antigen in conjugation with BSA and recombinant heavy chain of tetanus toxin, and synthetic hexasaccharide and synthetic hexasaccharide cluster conjugates have also been evaluated as vaccine antigens against V. cholera [21,28–30]. A virus like particle (VLP) based approach has a number of attractive features for vaccine applications since the highly ordered organization of the protein(s) in the VLPs is well recognized via pathogen- associated molecular patterns (PAMPs) [31]. VLPs can present antigens in an organized and polyvalent manner to crosslink B-cell receptors to induce intense cellular signaling for strong 50 immune activation. Bacteriophage Qβ VLP is a promising platform for organized display to induce antibody responses against a target antigen. For polysaccharide based conjugate vaccines, there are several coupling methods such as periodate activation for reductive amination, cyanylation and carbodiimide-mediated coupling. These approaches can suffer from incompatibility with some proteins and substrates and low selectivity. Squaric acid esters are favorable linker molecules in glyco-conjugate formation between amino-saccharides and proteins due to the amine-selectivity, high reactivity at room temperature, possibility of stoichiometric modification, and recovery of high-value unreacted (oligo)saccharid [32]. In the current study, we have prepared the squaric acid monoester derivative of V. cholerae O1 OSP core antigen utilizing the core amine group of the OSP and conjugated the monoester with VLP (carrier protein). While bacteriophage Qβ has been conjugated with glycan antigens [33–35], this is the first example of using a squarate linker for glycan conjugation with Qβ VLP. The squarate chemistry was highly efficient, leading to close to full derivatization of all free amines of Qβ coat proteins with a small glycan such as lactose 4. With squarate chemistry, the polysaccharide of V. cholerae O1 OSP was conjugated to the Qβ carrier protein via single point attachment due to the presence of single amino group per OSP molecule. The final construct may mimic native bacteria by presenting multiple OSP polysaccharide on the surface. OSP display on Qβ was in an immunologically relevant manner, which was recognized by convalescent sera of cholera infected humans but not of typhoid fever patients. The Qβ-OSP vaccine was immunogenic in mice, inducing persistent IgG responses against OSP. Such long term IgG production may assist with the long term protective goal of anti- cholera vaccination [36]. 51 The vaccine was administrated in the absence of an exogenous adjuvant. This in part may be due to inherent adjuvant properties of Qβ VLP, which can encapsulate E. coli RNA molecules, thus stimulating internal cellular signals via TLR7/8 in the antigen presenting cells [37]. The induced IgG antibodies recognized native V. cholerae LPS and had vibriocidal activities. Induction of vibriocidal antibodies correlate with protection against cholera [38], and these responses largely target V. cholerae OSP [38,39]. The mechanism of protection against V. cholerae in the intestinal lumen is currently unclear, but may involve inhibition of V. cholerae motility through the binding of OSP-specific antibodies [40,41]. The induction of low levels E. coli LPS-specific IgG antibody responses (Figure 2.10) may be due to the presence of trace amounts of residual E. coli LPS in Qβ VLP preps. Studies are ongoing to express Qβ VLPs in LPS deficient E. coli strains. Our study has several limitations. We focused on developing suitable coupling chemistry and analytical tools to synthesize and characterize the Qβ-OSP conjugate. We evaluated the immunogenicity of the conjugate, and future assessments could evaluate protective efficacy in a wild type V. cholerae challenge assay. We did not investigate the effect of adjuvant administration on anti-OSP IgG production, or the mechanism by which induced antibodies can provide protection. In addition to protective studies, the future work will analyze the effect of antibodies in inhibition of V. cholerae colonization in in vitro model using HT-29 cells, human colon carcinoma cell line, which have been widely used for interaction between host and enteric pathogens due to the similarity of structure and their function to human intestinal epithelial cells [42]. 2.4 Conclusions In this work, we have conjugated the V. cholerae O1 OSP polysaccharide to Qβ carrier for the first time. The MP technique yielded critical info on the level of OSP loading on Qβ. The Qβ- 52 OSP conjugate was recognized by sera from humans with cholera and was able to induce long lasting antibody production in the absence of adjuvant in a mice immunization study. The resulting antibodies exhibited vibriocidal activities. VLP-based display of bacterial OSP can be an attractive lead as a next generation anti-cholera vaccine. 53 2.5 Materials and methods 2.5.1 General experimental procedures and methods for synthesis All chemicals were reagent grade and were used as received from the manufacturer, unless otherwise noted. Protein concentration was measured using the Coomassie Plus Protein Reagent (Bradford Assay, Pierce) with BSA as the standard. OSP and BSA-OSP was produced as previously described [21]. 2.5.2 Qβ conjugation to lactoside 3 and purification A stock solution of 4.8 mg/mL Qβ in 0.1 M (pH 7.0) KPB buffer (208 mL) was placed in a Millipore Amicon Ultra-0.5 (10 kDa cut-off) ultrafiltration device and the content was ultrafiltered against 0.5 M pH 9.0 borate buffer for three times at 10°C to exchange the buffer to 0.5 M pH 9.0 borate buffer. The filtrates were discarded and the final retentate was transferred into a 0.5 mL V-shaped reaction vessel and the same buffer was added to adjust the overall volume to 200 mL. Lactose squarate 3 (0.6 mg, 0.986 mmol) was carefully added into the reaction mixture and the content of the vessel was stirred at r.t. for 20 h. SELDI-TOF-MS analysis showed that an average loading (lactose/Qβ monomer ratio) of ~4.5 was achieved (Figure 2.1b). Another 0.6 mg (0.986 mmol) of compound 3 was added and the reaction mixture was further stirred for 72 h. SELDI-TOF-MS showed that the average loading reached ~8.0. The reaction was worked up by ultrafiltering the reaction mixture in a Millipore Amicon Ultra-0.5 (30k Da cut-off) tube against pH 7.2 PBS (1x) buffer for 6 times to remove the unconjugated lactose derivatives. The final retentate was transferred into a conical tube for storage. 1 mL of the above reaction mixture was diluted with 10 mL of 0.5 M pH 7.0 phosphate buffer and then mixed with 11 mL of dithiothreitol (DTT) solution (0.1 M in water). The mixture 54 was incubated at 37°C for 30 min. 1 mL of the above solution was withdrawn from the mixture for SELDI analysis [19] using sinapinic acid (SPA) as matrix. 2.5.3 Qβ conjugation to OSP and purification V.cholerae O1 Inaba OSP 1 (5.4 mg, 0.0009 mmol) was converted to its squarate derivative by reacting with 3,4-dimethoxy-3-cyclobutene-1,2-dione (2, 2.56 mg, 0.018 mmol) in 0.5 M pH 7.0 phosphate buffer as described previously [20]. A white fluffy solid (5.4 mg, 0.00088 mmol, 98%) was obtained after work up. A stock solution of 6.7 mg/mL Qβ in 0.1 M (pH 7.0) KPB buffer (240 mL) was placed in a Millipore Amicon Ultra-0.5 (10k Da cut-off) ultrafiltration device and the content was ultrafiltered against 0.5 M pH 9.0 borate buffer for three times (at 10°C, for speed/rpm, time and volume of the concentrate, manufacturer’s suggestions were followed) to exchange the buffer to 0.5 M pH 9.0 borate buffer. The filtrates were discarded and the final retentate was transferred into a 1 mL V-shaped reaction vessel and the same buffer was added to adjust the overall volume to 225 mL. The squarate derivative obtained above (5.4 mg, 0.00088mmol) was added into the vessel and the clear solution formed was stirred at r.t. After 120 h, the reaction was worked up by ultrafiltering the reaction mixture in a Millipore Amicon Ultra- 4 (30k Da cut-off) tube against pH 7.2 PBS (1x) buffer for 6 times to remove the unconjugated antigen. The final retentate was transferred into a conical tube for storage. 2.5.4 MP procedure The 24x50 mm microscope coverslips (Fisher Scientific, Waltham, MA) and precut 2x2 silicon gasket wells (GBL103250, Sigma, MO) are cleaned and assembled as described in reference [44]. Measurements were performed on OneMP instrument (Refeyn, Oxford, UK) at room temperature. PBS (1x, pH 7.2) buffer was filtered through 0.22 μM filters before use. Ten microliters of buffer was loaded in gasket well to focus the objective on the coverslip surface. Qβ 55 conjugates stock were diluted 100 times in PBS (1x, pH 7.2) buffer and added to buffer in the well. The MP video was immediately recorded after the sample loading using the AcquireMP software (Refeyn, Oxford, UK). A one-minute video was recorded for each sample, and each sample was repeated twice. Data were processed using DiscoverMP software (Refeyn, Oxford, UK) with the threshold filter values of 5. The mass distribution was plotted as histograms with bin width of 50kD and fit with Gaussian peaks to obtain the average mass of different species. The contrast-to- mass calibration is performed using an unstained protein ladder (LC0725, Thermofisher, Wattham, MA) and empty AAV5 sample (Virovek, Hayward, CA). 2.5.5 Immunization All animal experiments were approved by and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Michigan State University. The animal usage protocol number is PROTO201900423. Female Swiss-Webster (3–5 week old) mice were used for studies, 5 for each group. Mice were injected intramuscularly on day 0, 21 and 42 with 0.06 mL Qβ-OSP construct (10 μg OSP, 65 μg Qβ per mouse) without an exogenous adjuvant. The control group was injected with the same amount of Qβ carrier (65 μg Qβ per mouse) as the experimental group. The final boost was given at day 265. Serum samples were collected on days 0 (before immunization), 7, 28, 49, 56, 86, 118, 170, 265, and 272. 2.5.6 Evaluation of antibody titers by ELISA The Nunc MaxiSorp® flat-bottom 96-well microtiter plates were coated with 1 μg mL-1 of the BSA-OSP conjugate (100 μL/well) [21] in NaHCO3/Na2CO3 buffer (0.05 M, pH 9.6) containing 0.02 % NaN3 by incubation at 4°C overnight. For ELISA study against LPS, plates were coated with 10 μg mL-1 of LPS, Inaba or E. coli, in PBS buffer overnight at room temperature. The coated plates were washed with PBS/0.5% Tween-20 (PBST) (4 × 200 μL) and blocked with 56 1 % BSA in PBS (200μL/well) at rt for 1 h. The plates were washed again with PBST (4 × 200 μL) and incubated with serial dilutions of mouse sera in 0.1 % BSA/PBS (100 μL/well, 2 wells for each dilution). The plates were incubated for 2 h at 37 °C and then washed with PBST (4 × 200 μL). A 1:2000 dilution of HRP-conjugated goat anti-mouse IgG or IgM (Jackson ImmunoResearch Laboratory, 115-035-003) in 0.1% BSA/PBS (100 μL) was added to the wells respectively to determine the titers of antibodies generated. The plates were incubated for 1 h at 37 °C and then washed with PBST (4 × 200 μL). A solution of enzymatic substrate 3,3',5,5'- tetramethylbenzidine (TMB, 200 μL) was added to the plates (for one plate: 5 mg of TMB was dissolved in 2 mL of DMSO plus 18 mL of citric acid buffer containing 20 μL of H2O2). Color was allowed to develop for 15 min and then quenched by adding 50 μL of 0.5 M H2SO4. The readout was measured at 450 nm using a microplate reader. The titer was determined by regression analysis with log10 dilution plotted with optical density and reported as the highest fold of dilution giving the optical absorbance value of 0.1 over those of the pre-immune control sera. 1:5000 serum dilution was used for LPS binding study as showed in figure 2.10. 2.5.7 Evaluation of Qβ-OSP conjugates using human serum To assess immunoreactivity of the OSP display on the Qβ conjugates, antigen specific ELISAs using sera collected from humans with cholera in Bangladesh were performed. The responses to human sera with cholera were compared to the response detected in humans with typhoid fever in Bangladesh. The plates coated with 100ng of Qβ or Qβ-OSP (based on protein mass) per well. After blocking and washing of plates, acute and convalescent phase sera from humans with cholera or typhoid (diluted 1:250 in 0.1% BSA in phosphate buffered saline-Tween) were added and incubated for 90 min at 37°C. HRP-conjugate anti-human IgG antibody at 1:5000 dilution in 0.1% BSA in phosphate buffered saline-Tween was used to detect antigen-specific 57 antibodies. After 90 min incubation at 37°C, 0.55 mg/ml solution of 2,2’-azinobis (3- ethylbenzothiazoline-6-sulfonic acid) (ABTS; Sigma) with 0.03% H2O2 (Sigma) were added to the plates and optical density was read at 405 nm for 5min at 30 s intervals. The maximum slope for an optical density change of 0.2 U was reported as millioptical density units per minute (mOD/min). 2.5.8 Serum vibriocidal responses The vibriocidal antibody titers against V. cholerae O1 El Tor Inaba strain PIC018 were assessed in a micro-assay as previously reported [21]. The endogenous complement activity of mouse serum was inactivated by heating it for 30 min at 56°C. 50 μl aliquots of serial dilution of heat-inactivated sera in 0.15 M saline were added to wells of sterile 96-well tissue culture plates containing 25 μl/well of V. cholerae O1 El Tor Inaba strain PIC018 (OD 0.3) in 0.15 M saline and 22% guinea pig complement. After 1 hr incubation at 37°C, 150 μl of brain heart infusion broth was added to each well, and plates were incubated for an additional 2h at 37°C. The optical density of plates was then measured at 595nm. A responder was defined as having a 4-fold increase of vibriocidal titer at day 56 compared with baseline day 0 titer. 58 APPENDIX 59 a) Zoom b) Zoom Figure 2. 13 SELDI-TOF MS result of conjugation of a) wild type Qβ or b) mutant Qβ to squarate- OSP. The broad weak peak could be observed at ~20 KDa which correlates to conjugate 6 with loading 1 OSP. 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Pancreatic cancer incidence and mortality has been predicted to take over other cancer types by 2030, due to the complexity of screening and early detection of disease along with the absence of efficacious and less toxic therapeutic agents[3]. The glycan carbohydrate antigen 19-9 (CA19-9), also called sialyl Lewisa (sLea), is an important and widely used biomarker for pancreatic cancer diagnosis [4]. CA 19-9 was first described as a tumor antigen in 1979 by its recognition with monoclonal antibody NS19-9, which was developed by using human colorectal cancer cell line SW1116 as an immunizing antigen [5]. 10 to 30% of pancreatitis patients and 75% of pancreatic cancer patients have shown elevated serum levels of CA19-9. CA19-9 elevation is also detected in other gastrointestinal diseases like pancreatic intraepithelial neoplasms (PanINs), which are precursors to pancreatic ductal adenocarcinoma (PDAC) [6]. CA19-9 has been investigated as a biomarker, predictor, and promoter in pancreatic cancer [7]. Although CA19-9 has approximately 80% sensitivity for diagnosis, its application for mass screening is limited due to the false positive results in benign diseases such as pancreatitis and nonpancreatic cancer conditions and false negative result in Lewis-negative individuals which counts for 5–10% of population [8–10]. CA19-9 as a predictor correlates strongly with tumor stage, following patients with known disease, treatment response, recurrence and overall survival [7,11]. The role of CA19-9 in promoting pancreatic cancer was recently demonstrated, suggesting a glycosylation linkage between pancreatitis and pancreatic cancer via CA19-9 biosynthesis [6]. Importantly, antibodies against CA19-9 were able to reverse pancreatitis in the Kras G12D mouse model [6]. The over expression of sLea correlates with 68 metastasis and poor survival, promotes cancer development and progression by facilitating tumor cell adhesion, angiogenesis, tumor vascularization and mediating adhesion of circulating cancer cells to endothelial cells resulting in extravasation and metastasis formation [8,12–14]. sLea is a tetrasaccharide (NeuAcα2,3Galβ1,3[Fucα1,4]GlcNAc-R) [15] and is a type of Lewis blood group on the cell membrane surface in which a sialic acid is added to the Lewis a sugar chain (Lea) [16–18]. sLea is predominantly expressed in pancreatic ducts and islet cells, while the natural counterpart, disialyl-Lewis a, is found in nonmalignant epithelial cells [14]. sLea is a structural isomer of sLex, another common tumor antigen overexpressed in carcinoma cells [19], with alternated attachment of L-fucose and D-galactose to the glucosamine moiety. Although many tumor-associated carbohydrate antigens (TACAs) have been identified and routinely used in the clinic to monitor tumor progression and response to treatment, the development of an effective carbohydrate-targeting antitumor vaccine or antibody is extremely challenging [20,21]. To date, only one TACA-targeting antibody, anti-GD2 monoclonal antibody dinutuximab [22], has been FDA approved. A major obstacle is due to the T-cell independency of glycans, which makes them unable to induce the production of long-lived protective antibodies and does not establish immunological memory if immunized with the glycan antigen alone. Glycans can be recognized by B cells and trigger the secretion of low-avidity IgM antibodies. To overcome this challenge, glycan epitopes have been conjugated to an immunogenic carrier ranging from proteins, to peptides, oligonucleotides, lipids, zwitterionic polysaccharides, and nanoparticles to boost the anti-glycan antibody responses [23]. While the sLea as an attractive therapeutic target for cancer therapy [18,24,25], only one study investigated the sLea based vaccine with KLH as the carrier [26]. The 5B1 monoclonal antibody obtained from patients immunized with KLH-sLea conjugate had good affinity and 69 specificity for sLea [27] and was under investigation for cancer imaging in clinical trials [28]. Building on our previous studies which we showed the superiority of bacteriophage Qβ over KLH in ease of preparation, characterization and the ability to elicit high levels of antibodies [29,30], we became interested in testing whether the virus-like particle (VLP) platform could potently induce antibody response against sLea. We have also developed an efficient approach for chemical synthesis of sLea. Our data suggests that anti-sLea vaccination has the potential for treatment of vulnerable population at risk of pancreatic cancer. 3.2 Results 3.2.1 Synthesis of Qβ-sLea conjugate vaccine and mouse immunization sLea tetrasaccharide 1 with the isothiocyanate moiety at the reducing end was chemically synthesized by Dr. Sherif Ramadan. Isothiocyanate can provide an efficient conjugation method between the sLea tetrasaccharide and Qβ protein. Moreover, the valuable unconjugated sugar can be recovered as the free amine form from the reaction mixture and the isothiocyanate group can be regenerated, which adds to the practical advantage of this chemistry. The Qβ-sLea conjugation was readily performed by mixing the isothiocyanate functionalized sLea 1 (Scheme 3.1a) and Qβ in buffered solution overnight at 37°C. The sLea / Qβ epitope ratio of the sLea conjugate was determined using mass spectrometry. There were on average 300 sugar moieties per Qβ capsid (Appendix, Figure 3.12). KLH-sLea conjugate was also prepared for comparison studies and the total loading was 400 sLea per KLH molecule (Scheme 3.1b). BSA-sLea conjugate was prepared by a similar method and was characterized by MALDI-TOF MS, which showed conjugation of four sLea per BSA molecule (Appendix, Figure 3.13). The BSA-sLea conjugated was used in Enzyme Linked Immunosorbent Assay (ELISA) to analyze the anti-sLea antibodies generated in 70 post-immune sera without the interference of anti-Qβ antibodies. Qβ-sLea conjugate and physical mixture of sLea and Qβ were used for mouse immunization. Groups of five C57BL/6 female mice were injected subcutaneously under the scruff on days 0, 14 and 28 with 0.2 mL Qβ constructs (containing 4 μg carbohydrate) mixed with 20 µg MPLA as the adjuvant. Mice were bled at day35 and different time points throughout the study (Figure 3.2). OH OH HO O OH OH OH OH CO2- S HO OH O O C O O N AcHN O O O OH NHAc HO sLea-isothiocyanate 1 Figure 3. 1 Structure of sLea-isothiocyanate (sLea-NCS) Scheme 3. 1 synthesis of a) Qβ-sLea conjugate. To a solution of Qβ (1 mg, 10 mg/ml concentration, 0.4 nmol subunit, 0.36 μmol reactive amine) in 0.1 M potassium phosphate (KPB) buffer pH 8.5, was added sLea-NCS (2.5 equivalent to reactive amine) and the reaction was incubated at 37 ℃ overnight and worked up by ultrafiltering the reaction mixture against 0.1 M KPB buffer (pH 7.0, 0.1 M). b) KLH-sLea conjugate was synthesized under a similar condition. 71 Figure 3. 2 Immunization and blood collection schedule. Groups of five C57BL/6 female mice received 3 immunizations of Qβ-sLea conjugate or sLea and Qβ mixture two weeks apart with blood collected at days 0, 35, 65, 95, 125, 185, 277, and 356. At day 365, both groups were inoculated i.v. with 5 × 105 B16-FUT3 tumor cells and lungs were collected 14 days later. 3.2.2 Qβ-sLea conjugate elicited high titers of IgG antibodies titers and longer lasting anti-sLea IgG antibodies in mice compared with the KLH-sLea conjugate as well as the admixture of Qβ and sLea The peak anti-sLea ELISA titers were observed on day 35 after 1st vaccination with Qβ- sLea conjugate, which reached 2,137,962 ELISA units (Figure 3.3a). No evidence of significant antibody induction above background was observed after immunization with the sLea and Qβ mixture. The antibody level induced by Qβ-sLea conjugate remained significantly elevated up to 277 days. The average level of KLH-sLea conjugate induced antibodies was significantly lower than that by Qβ-sLea conjugate and was not detectable at any time points following immunization (Figure 3.3b). The IgM antibody was not detected for day 35 sera at 1:100,000 dilution. 72 **** a) 10 7 10 6 Qβ-sLea Conjugate 10 5 Qβ+sLea mixture 10 4 KLH-sLe a Titer Day 0 10 3 10 2 10 1 10 0 b) 10 7 Qβ-sLea Conjugate 10 6 a Qβ+sLe mixture Anti-sLea titer 10 5 a 10 4 KLH-sLe 10 3 10 2 10 1 10 0 35 65 95 125 185 277 356 379 Days Figure 3. 3 a) Titers of anti-sLea IgG antibodies from mice immunized with the Qβ-sLea conjugate against BSA-sLea (each symbol represents one mouse, n=5 mice for Qβ-sLea or sLea and Qβ mixture group and n=3 for KLH-sLea group). Pooled sera from 5 mice were used for Qβ and day 0. The statistical significance was determined through an unpaired two tailed t-test using GraphPad Prism. **** p<0.0001. b) Changes of the titers of anti-sLea IgG antibodies from Qβ-sLea immunized mice over time. The IgG titers were determined with pooled sera. 3.2.3 Qβ-sLea conjugate elicited antibodies capable of binding with sLea expressing tumor cells To assess whether the induced antibodies after vaccination could bind to natural sLea, three mouse cell lines were investigated. The mouse pancreatic cancer cells are represented by neo FC1199, FC1242, and FC1245. As mouse cells do not endogenously express sLea due to the lack of fucosyl transferase, transfection of these pancreatic cancer cells with the genes encoding both Fucosyltransferase 3 (FUT3) and 1,3-galactosyltransferase 5 (b3GALT5) led to cell surface production of CA19-9 on FC1199, FC1242, and FC1245 FB cells at levels equivalent to those 73 observed in human cancer cell lines. The results of flow cytometry testing on these cancer cell lines are demonstrated in figure 3.4a Striking increases in IgG reactivity against FC1245FB cells were seen for all mice immunized with Qβ-sLea conjugate while no reactivity above the background was detected in the sera from mice receiving the mixture of Qβ and sLea. Mouse anti-sLea antibody was used as the positive control (clone 121SLE) in the flow cytometry testing. All 5 sera from Qβ-sLea vaccinated group showed stronger binding to sLea expressing cells compared to monoclonal antibody 121SLE. The cell surface binding was not observed in neo cell lines, which did not express sLea, neither with monoclonal antibody or post-immune sera, confirming that serum binding to FB cells was specific to sLea expression (Figure 3.4a). Cell surface reactivity against fucosyl transferase reprogrammed 1199 FB cells was also demonstrated by complement dependent cytotoxicity (CDC) assays using rabbit complement and mouse sera from different groups (Figure 3.4b). Both 121SLE antibody and sera from sLea-Qβ immunized group showed close to 80% killing activities, while sera from group receiving the mixture had little killing activities, similar to pre-immunization sera. The 121SLE mAb showed similar CDC potency to sera of Qβ-sLea immunized group although its binding was weaker in FACS study. Since 121SLE is an IgM isotype, its higher cytotoxicity could be related to the higher efficiency of IgM in complement activation. 74 Figure 3. 4 Recognition of cell surface expression of sLea with FACS (a) and CDC (b) of 1199FB cells in presence of Qβ-sLea antisera. a) 3x105 cells were incubated with mouse sera dilution (1:20), or 10 μg/ml of anti-sLea 121SLE mAb for 0.5h at 4°C and washed with FACS buffer. The sera binding to cells were assessed using PE conjugated anti-mouse IgG or IgM (121SLE) secondary Antibodies. b) 3x104 1199FB cells were incubated with mouse sera dilution (1:20), or 10 μg/ml of anti-sLea 121SLE mAb for 1h at 4°C. Then cells were washed, and rabbit sera complement at 1:10 dilution was added and further incubated at 37°C for 3h. The cell viability was tested with MTS assay. **** p<0.0001. The p values were determined through a two-tailed unpaired t test using GraphPad Prism. 3.2.4 Antibodies induced by the Qβ-sLea conjugate were highly selective toward human pancreatic ductal adenocarcinoma tissues To test the translational potential of Qβ-sLea conjugate vaccine to human patients, we obtained pancreatic ductal adenocarcinoma and pancreatic cancer tissue microarray containing cancer tissues from different patients. Pooled sera from Qβ-sLea conjugate vs mixture immunized mice were used to stain the tissue microarrays at 1:1000 dilution. In the pancreatic ductal adenocarcinoma array, 16 cores out of 24 different core samples (67% positive staining) showed strong binding with sera from Qβ-sLea group while there was not any staining for sera from mice immunized with the mixture of Qβ and sLea (Figure 3.5a, 3.5b, Appendix Figure 3.15 and Figure 3.16). In a separate experiment, pancreatic cancer tissue microarray contains normal and metastatic cancer tissue from 48 different patients was stained with pooled sera from Qβ-sLea group. In this array, 26 cores out of 31 pancreatic adenocarcinoma were stained in remarkable contrast to normal tissues. The staining was also observed for squamous cell and adenosquamous carcinoma but not for metastatic or neuroendocrine tumor (Figure 3.6, Appendix Figure 3.17). 75 Figure 3. 5 Individual XPAN024 microarray slides stained with pooled serum of 5 mice immunized with Qβ-sLea conjugate(a), sLea and Qβ mixture (b) at day 35 at 1:1000 serum dilution or with 5B1 recombinant antibody at 1.08 μg/ml final concentration (c). Similar cores have been shown here to compare the intensity of staining. Characterization of each core has been provided in appendix, table 3.1 based on their map ID and the pictures of all the cores in appendix, figure 3.15 and 3.16. The brown/red color was due to antibody binding to tissues. The lack of brown/red staining indicates little binding of antibodies to the tissues. Scale bar is 100μm. Figure 3. 6 XPAN048 microarray stained with pooled serum of 5 mice immunized with Qβ-sLea conjugate at day 35 at 1:1000 serum dilution. Selected cores: N2 and N3 are normal pancreatic tissue, N4 metastatic pancreatic malignant islet cell tumor, N11 Neuroendocrine tumor, N14 Squamous cell carcinoma, N15 Adenosquamous carcinoma, N23 and N32 Adenocarcinoma. Characterization of each core has been provided in appendix, table 3.2 based on their core number and the pictures of all the cores in appendix, figure 3.17. The brown/red color was due to antibody 76 binding to tissues. The lack of brown/red staining indicates little binding of antibodies to the tissues. Scale bar is 100μm. 3.2.5 Antibodies induced by the Qβ-sLea conjugate were highly selective toward sLea binding based on glycan microarray analysis The carbohydrate specificity of induced antibodies at day 35 was probed in glycan array analysis against 815 glycans. The sera were tested individually at 1:100 dilution. The results confirmed the high specificity of sera from mice group immunized with Qβ-sLea conjugate with selective recognition of the sLea tetrasaccharide. There was no binding to closely related antigens that were present in the array, including sLex, Lea, Lex, Ley or GD2, GD3, GM2 and GM3 gangliosides. We observed some binding to sLec antigens, but the result was not consistent in all sera samples (Appendix, Table 3.3). 3.2.6 Vaccine activity in animal model for metastasis In order to study the vaccine efficacy against sLea in an immunocompetent environment, two murine cell lines (i.e., B16 melanoma and EL4 lymphoma murine tumor cell lines) that stably express sLea were obtained [31]. These two cell lines are well-established model systems for studying the antitumor activities of antibodies, which are transduced to express human fucosyltransferase III (FUT3). FUT3 is responsible for sLea synthesis in cells. To set up a positive control for our in vivo study, we used the well-studied sLea monoclonal antibody, 5B1, which was generated from patients immunized with KLH-sLea. 5B1 has shown high specificity for sLea antigen and low cross reactivity with similar carbohydrates. The variable regions of human 5B1 mAb, were expressed as murine IgG2a to generate chimeric recombinant antibody with murine Fc portion. In ELISA experiment, 5B1-IgG2a gave titer of 331,131 comparing to the sera from Qβ- sLea conjugate at day35 which gave average IgG titer of 2,137,962 ELISA units. Tissue staining with 5B1-IgG2a gave comparable staining to the pooled sera from Qβ-sLea conjugate. 77 The expression of sLea was confirmed in both EL4-FUT3 and B16-FUT3 cell lines (Figure 3.7a and Figure 3.7b) with mouse anti-Sialyl Lewisa antibody (121SLE). While 5B1 has comparable binding to EL4-FUT3 cell line with 121SLE antibody at 10 μg/ml concentration, its binding was lower than the binding by serum of mice immunized with Qβ-sLea conjugate at 1:20 serum dilution (Figure 3.7a). Moreover, monoclonal antibodies and sera from immunized group showed binding to the EL4-FUT3 cell line and there was not any binding against EL4 parent cell line. 4500 a) 4000 b) 3500 EL4-FUT3 250 EL4 200 MFI 150 100 50 0 121SLE 1ug/ml 121SLE 10ug/ml 5B1 1ug/ml 5B1 10ug/ml 121SLE 1ug/ml 121SLE 10ug/ml 5B1 1ug/ml 5B1 10ug/ml Qß-sLea d35 Qß + sLea d35 Qß-sLea d35 Qß + sLea d35 Cell Cell Figure 3. 7 Expression of sLea confirmed through FACS experiment by using a) 121SLE mAb and chimeric recombinant antibody 5B1 at 10ug/ml concentration in EL4-FUT3 cells. Pooled serum from group immunized with Qβ-sLea conjugate showed significantly higher binding toward EL4-FUT3 cell line. The absence of non-specific binding was confirmed by using EL4 parent cell line. b) 121SLE mAb at different concentration and pooled sera from Qβ-sLea conjugate in B16- FUT3 cells which showed binding to sLea expressed on cell surface. PE-anti mouse IgG or IgM was used as secondary antibody. To mimic clinical conditions for cancer treatment, we evaluated the vaccine efficacy in a therapeutic setting in two different models I) immunization of mice followed by challenge by tumor cell injection. This model will provide information if active immunization can protect mice from tumor metastasis; and II) the effect of anti- sLea generated antibodies in preventing tumor metastasis in a passive protection model. 78 We validated the progress of tumor models with both EL4-FUT3 and B16-FUT3 cells in naïve mice. WT C57BL/6 immunocompetent mice were inoculated i.v. either with B16-FUT3 or EL4-FUT3 tumor cells (which express sLea) (Figure 3.8a) and treated with 5B1-mIgG2a. Treatment with anti-sLea antibody increased the overall survival of mice inoculated with EL4- FUT3 cells (Figure 3.8b) or dramatically reduced metastatic colonization of B16-FUT3 cells in lung (Figure 3.8c). a) b) c) Figure 3. 8 a) WT C57BL/6 mice were inoculated i.v. with 5 × 105 B16-FUT3 (n=3) or EL4-FUT3 (n=6) tumor cells. 100μg of anti-sLea 5B1 Ab was administered i.p. on days 1, 4, 7, and 11 and survival was assessed daily for EL4-FUT3 group (b), p= 0.0448 and for B16-FUT3 group, mice were euthanized, lungs were excised and fixed fourteen days after cell inoculation (c). To examine the potency of vaccine in preventing pancreatic cancer in the therapeutic model I, the mice immunized with Qβ-sLea conjugate were challenged with B16-FUT3 cell line at day 365 post immunization. The serum obtained at day 356 post immunization was still able to recognize the sLea expression on B16-FUT3 cells (Figure 3.9). Mice immunized with Qβ-sLea 79 conjugate or sLea and Qβ mixture were inoculated i.v. with 5 × 105 B16-FUT3 tumor cells. Fourteen days after inoculation, mice were euthanized, lungs were excised and fixed, and metastatic foci were counted. There were 3 mice in Qβ-sLea conjugate vaccinated group which showed lower tumor development trend and tumor burden remained below 10% of total lung area (Figure 3.9, Appendix Figure 3.20). 600 d35 d95 d185 MFI FITC 400 d356 121 SLE mAb (10ug/ml) Cell+ Secondary Ab 200 cell 0 a a (10u Qβ Co -S nt g/ m Le ro l) ci lQ m ne β+ Ab Va SL c e SL E 12 1 Figure 3. 9 The binding of pooled mice sera from day 35, 95, 185 and 356 against sLea antigen at 1:10 dilution with B16-FUT3 cell line were analyzed with flow cytometry with two biological replicates. 356 days after immunization the sera from Qβ-sLea conjugate vaccinated group showed binding toward cell surface expression of sLea antigen. 40 Qβ-sLea Conjugate % Metastatic foci/ a 30 Qβ + sLe mixture 20 Lung area 10 0 Figure 3. 10 Total tumor area ratio to lung tissue after analyzing the pictures with ImageJ. The tumor area in Qβ-sLea conjugate vaccinated group had lower trending compared with group vaccinated with sLea and Qβ mixture. 80 3.2.7 Ongoing experiments Building on the results we obtained in previous mice protection study, an expanded experiment is undergoing. In this study, 2 group of C57BL/6 mice (WT, n=10) are immunized with Qβ-sLea conjugate or sLea and Qβ mixture biweekly at days 0, 14, 28 (containing 4 μg carbohydrate) mixed with 20 μg MPLA as adjuvant. Mice will be bled at day35 and will be challenged at day 42 with B16-FUT3 cells. Lung tumor metastasis will be analyzed fourteen days after tumor cell inoculation. Two groups of 5 mice (n=5) are considered as controls which will be treated as vehicle or 5B1-mIgG2a following the protocol discussed previously (Figure 3.9a). We are also interested in developing the therapeutic model II to address the question whether induced anti-sLea antibodies can prevent tumor progression, which can have clinical application in terms of passive immunization and monoclonal antibody development. Two rabbits are being immunized with (containing 8 μg for the first dose and 4 μg carbohydrate for subsequent immunizations) mixed with CFA or IFA adjuvant (Figure 3.11). The sera collected at day 63 will be used to prevent tumor development in mice challenged with B16-FUT3 or EL4-FUT3 in the set up discussed in figure 3.9a. Figure 3. 11 Two New Zealand rabbits were injected subcutaneously on day 0 with 0.1 mL Qβ- sLea constructs (at 8µg glycan) as emulsions in Complete Freund’s Adjuvant according to manufacturer’s instructions. Boosters were given subcutaneously on days 14, 28 and 42 (at 4µg glycan) mixed with Incomplete Freund’s Adjuvant. Serum samples were collected on days 0 (before immunization), 35, 49 and 63. 81 3.3 Discussion Changes in carbohydrate expression is often associated with tumor development. These changes are in the carbohydrate core structure and/or terminal carbohydrate structure such as incomplete synthesis and modification of normal cell surface carbohydrates [27]. The incomplete synthesis of the cell surface carbohydrates results in an increased expression of the precursor structures such as sLea, one of the blood group-related antigens [13]. sLea is a well-stablished biomarker for pancreatic cancer and other malignant stomach, lung, colon, breast, ovary, and uterus tumor cells [10]. Targeting carbohydrate antigens has been hampered due to the complexity of carbohydrate chemistry and biology. sLea is a well-recognized and the most extensively studied biomarker in diagnosis of pancreatic cancer in symptomatic patients, and can be monitored for information regarding prognosis, overall survival and prediction of recurrence inpatients with established disease [9]. Although the proteins carrying sLea are secreted into blood, the significant higher concentration of antigen in the tumor microenvironment than in serum lead to accumulation of antibodies in the tumor tissue [28,32]. The result from KLH-sLea conjugate vaccine in human was promising for developing monoclonal antibody targeting sLea [27]. The KLH-sLea conjugate vaccine was not effective in our study and this could be due to the lower loading, which reported the loading of 874 sLea per KLH molecule and different adjuvant that we have used [26]. Here we showed that sLea antigen was conjugated with a powerful protein carrier, bacteriophage Qβ, and this conjugate was superior in eliciting antibody response corresponding to the KLH conjugate. Considering the differences in antibodies produced by Qβ-sLea versus KLH- sLea in ELISA experiment, Qβ-sLea conjugate was selected for further in vitro and in vivo studies. Mice immunized with Qβ-sLea were able to generate significantly higher levels of IgG antibodies as compared to those immunized with sLea and Qβ mixture and the induced antibody responses 82 were long lasting up to 277 days. Furthermore, Qβ-sLea induced antibodies showed selective binding in glycan microarray analysis and sLea specific tissue staining. The generated anti-sLea antibodies were able to lyse 80% of sLea expressing cells in CDC assay and had strong binding to multiple sLea expressing cells. This result suggested that the induced antibodies can recognize the sLea antigen either as synthetic conjugated form (in ELISA and glycan micro array) or as natural antigen on cancer cell surface. Building on the encouraging in vitro result obtained, further in vivo studies have been designed to confirm the efficacy of Qβ-sLea conjugate vaccine which include the mouse protection model study after mice have been challenged with sLea expressing tumor cells either in active immunization or passive antibody treatment. 83 3.4 Materials and methods 3.4.1 General experimental procedures and methods for synthesis All chemicals were reagent grade and used as received from the manufacturer unless otherwise noted. Centrifugal filter units of 10,000 and 100,000 molecular weight cut-off (MWCO) were purchased from EMD Millipore. For characterization of Qβ-sLea conjugates, liquid chromatography-mass spectrometry (LCMS) analysis was performed. The samples for LCMS were prepared as follows: 1:1 v/v of 40 μg mL−1 of Qβ-sLea stock solution and an aqueous solution of 100 mM DTT was mixed and incubated in a water bath at 37 ℃ for 30 min. One drop of 50% formic acid was added to the mixture. LCMS was performed on Waters Xevo G2-XS quadrupole/time-of-flight UPLC/MS/MS. The liquid chromatography was done on Hypersil GOLD™ Cyano HPLC Column, 1mm x 10mm (3um particle size) with 1 mm I.D. Uniguard™ direct-connection guard cartridge holder using gradient eluent from 95% 0.1% formic acid in water to 95% 0.1% formic acid in CH3CN (0.3 mL min−1 flowrate). The multiple charge mass spectra were transformed to a single charge by using the algorithm MaxEnd148a. The average number of sLea on each Qβ subunit was analyzed by the signal intensities of the respective peaks in mass spectrum. For characterization of BSA-sLea conjugates, MALDI-TOF MS analysis was performed. The samples for MALDI-TOF were prepared as follows: 1:1 v/v of 2 mg mL−1 of BSA- sLea conjugates and 100 mM DTT was mixed and incubated in a water bath at 37 ℃ for 30 min. After desalting using Cleanup C18 Pipette Tips (Agilent Technologies), the sample (5 μL) and matrix solution (5 μL, 10 mg mL−1 sinapic acid in 50/50/0.1 CH3CN/H2O/TFA) was mixed and spotted on a MALDI plate, air-dried (3 rounds) and then analyzed by MALDI-TOF mass spectrometry (Applied Biosystems Voyager DE STR). Protein concentration was measured using 84 the Coomassie Plus Protein Reagent (Bradford Assay, Pierce) with bovine serum albumin (BSA) as the standard. For cell studies, EL4 lymphoma cells were from ATCC and grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% horse serum, 100 U mL−1 penicillin/100 μg mL−1 streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate. 1199, 1242, 1245 FB and Neo cell lines were generously provided by Dr. Dannielle D. Engle’s laboratory and cultured in DMEM with 10% FBS and 1%PS. B16-FUT3 and EL4-FUT3 cells were kindly provided by Dr. Jeffrey V. Ravetch’s laboratory and maintained in DMEM with 10% FBS and 1%PS. Monoclonal anti-sLea antibody produced in mouse, clone 121SLE, was obtained from sigma (SAB4700773). Low-Tox®-M Rabbit Complement (CL3051) was obtained from Cedarlane. Peroxidase-conjugate AffiniPure Goat Anti-Mouse IgG (H+L) (115-035-003) was obtained from Jackson ImmunoResearch. 5B1 recombinant antibody was constructed at Sino Biological through HEK293 expression using mouse IgG2a heavy chain and mouse kappa light chain backbone. The gene sequence for 5B1 rAb: Heavy chain MEFGLSWLFLVAILKGVQVQLVESGGGSVQPGRSLRLSCEASGFTFEAYAMHW VRQPPGKGLEWVSSINWNSGRIAYADSVKGRFTISRDNARNSLYLQMNSLRLEDTAFYY CAKDIRRFSTGGAEFEYWGQGTLVTVSSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVK GYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDD PDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKD LPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGK 85 TELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRT PGK Light chain MAGFPLLLTLLTHCAGSWAQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNFVYW YQQLPGTAPKLLIYRNNQRPSGVPDRFSGSRSGTSASLAISGLRSEDEADYYCAAWDDS LGGHYVFGTGTKVTVFSGSRSGTSASLAISGLRSEDEADYYCAAWDDSLGGHYVFGTG TKVTVRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNS WTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 3.4.2 Synthesis of Qβ-sLea conjugate To a solution of Qβ (1 mg, 5 mg/ml concentration, 0.4 nmol subunit, 0.36 μmol reactive amine) in 0.1 M K-Phos buffer pH 8.5, was added 3.28 mg of sLea-NCS (2.5 equivalent to reactive amine, 3.6 μmol). The reaction was incubated at 37 ℃ overnight and was gently inverted several times to ensure that the reactants were mixed. The unreactive tetrasaccharides were separated from the conjugate by centrifugal filtration against 0.1 M KPB buffer (pH 7.0, 0.1 M). The extent of particle modification was determined by ESI-TOF LC-MS and electrophoretic analysis. The total protein concentration was determined by Bradford assay against BSA standards. Percent protein recovery was 50–70 %. 3.4.3 KLH-sLea conjugation 1.5 mg of KLH was washed with 0.1 M KPB buffer, pH=8.6 for several rounds and the final concentration was determined with Bradford assay. 8000 equivalents of sLea-NCS sugar was added to KLH solution and the mixture was incubated at 37°C overnight. The reaction was purified by ultracentrifugation using 100 KDa cut off filter. The protein content was determined with the 86 Bradford assay and the sugar loading was quantified using anthrone assay10. Total carbohydrate content was 69 ug/ml, which corresponds to 400 copy of sLea per KLH molecule. A group of three C57BL/6 female mice was injected subcutaneously under the scruff on day 0, 14 and 28 with KLH-sLea constructs mixed with 20 ug MPLA as the adjuvant. 3.4.4 Synthesis of BSA-sLea conjugate For the synthesis of BSA-sLea conjugate, a solution of BSA (0.5 mg, 25 mg/ml) in 0.1 M KPB buffer pH 8.5, and sLea-NCS (20 equivalent, 0.137 mg, in 0.1 M KPB buffer pH 8.5) were added to the BSA solution (1.5 mg). The reaction was inverted and incubated at 37°C overnight. The product was purified by an Amicon Ultra 10 kDa MW cut-off against 0.1 M KPB. Total protein content was quantified by the Bradford assay against BSA standards. The extent of modification was determined by MALDI-TOF MS. 3.4.5 Mouse immunization Pathogen-free C57BL/6 female mice aged 6–10 weeks were maintained in the University Laboratory Animal Resources facility of Michigan State University. All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Michigan State University (approved protocol #: PROTO201900423). For evaluation of Qβ-sLea and KLH-sLea, C57BL/6 mice were subcutaneously injected under the scruff on day 0 with 0.2 mL of various Qβ-sLea / sLea + Qβ mixture / KLH-sLea vaccines in PBS containing MPLA (20 μL, 1 mg mL−1 in DMSO) for each mouse. For the Qβ-sLea conjugate or sLea + Qβ mixture, 5 mice immunized in each group and for KLH-sLea, 3 mice immunized. Boosters were given subcutaneously with the same amounts of vaccines with MPLA under the scruff on days 14 and 28. All Qβ-sLea /sLea + Qβ mixture / KLH-sLea conjugates administered have the same 87 amounts of sLea (4μg). Sera samples were collected on days 0 (before immunization), 35, 65, 95, 125, 185 and 277. 3.4.6 Rabbit immunization Rabbit immunization was performed by ProSci Incorporated (Poway, CA) with 2 New Zealand rabbits. Each rabbit was immunized subcutaneously on day 0 with Qβ-sLea (100 μl, 8 µg of sLea) as emulsions in Complete Freund’s Adjuvant according to the manufacturer’s instructions. Boosters were given subcutaneously under the scruff on days 14, 28 and 42 mixed with Incomplete Freund’s Adjuvant and 4 µg of sLea. Serum samples were collected on days 0 (before immunization), 49 and day 63 for the final bleed. 3.4.7 Tumor challenge and antibody treatments B16-FUT3 and EL4-FUT3 (5 × 105 cells/mouse) cells were inoculated i.v. into the lateral tail vein in 200 μL PBS. Mice were given intraperitoneal injections of 100 μg of 5B1 sLea recombinant antibody, rabbit sera (the amount for rabbit sera will be determined based on ELISA result) or PBS serving as control, on days 1, 4, 7, and 11 after inoculation. For the B16-FUT3 lung colonization model, the lungs were harvested on day 14 and analyzed for the presence of surface metastatic foci. For EL4-FUT3 tumor model, survival was assessed daily. 3.4.8 Evaluation of antibody titers by ELISA The Nunc MaxiSorp® flat-bottom 96-well microtiter plates were coated with 1 μg mL−1 of the BSA-sLea conjugate (100 μL/well) in NaHCO3/Na2CO3 buffer (0.05 M, pH 9.6) containing 0.02 % NaN3 by incubation at 4 oC overnight. The coated plates were washed with PBS/0.5% Tween-20 (PBST) (4 × 200 μL) and blocked with 1 % BSA in PBS (200 μL/well) at rt for 1 h. The plates were washed again with PBST (4 × 200 μL) and incubated with serial dilutions of 5B1 88 recombinant antibody mice or rabbit sera in 0.1 % BSA/PBS (100 μL/well, 3 wells for each dilution). The plates were incubated for 2 h at 37 ℃ and then washed with PBST (4 × 200 μL). A 1:2000 dilution of HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratory) in 0.1% BSA/PBS (100 μL) or peroxidase conjugated rabbit anti-rabbit IgG (SigmaAldrich, A6792) in 0.1% BSA/PBS (100 μL) was added to the wells respectively to determine the titers of antibodies generated. The plates were incubated for 1 h at 37 ℃ and then washed with PBST (4 × 200 μL). A solution of enzymatic substrate 3,3',5,5'-tetramethylbenzidine (TMB, 200 μL) was added to the plates (for one plate: 5 mg of TMB was dissolved in 2 mL of DMSO plus 18 mL of citric acid buffer containing 20 μL of H2O2). Color was developed for 15 min and quenched by adding 50 μL of 0.5 M H2SO4. The readout was measured at 450 nm using a microplate reader. The titer was determined by regression analysis with log10 dilution plotted with optical density and reported as the highest fold of dilution giving the optical absorbance value of 0.1 over those of the pre-immune control sera (OD ~ 0.1). All samples were performed in three replicates. 3.4.9 Detection of antibody binding to cells by FACS 1199, 1242, 1245 FB and Neo cell lines, B16-FUT3, EL4-FUT3 and EL4 cells were respectively cultured at 37 °C under 5% CO2 in cell growth medium (DMEM with 10% FBS and 1% P.S.). The number of cells was determined using a hemocytometer. Suspensions of 3.0 × 105 cells were added to each FACS tube, then centrifuged at 300X g for 5 min to remove the supernatant. The cell pellets were washed with FACS buffer (1% BSA in PBS with 0.1 % NaN3) and incubated with 1:20 dilution of mouse sera or different concentration of 5B1 or 121 SLE anti- sLea mAb in FACS buffer (100 μL) for 30 min on ice. The incubated cells were washed twice with FACS buffer, followed by incubation with PE conjugated goat anti-mouse IgG (minimal x- reactivity) antibody (Biolegend, 1 μL, 0.2 mg mL−1) for 30 min on ice. The cells were washed 89 twice, re-suspended in FACS buffer, and analyzed by LSR II (BD Biosciences). Data was processed by FlowJo software. 3.4.10 Complement dependent cytotoxicity 1199 FB cells (30,000 cells/well) were added in 96 well plate in DMEM. The culture medium was centrifuged at 1,600 rpm for 5 min and then removed carefully. Various dilutions of mouse sera (starting dilution is 20 fold) from different groups of immunized mice in 50 μL of DMEM were respectively added to the plate and incubated for 1h at 4°C. After 1h, the cells were washed twice with 1% BSA in PBS and supernatant discarded. Then rabbit serum complement at a dilution (1:10 dilution) in 100 μL of DMEM was added and incubated at 37 °C for 3 h. MTS (CellTiter 96® AQueous One Solution Cell Proliferation Assay; Promega, 20 μL) was added into each well and further incubated at 37 °C for 3 h. The optical absorption of the MTS assay was measured at 490 nm. Cells treated alone complement only were used as a positive control (maximum OD), and 5% Triton X-100 treated cells were used as a negative control (minimum OD). All data were performed in three replicates. Cytotoxicity was calculated as follows: Cytotoxicity (%) = (OD positive control – OD experimental) / (OD positive control – OD negative control) × 100. 3.4.11 Immunochemistry staining of cancer tissue microarrays Tissue arrays were purchased from US Biolab (XPan024 and XPan048) and stained at investigative Histopathology laboratory at Michigan State University using IP FLX background punisher (IP974), MACH3 mouse probe (MP530H), MACH3 mouse HPR-Poly (MH530H), Romulin AEC (RAEC810), and Cat Hematoxylin (CATHE) from BioCare Medical. Pooled sera from 5 mice (1:1000 dilution) from each group of Qβ-sLea or sLea + Qβ mixture at day 35 and 90 5B1 recombinant antibody at 1.08 µg/ml final concentration were used to stain the tissue arrays. Images were obtained with Nikon upright microscope, ECLIPSE Ci-L, with 10x magnitude. 3.4.12. Active tumor protection model C57BL/6 mice were subcutaneously injected under the scruff on day 0 with 0.2 mL of various Qβ-sLea / sLea + Qβ mixture vaccines in PBS containing MPLA (20 μL, 1 mg mL−1 in DMSO) for each mouse. For the Qβ-sLea conjugate or sLea + Qβ mixture, 10 mice immunized in each group. Boosters were given subcutaneously with the same amounts of vaccines with MPLA under the scruff on days 14 and 28. All Qβ-sLea /sLea + Qβ mixture administered have the same amounts of sLea (4μg). Serum samples were collected on days 0 (before immunization), 23, 35. At day 35, B16-FUT3 (5 × 105 cells/mouse) cells were inoculated i.v. into the lateral tail vein in 200 μL PBS. Two control groups (5 mice per group) will receive PBS or 100 μg of 5B1 IgG2a on days 1, 4, 7, and 11 after inoculation. The lung will be harvested on day 14 and analyzed for the presence of surface metastatic foci. 3.4.13 Passive tumor protection model B16-FUT3 and EL4-FUT3 (5 × 105 cells/mouse) cells will be inoculated i.v. into the lateral tail vein in 200 μL PBS. Mice will be given intraperitoneal injections of 100 μg of 5B1 recombinant antibody, rabbit sera (the amount for rabbit sera will be determined based on ELISA result) or PBS serving as control, on days 1, 4, 7, and 11 after inoculation. For the B16-FUT3 lung colonization model, the lungs will be harvested on day 14 and analyzed for the presence of surface metastatic foci. For EL4-FUT3 tumor model, survival will be assessed daily. 91 APPENDIX 92 Figure 3. 12 ESI-TOF HRMS spectrum for Qβ-sLea conjugate. Mass spectrometry analysis of the Qβ-sLea conjugate showed average loading of 300 tetrasaccharides on viral capsid. Each peaks shows the addition of sLea with MW of 918 Da. The average loading was calculated based on ratio of sum of peaks loading multiplied by their intensity to total intensity of all peaks. Figure 3. 13 MALDI-TOF characterization of BSA-sLea. The molecular weight of BSA shifted from 66,417 Da to 70,146 Da after conjugation. The difference of MW before and after conjugation is divided to the sLea MW (918) to obtain the average loading of four sLea sugar per BSA molecule. 93 **** **** 6× 10 3 a Qβ-sLe Conjugate Qβ + sLe a mixture MFI PE 4× 10 3 a Anti-sLe mAb Cell+ 2nd Ab 2× 10 3 Cell 0 ls ce ce l lls eo FB N 2 2 12 12 4 4 *** **** 3× 10 3 a Qβ-sLe Conjugate a Qβ + sLe mixture 2× 10 3 Anti-sLea mAb MFI PE Cell+ 2nd Ab Cell 1× 10 3 0 ce lls lls ce Neo FB 45 12 12 45 Figure 3. 14 Sialyl Lewis a expression in 1242 and 1245 FB cell line versus neo cells was analyzed with flow cytometry. Sera from mice immunized with Qβ-sLea conjugate bind to FB cells very strongly, while binding to Neo cells was similar to the cells without addition of serum in presence or absence of secondary Ab which served as negative control cells. The sLea and Qβ mixture did not show binding to FB or neo cell lines. Anti-Sialyl Lewis a mAb, 121 SLE, served as positive control. ***P = 0.0004, ****P < 0.0001 (unpaired two tailed t test). (The experiments for 1242 and 1245 were done separately with different instrument setting which resulted in different background level) 94 No. Map ID Sex Age Organ Pathology Diagnosis Grade Stage TNM 1 A1 M 50 Pancreas Ductal adenocarcinoma 3 IIB T3N1M0 2 A2 F 62 Pancreas Ductal adenocarcinoma 3 IIA T3N0M0 3 A3 F 23 Pancreas Ductal adenocarcinoma 3 IIA T3N0M0 4 A4 F 46 Pancreas Ductal adenocarcinoma 3 III T4N0M0 5 A5 F 47 Pancreas Ductal adenocarcinoma 3 IIB T3N1M0 6 B1 F 61 Pancreas Ductal adenocarcinoma 1--2 IB T2N0M0 7 B2 M 49 Pancreas Ductal adenocarcinoma 3 IIB T3N1M0 8 B3 M 64 Pancreas Ductal adenocarcinoma 3 IIA T3N0M0 9 B4 F 55 Pancreas Ductal adenocarcinoma 3 IB T2N0M0 10 B5 F 76 Pancreas Ductal adenocarcinoma 2--3 IIA T3N0M0 11 C1 F 51 Pancreas Ductal adenocarcinoma 2 IB T2N0M0 12 C2 M 44 Pancreas Ductal adenocarcinoma 2 IIA T3N0M0 13 C3 M 44 Pancreas Ductal adenocarcinoma 3 IIA T3N0M0 14 C4 M 74 Pancreas Ductal adenocarcinoma 2 IIA T3N0M0 15 C5 M 54 Pancreas Ductal adenocarcinoma 2--3 IIA T3N0M0 16 D1 M 49 Pancreas Ductal adenocarcinoma 2 IB T2N0M0 17 D2 M 41 Pancreas Ductal adenocarcinoma 2 III T4N1M0 18 D3 F 58 Pancreas Ductal adenocarcinoma 2 IIB T3N1M0 19 D4 F 64 Pancreas Ductal adenocarcinoma 2 IIA T3N0M0 20 D5 M 54 Pancreas Ductal adenocarcinoma 2 IB T2N0M0 21 E1 M 55 Pancreas Ductal adenocarcinoma 2 IIA T3N0M0 22 E2 M 65 Pancreas Ductal adenocarcinoma 2 IIB T3N1M0 23 E3 M 42 Pancreas Ductal adenocarcinoma 2 IIA T3N0M0 24 E4 F 42 Pancreas Ductal adenocarcinoma 2 IIA T3N0M0 Table 3. 1 Human pancreatic ductal adenocarcinoma tissue microarray (XPAN024-01) specification which contains 24 cores and 24 cases. 1 core per case. Each core has 1.5 mm diameter with 5μm thickness and fixed with formalin. 95 No. Map ID Sex Age Organ Pathology Diagnosis Grade Stage TNM 1 A1 M 40 Pancreas Pancreatic tissue 2 A2 M 45 Pancreas Pancreatic tissue 3 A3 F 42 Pancreas Pancreatic tissue 4 A4 M 48 Pancreas Metastatic pancreatic malignant islet cell tumor 5 A5 M 54 Pancreas Metastatic pancreatic papillary adenocarcinoma 6 A6 F 45 Pancreas Neuroendocrine tumor IIA T3N0M0 7 A7 M 29 Pancreas Neuroendocrine tumor (myeloid) G3 IIB T3N1M0 8 A8 F 37 Pancreas Neuroendocrine tumor 2 IB T2N0M0 9 B1 M 28 Pancreas Neuroendocrine tumor 1 IIB T2N1M0 10 B2 F 30 Pancreas Mixed acinar-endocrine tumor - IB T2N0M0 11 B3 M 62 Pancreas Neuroendocrine tumor - IIA T3N0M0 12 B4 F 47 Pancreas Neuroendocrine tumor - IIA T3N0M0 13 B5 M 62 Pancreas Squamous cell carcinoma 2--3 IIA T3N0M0 14 B6 F 49 Pancreas Squamous cell carcinoma 2 IIB T3N1M0 15 B7 M 52 Pancreas Adenosquamous carcinoma - IIA T3N0M0 16 B8 M 64 Pancreas Adenosquamous carcinoma - IIA T3N0M0 17 C1 F 62 Pancreas Adenosquamous carcinoma - IIA T3N0M0 18 C2 M 50 Pancreas Adenocarcinoma 3 IIA T3N0M0 19 C3 M 60 Pancreas Adenocarcinoma 3 IIA T3N0M0 20 C4 M 52 Pancreas Adenocarcinoma 3 IB T2N0M0 21 C5 M 62 Pancreas Adenocarcinoma 3 IIA T3N0M0 22 C6 F 66 Pancreas Adenocarcinoma 3 IIB T2N1M0 23 C7 M 50 Pancreas Adenocarcinoma 3 IIB T3N1M0 24 C8 M 45 Pancreas Adenocarcinoma 3 IIA T3N0M0 25 D1 F 46 Pancreas Adenocarcinoma 3 III T4N0M0 26 D2 F 23 Pancreas Adenocarcinoma 3 IIA T3N0M0 27 D3 F 62 Pancreas Adenocarcinoma 3 IIA T3N0M0 28 D4 F 47 Pancreas Adenocarcinoma 3 IIB T3N1M0 29 D5 F 61 Pancreas Adenocarcinoma 2--3 IB T2N0M0 30 D6 M 49 Pancreas Adenocarcinoma 2--3 IIB T3N1M0 31 D7 F 55 Pancreas Adenocarcinoma 2--3 IIA T3N0M0 32 D8 M 64 Pancreas Adenocarcinoma 2--3 IIA T3N0M0 33 E1 M 55 Pancreas Adenocarcinoma 2--3 IIA T3N0M0 34 E2 F 76 Pancreas Adenocarcinoma 2 IIA T3N0M0 35 E3 F 51 Pancreas Adenocarcinoma 2 IB T2N0M0 36 E4 M 44 Pancreas Adenocarcinoma 2 IIA T3N0M0 37 E5 M 74 Pancreas Adenocarcinoma 2 IIA T3N0M0 38 E6 M 54 Pancreas Adenocarcinoma 2 IIA T3N0M0 39 E7 M 44 Pancreas Adenocarcinoma 2 IIA T3N0M0 40 E8 M 49 Pancreas Adenocarcinoma 2 IB T2N0M0 41 F1 M 41 Pancreas Adenocarcinoma 2 III T4N1M0 42 F2 F 58 Pancreas Adenocarcinoma 2 IIB T3N1M0 43 F3 M 55 Pancreas Adenocarcinoma 2 IIA T3N0M0 44 F4 F 64 Pancreas Adenocarcinoma 2 IIA T3N0M0 45 F5 M 54 Pancreas Adenocarcinoma 1--2 IIA T3N0M0 46 F6 M 65 Pancreas Adenocarcinoma 1 IIB T3N1M0 47 F7 M 42 Pancreas Adenocarcinoma 1 IIA T3N0M0 48 F8 F 42 Pancreas Adenocarcinoma 1 IIA T3N0M0 Table 3. 2 Human pancreatic cancer tissue microarray (XPAN048-01) specification which contains 48 cores and 48 cases. 1 core per case. It contains 3 cases of normal tissue, 2 cases of metastatic cancer, 2 cases of squamous cell carcinoma, 2 cases of adenosquamous carcinoma, and 31 cases of pancreatic adenocarcinoma. 96 Figure 3. 15 XPAN024 microarray stained with pooled serum of 5 mice immunized with Qβ-sLea conjugate at day 35 at 1:1000 serum dilution. Characterization of each core has been provided in table 1 based on their map ID. The brown/red color was due to antibody binding to tissues. The lack of brown/red staining indicates little binding of antibodies to the tissues. Scale bar is 100μm. 97 Figure 3. 16 XPAN024 microarray stained with pooled serum of 5 mice immunized with sLea and Qβ mixture at day35 at 1:1000 serum dilution. Characterization of each core has been provided in table 1 based on their map ID. The brown/red color was due to antibody binding to tissues. The lack of brown/red staining indicates little binding of antibodies to the tissues. Scale bar is 100μm. 98 Figure 3. 17 XPAN048 microarray stained with pooled serum of 5 mice immunized with Qβ-sLea conjugate at day 35 at 1:1000 serum dilution. Characterization of each core has been provided in table 2 based on their number. The brown/red color was due to antibody binding to tissues. The lack of brown/red staining indicates little binding of antibodies to the tissues. Scale bar is 100μm. 99 Figure 3.17 (cont’d) 100 Figure 3. 18 XPAN024 microarray hematoxylin and eosin (H&E) staining. Pictures were obtained to determine the inflammatory stage of tissues. Areas with dense nucleus population (dark blue spots) suggests the presence of macrophages. Characterization of each core has been provided in table 1 based on their map ID. Scale bar is 100μm. 101 Figure 3. 19 XPAN024 microarray stained with 5B1 recombinant antibody at 1.08 µg/ml final concentration. Characterization of each core has been provided in table 1 based on their map ID. The brown/red color was due to antibody binding to tissues. The lack of brown/red staining indicates little binding of antibodies to the tissues. Scale bar is 100μm. 102 Figure 3. 20 Images of lung tissues obtained from the mice immunized with a) sLea and Qβ mixture, and b) Qβ-sLea conjugate which challenged with B16-FUT3 cell line 356 days post immunization. Figure 3. 21 ESI-TOF HRMS spectrum for the 2nd batch of Qβ-sLea conjugate that used for rabbit study. Mass spectrometry analysis of the Qβ-sLea conjugate showed that the number of tetrasaccharides on viral capsid were 455 on average. 103 Qβ-sLea conjugate Qβ + sLea mixture IgG IgG Family a81 Abbreviation Description S-LR S-N S-L S-R S-2L average C-R C-2L C-LR C-N C-L 5 Lewis 48 3'Neu5Ac-LeA -12 Neu5Aca2-3Galb1-3[Fuca1-4)GlcNAcb1-3Galb1-APD- 213 6 9 0 6 HAS; Sialyl Lewis A; SLeA 5447 32545 32206 26581 25451 24446 Lewis 281 3'Neu5Ac-LeC-Sp - Neu5Aca2-3Galb1-3GlcNAcb-Sp-BSA 286 105 159 -54 8 0 -30 0 12 65479 65357 26277 Lewis 284 3'Neu5Ac-LeC-Sp - Neu5Aca2-3Galb1-3GlcNAcb-Sp-BSA 122 50 79 -57 11 12 -32 3 05 65476 65360 26217 Lewis 303 3'Neu5Gc-LeC-Sp - Neu5Gca2-3Galb1-3GlcNAcb-Sp-BSA 124 6 39 -24 7 19 -20 5 12 54451 55663 22057 Lewis 233 3'Neu5Ac(9Ac)-LeC- Neu5Ac(9Ac)a2-3Galb1-3GlcNAcb-Sp-BSA 266 2 150 -54 8 0 -16 0 Sp - 12 17534 15768 6744 Lewis 310 3'Neu5Ac(9Ac)-LeC- Neu5Ac(9Ac)a2-3Galb1-3GlcNAcb-Sp-BSA 126 -43 93 -82 0 0 -40 0 Sp - 05 14805 16309 6258 Lewis 268 3'KDN-LeC-Sp - 12 KDNa2-3Galb1-3GlcNAcb-Sp-BSA 0 -34 0 -63 5 6 -29 0 61039 46803 21561 Lewis 316 3'Neu5Gc-LeC-Sp - Neu5Gca2-3Galb1-3GlcNAcb-Sp-BSA 34 -20 11 -60 5 6 -34 0 05 43014 48599 18328 Lewis 263 3'KDN-LeC-Sp - 04 KDNa2-3Galb1-3GlcNAcb-Sp-BSA 5 -31 0 -45 5 6 -25 0 31708 23708 11078 glycolipid 659 GD2 tetra (Kdn, Gc) Kdna2-8Neu5Gca2-3(GlcNAcb1-4)Galb1- -3 57 -40 0 265 -40 32 0 -20 3 1309 glycolipid 644 3'Neu5Ac-LNnT Neu5Aca2-3Galb1-4GlcNAcb1-3Galb1-4 887 -20 42 -57 0 170 -65 20 3 -40 0 glycolipid 647 GD3 tri (Ac-Kdn) Neu5Aca2-8Kdna2-3Galb1- 710 -10 34 -32 0 141 -37 12 0 -23 0 non-human 671 laminarihexaose - Glcb1-3Glcb1-3Glcb1-3Glcb1-3Glcb1-3Glcb1- 771 179 42 -59 130 212 -12 17 91 -42 3 05 non-human 547 Glcb1-3Glcb- 26 Glcb1-3Glcb- 0 332 11 535 402 3 74 -31 0 1132 2712 Blood Group 294 Globo A - 09 GalNAca1-3(Fuca1-2)Galb1-3GalNAcb1-3Gala1-4Galb1- 45 32 A BSA 28156 3809 31608 5882 19376 17766 10741 21254 1791 Blood Group 63 BG-A4-14 GalNAca1-3(Fuca1-2)Galb1-3GalNAcb1-linker-BSA 388 22 -65 221 A 21755 1428 19453 18890 12383 5244 7843 Blood Group 58 BG-A3-14 GalNAca1-3(Fuca1-2)Galb1-3GalNAcα1-linker-BSA 574 310 564 28 -54 279 A 17664 8961 12985 8099 16648 Blood Group 94 Globo A - 03 GalNAca1-3(Fuca1-2)Galb1-3GalNAcb1-3Gala1-4Galb1- 133 85 351 20 -66 204 A BSA 5375 4452 5158 3040 2140 y- 363 PSM A pos porcine submaxillary mucin (A+) 14 206 -9 93 9 20 247 -45 125 glycoprotein 1831 glycolipid- 31 Gala1-4Galb - 11 Gala1-4Galb-CETE-BSA 574 45 105 3 2 11 168 70 25 102 neutral Blood Group 62 BG-A4-05 GalNAca1-3(Fuca1-2)Galb1-3GalNAcb1-linker-BSA 164 117 -34 68 3 -49 11 A 1630 1128 1484 non-human 13 Rha-b - 21 Rha-b - BSA 34 39 139 0 5 -23 40 108 -3 68 Blood Group 71 BG-B3-17 Gala1-3(Fuca1-2)Galb1-3GalNAcα1-linker-BSA 6 -34 102 -9 5 -119 11 159 -51 136 B Blood Group 112 Globo B - 12 Gala1-3(Fuca1-2)Galb1-3GalNAcb1-3Gala1-4Galb1-BSA 6 -31 140 -33 17 -91 3 105 -57 42 B non-human 619 Rha-a-23 Rha-a – BSA 572 6 551 0 177 -12 322 788 15 506 non-human 12 Rha-a - 18 Rha-a – BSA 402 -3 538 -14 430 -33 278 -3 707 y-other 481 DNP-BSA dinitrophenylated lysines on BSA 19 110 226 314 262 646 1616 26 159 25 5134 y-other 401 dsDNA double stranded calf thymus DNA 6 -3 746 0 11 -3 3 40 -3 5 peptide-Tn 173 Ac-P-Tn(Thr)-T-G - Ac-Pro-(GalNAca)Thr-Thr-Gly-Hex-BSA (muc2) 6 -25 143 -51 0 -31 5 103 -32 6 22 non-human 692 Xylb5 - 05 Xylb1-4Xylb1-4Xylb1-4Xylb1-4Xylb1-BSA 420 -18 28 -40 0 950 12 6 -51 0 carb-GlcNAc 266 3'GN type1-Sp - 16 GlcNAcb1-3Galb1-3GlcNAcb-Sp-BSA 133 -6 200 -11 0 -39 0 -3 -27 62 carb-GlcNAc 286 3'GN-LacNAc GlcNAcb1-3(Galb1-4GlcNAcb1-3)2b-Sp-BSA 43 -26 113 -57 0 -93 0 3 -59 79 (dimeric)-Sp - 14 carb-GalNAc 719 GalNAca-phenyl GalNAca-O-phenyl-amide linked 43 -12 7 -28 0 -25 0 0 -17 3 amide- high carb-GlcNAc 320 3'GN-LacNAc GlcNAcb1-3(Galb1-4GlcNAcb1-3)2b-Sp-BSA 17 -9 171 -37 0 -46 -3 6 -31 94 (dimeric)-Sp - 06 carb-GalNAc 5 GalNAc-a - 22 GalNAc-a - BSA 6 -9 8 -31 8 -40 3 9 -26 0 peptide-core 338 Ac-S-Thr(core 3)-S- Ac-Ser-(GlcNAcb1-3GalNAca)Thr-Ser-Gly-Hex-BSA 3 -40 197 -40 0 -66 5 6 -35 20 3 G - 21 peptide- 518 AzHex-G-Y(GalNAc-a)-A-NH2 BSA-AzHex-G-Y(GalNAc-a)-A-amide - 04 3 -42 0 -22 0 -32 2 0 -17 0 GalNAc Tyr Blood Group 350 BG-H6-21 Fuca1-2Galb1-4Glcb1-linker-BSA 3 -10 191 -51 0 -54 0 6 -49 3 H carb-GlcNAc 255 GNLacNAc-Sp - 16 GlcNAcb1-3Galb1-4GlcNAcb-Sp-BSA 3 -26 221 -48 0 -95 8 29 -63 193 glycolipid- 80 P1 - 09 Gala1-4Galb1-4GlcNAc-BSA 3 -29 3 -45 0 -80 -1 0 -43 0 neutral non-human 45 X3Glc3 - 15 Xyla1-6Glcb1-4(Xyla1-6)Glcb1-4(Xyla1-6)Glcb1-BSA 3 -26 3 -43 4 -63 0 2 -46 0 glycolipid- 124 GA2di - 37 GalNAcb1-4Galb - BSA (aka: asialo-GM2) 2 -27 11 -31 3 -54 3 196 -43 0 neutral glycolipid- 164 GA2di (accurate) - GalNAcb1-4Galb - BSA (aka: asialo-GM2) 0 -32 3 -40 0 -57 5 168 -37 0 neutral 28 Table 3. 3 Glycopeptide microarray screening results of antisera induced by Qβ-sLea or Qβ and sLea mixture. 104 Table 3.3 (cont’d) non-human 512 AX3 - 04 3 0 -22 5 -22 0 -4 0 -49 0 Xylβ1-4(Arafα1-2)Xylβ1-4Xylβ-]; 3 -α-L- 1750 Arabinofuranosyl-xylotriose non-human 801 Xylb2 - 26 Xylb1-4Xylb1-BSA 0 -3 11 6 0 -3 6 -15 9 non-human 769 Xylb3 - 05 Xylb1-4Xylb1-4Xylb1-BSA 0 -20 5 -17 0 1426 0 6 -29 0 carb-Sia 274 6'Neu5Ac-LDN-Sp - Neu5Aca2-6GalNAcb1-4GlcNAcb-Sp-BSA 19 -8 0 -31 0 2566 -47 0 6 -29 0 13 Carb-Glc 736 Glca1-6Glca -15 Glca1-6Glca -BSA 0 -22 2 -46 0 -71 -3 3 -40 -3 Blood Group 61 BG-B4-06 Gala1-3(Fuca1-2)Galb1-3GalNAcb1-linker-BSA 8 -29 28 -56 8 -108 0 27 -57 0 B non-human 674 chitotriose - 04 GlcNAcb1-4GlcNAcb1-4GlcNAcb1- 911 -14 45 -41 0 -55 20 0 -42 0 carb-Glc 686 Isomaltotriose - 05 Glca1-6Glca1-6Glca1- 895 -20 59 -78 0 -102 20 0 -59 3 carb-GlcNAc 287 LNT-2-Sp - 06 GlcNAcb1-3Galb1-4Glcb-Sp-BSA 684 105 98 -37 0 -57 3 3 -37 9 glycolipid 650 GD3 tri (Kdn-Kdn) Kdna2-8Kdna2-3Galb1- 655 -3 34 -23 0 -20 17 0 -14 -6 glycolipid 653 GD3 tri (Ac-Gc) Neu5Aca2-8Neu5Gca2-3Galb1- 649 -3 31 -28 0 -29 20 3 -17 -3 carb-Man 641 Mana1-6Man-a - 14-HSAMana1-6Man-a - HSA 622 -3 28 -20 0 -23 23 0 -8 -3 glycolipid 662 GD2 tetra (Gc, Kdn) Neu5Gca2-8Kdna2-3(GlcNAcb1-4)Galb1- 618 -5 28 -9 0 -11 15 0 -12 0 non-human- 290 alpha-Gal tetra - 17 Gala1-3Galb1-4GlcNAcb1-3Galb1-BSA 567 54 0 -29 0 -48 3 9 -29 3 aGal glycolipid 665 GD1a (Gc, Gc) Neu5Gca2-8Neu5Gca2-3(Galb1-3GlcNAcb1-4)Galb1- 544 -8 28 -22 0 -26 23 0 -18 3 glycolipid 656 GD2 tetra (Kdn, Kdn)Kdna2-8Kdna2-3(GlcNAcb1-4)Galb1- 513 -8 27 -23 0 -26 26 0 -14 0 Lewis 668 LeY tetra-10 Fuca1-2Galb1-4[Fuca1-3)GlcNAcb1- 500 -11 31 -17 0 -27 15 0 -23 6 carb-Gal 683 Galb1-6Galb1 - 05 Galb1-6Galb1- 394 6 39 -43 0 -63 12 0 -40 14 glycolipid 695 GD2-NHAc (Xuefei) Neu5Ac9NHAca2-8Neu5Aca2-3(GlcNAcb1-4)Galb1-Glc- 371 12 34 0 14 -26 15 3 -14 11 glycolipid 280 GQ2-Sp - 06 Neu5Aca2-8Neu5Aca2-8Neu5Aca2-8Neu5Aca2- 366 145 0 -17 0 -48 3 0 -26 3 3[GalNAcb1-4]Galb1-4Glcb-Sp-BSA peptide- 306 Ac-S-Ser(GlcNAc-b)- Ac-Ser-(GlcNAcβ)Ser-Ser-Gly-Hex-BSA 346 15 17 -29 5 -46 -3 14 -27 0 GlcNAcb S-G - 07 carb-Glc 680 maltotriose - 21 Glca1-4Glca1-4Glca1- 298 -10 22 -26 0 -29 5 3 -20 5 carb-Glc 677 maltoheptaose - 21 Glca1-4Glca1-4Glca1-4Glca1-4Glca1-4Glca1-4Glca1- 238 -9 8 -21 0 -20 6 3 -17 6 carb-Sia 319 6'Neu5Ac-LacNAc- Neu5Aca2-6Galb1-4GlcNAcb-Sp-BSA 199 54 0 -17 0 -23 0 0 -10 3 Sp - 11 Blood Group 293 Globo H - 10 Fuca1-2Galb1-3GalNAcb1-3Gala1-4Galb1-BSA 193 35 3 -40 0 -51 3 6 -31 0 H carb-Glc 701 Glcb1-4Glcb1-4Glc - Glcb1-4Glcb1-4Glcb1- 190 -8 17 -37 0 -40 9 2 -23 3 04 carb-Sia 297 3'Neu5Ac-LacNAc Neu5Aca2-3(Galb1-4GlcNAcb1-3)2b-Sp-BSA 171 -9 142 -37 110 -40 0 79 -35 17 (dimeric)-Sp - 13 non-human- 309 alpha-Gal tetra - 04 Gala1-3Galb1-4GlcNAcb1-3Galb1-BSA 167 9 26 -26 5 -40 0 11 -17 3 aGal Blood Group 300 TFiLNO(1-2,1-2,1- Fuca1-2Galb1-3GlcNAcb1-3Galb1-4GlcNAcb1-6[Fuca1- 164 -11 74 -34 28 -57 0 34 -39 14 H 4) - 06 2Galb1-3(Fuca1-4)GlcNAcb1-3]Galb-BSA carb-Glc 698 maltopentaose - 17 Glca1-4Glca1-4Glca1-4Glca1-4Glca1- 153 -8 20 -22 0 -34 6 9 -23 0 carb-Gal 707 Galb1-3Galb1 - 24 Galb1-3Galb1- 133 -20 11 -57 0 -65 6 0 -43 3 carb-Glc 710 maltohexaose - 20 Glca1-4Glca1-4Glca1-4Glca1-4Glca1-4Glca1- 131 -5 14 -20 0 -22 6 6 -23 3 carb-type 1 313 MFiLNO(1-3) - 09 Galb1-3GlcNAcb1-3Galb1-4(Fuca1-3)GlcNAcb1-6 105 -12 5 -59 0 -82 0 0 -45 0 (Galb1-3GlcNAcb1-3)Galb1-BSA glycolipid 288 GT2-Sp - 08 Neu5Aca2-8Neu5Aca2-8Neu5Aca2-3[GalNAcb1- 102 -3 3 -17 0 -23 0 3 -14 0 4]Galb1-4Glcb-Sp-BSA carb-Glc 716 maltotetraose - 05 Glca1-4Glca1-4Glca1-4Glca1- 80 -14 6 -32 0 -31 0 6 -21 3 carb-GalNAc 713 GalNAcb-phenyl Dz- GalNAcb-O-phenyl-diazirine linker 79 -5 5 -20 0 -34 3 0 -11 3 high glycolipid 271 GD3-Sp - 04 Neu5Aca2-8Neu5Aca2-3Galb1-4Glcb-Sp-BSA 71 0 3 -20 0 -29 0 0 -12 0 Carb-Glc 794 Glcb1-4Manb1 - 15 Glcb1-4Manb1 - BSA 68 -20 17 -59 0 -68 6 0 -34 -3 Carb-Glc 809 Glca1-6Glca1-6Glca1-6Glca1-6Glca1-6Glca Glca1-6Glca1-6Glca1-6Glca1-6Glca1-6Glca -09 -BSA 54 0 8 -7 0 -40 3 0 -20 0 non-human 737 PNAG 16 (10000) GlcNAcb1-6GlcNb1-6GlcNb1-6GlcNb1-6GlcNb1- 54 3 5 -8 0 -23 0 0 -15 3 PNAG glycolipid 236 GD1a-Sp - 10 Neu5Aca2-3[Neu5Aca2-3Galb1-3GalNAcb1-4]Galb1- 51 3 0 -11 0 -8 0 3 3 0 4Glcb-Sp-BSA glycolipid 312 GM2-Sp - 07 Neu5Aca2-3[GalNAcb1-4]Galb1-4Glcb-Sp-BSA 51 -11 11 -28 5 -63 3 11 -43 5 Blood Group 269 B tetra type 2-Sp - Gala1-3[Fuca1-2]Galb1-4GlcNAcb-Sp-BSA 48 -3 74 -34 0 -42 0 -3 -29 -3 B 20 Blood Group 99 BG-A1- 05 GalNAca1-3(Fuca1-2)Galb1-3GlcNAcb1-3Galb1-4(Glc)- 47 156 141 105 113 -77 -3 3 -34 0 A APD-HSA non-human 746 PNAG 25 (11001) GlcNAcb1-6GlcNAcb1-6GlcNb1-6GlcNb1-6GlcNAcb1- 46 -9 3 -17 0 -20 3 3 -3 3 PNAG non-human 743 Ara3 - 06 Araa1-5Araa1-5Ara-BSA 45 -12 0 -31 79 -88 0 23 -60 8 glycolipid 322 GM3(Gc)-Sp - 05 Neu5Gca2-3Galb1-4Glcb-Sp-BSA 45 -7 0 -42 0 -70 0 0 -45 0 non-human 740 PNAG 19 (10011) GlcNAcb1-6GlcNb1-6GlcNb1-6GlcNAcb1-6GlcNAcb1- 40 -3 3 -20 0 -31 3 0 -19 -3 PNAG non-human 779 PNAG 27 (11011) GlcNAcb1-6GlcNAcb1-6GlcNb1-6GlcNAcb1-6GlcNAcb1- 40 0 8 -8 8 -9 0 6 -6 6 PNAG carb-Sia 296 3'Neu5Gc-LacNAc- Neu5Gca2-3Galb1-4GlcNAcb-Sp-BSA 39 3 5 -31 0 -54 0 6 -31 0 Sp - 10 105 Table 3.3 (cont’d) non-human 791 4-Me-GlcAa1-2Xylb1-4Xylb- 4-Me-GlcAa1-2Xylb1-4Xylb- 19 BSA 37 -9 5 -23 0 -23 0 -3 -9 -6 carb-Sia 315 6'Neu5Ac-LacNAc Neu5Aca2-6[Galb1-4GlcNAcb1-3)2b-Sp-BSA 37 -17 11 -42 5 -83 0 6 -48 3 (dimeric)-Sp - 05 carb-GalNAc 728 GalNAca-phenyl Dz- GalNAca-O-phenyl-diazirine linker 37 -3 3 -12 0 -20 2 0 -8 3 low non-human 770 PNAG 20 (10100) GlcNAcb1-6GlcNb1-6GlcNAcb1-6GlcNb1-6GlcNb1- 36 -5 5 -17 22 -26 0 9 -16 5 PNAG non-human 785 PNAG 31 (11111) GlcNAcb1-6GlcNAcb1-6GlcNAcb1-6GlcNAcb1-6GlcNAcb1- 34 0 5 -11 34 -13 0 6 -6 0 PNAG Blood Group 66 BG-B1-05 Gala1-3(Fuca1-2)Galb1-3GlcNAcb1-linker-BSA 34 -6 32 -23 61 -37 3 23 -20 3 B non-human 788 PNAG 1 (00001) GlcNb1-6GlcNb1-6GlcNb1-6GlcNb1-6GlcNAcb1- 31 -6 5 -11 39 -1 6 11 -3 0 PNAG carb-GalNAc 731 GalNAcb-phenyl GalNAcb-O-phenyl-amide linked 28 0 5 -8 0 -12 6 3 -14 -2 amide- low glycolipid 676 GD2 (Xuefei) Neu5Aca2-8Neu5Aca2-3(GlcNAcb1-4)Galb1-Glc- 28 19 22 15 14 -25 3 6 -12 20 non-human 782 PNAG 7 (00111) GlcNb1-6GlcNb1-6GlcNAcb1-6GlcNAcb1-6GlcNAcb1- 27 -8 5 -15 0 -18 5 3 -14 0 PNAG carb-Sia 308 3'Neu5Ac-LacNAc Neu5Aca2-3(Galb1-4GlcNAcb1-3)2b-Sp-BSA 26 -5 5 -42 0 -37 0 0 -28 0 (dimeric)-Sp - 06 y- 359 CEA carcinoembryonic antigen isolated from human 26 40 54 45 28 37 20 39 32 25 glycoprotein metastatic liver non-human 539 KDOa2-8KDOa2-4KDOa KDOa2-8KDOa2-4KDOa-APTE-BSA - 06 26 28 25 46 11 66 26 6 29 14 non-human 800 Manb2 - 18 Manb1-4Man 26 -6 14 -39 0 128 6 0 -46 0 non-human 749 PNAG 4 (00100) GlcNb1-6GlcNb1-6GlcNAcb1-6GlcNb1-6GlcNb1- 26 -5 5 -21 0 -11 3 0 -12 3 PNAG non-human 734 PNAG 0 (00000) GlcNb1-6GlcNb1-6GlcNb1-6GlcNb1-6GlcNb1- 25 -3 7 -8 0 -3 3 5 -6 0 PNAG glycolipid 651 GM2 tri (8MeAc) Neu5Ac8Mea2-3(GlcNAcb1-4)Galb1- 25 -16 22 -49 0 -85 23 37 -29 31 non-human 803 Arafa1-3(Arafa1-2)Xylb1-4Xylb Arafa1-3(Arafa1-2)Xylb1-4Xylb - 17 -BSA 23 0 5 -8 0 -11 0 3 -9 -3 Blood Group 351 BG-A5-16 GalNAca1-3(Fuca1-2)Galb1-3Galb1-linker-BSA 23 -17 16 -40 20 -54 0 12 -40 0 A non-human 97 Cellotriose - 13 Glcb1-4Glcb1-4Glcb-BSA 23 -14 21 -34 0 -31 6 40 -34 5 non-human 289 Forssman Tetra- GalNAca1-3GalNAcb1-3Gala1-4Galb-BSA 23 -17 0 -45 0 -85 0 6 -45 0 BSA - 13 non-human 51 G2M4 - 07 Manb1-4(Gala1-6)Manb1-4(Gala1-6)Manb1-4Manb1- 23 -3 125 -20 184 66 3 0 -34 3 BSA glycolipid 325 GT3-Sp - 07 Neu5Aca2-8Neu5Aca2-8Neu5Aca2-3Galb1-4Glcb-Sp- 23 3 3 -6 0 -12 3 3 -9 3 BSA glycolipid- 305 Gb5/SSEA3 - 12 Galb1-3GalNAcb1-3Gala1-4Galb1-BSA 20 6 3 -32 0 -34 0 0 -14 12 neutral glycolipid 285 GM3-Sp - 04 Neu5Aca2-3Galb1-4Glcb-Sp-BSA 20 -13 3 -43 0 -51 0 0 -32 3 carb-type 1 302 LNT-Sp - 15 Galb1-3GlcNAcb1-3Galb1-4GlcNAcb-Sp-BSA 20 3 5 -29 0 -37 3 6 -9 3 Peptide 722 TSSASTGH-BSA N-terminus Muc4 – TSSASTGH-BSA 20 -3 0 -22 0 -28 0 0 -20 -3 carb-GlcNAc 328 3'GN type1-Sp - 04 GlcNAcb1-3Galb1-3GlcNAcb-Sp-BSA 17 -9 71 -40 0 -57 3 6 -37 11 Lewis 812 3'Neu5Ac-LeX Neu5Aca2-3Galb1-4[Fuca1-3)GlcNAc – HSA 17 -8 5 -12 0 -9 3 0 -6 0 (Sialyl LeX) - HSA carb-Sia 324 6'Neu5Ac-LDN-Sp - Neu5Aca2-6GalNAcb1-4GlcNAcb-Sp-BSA 17 -10 5 -34 0 -40 3 3 -23 0 05 glycolipid- 291 Gb5/SSEA3 - 04 Galb1-3GalNAcb1-3Gala1-4Galb1-BSA 17 -8 5 -34 0 -43 3 0 -22 0 neutral glycolipid 318 GD1b - 05 Neu5Aca2-8Siaa2-3(Galb1-3GalNAcb1-4)Galb1-4-BSA 17 -17 5 -32 3 -57 2 6 -40 0 N-linked 321 Man1 - 12 Manβ1-4GlcNAcβ1-4GlcNAcβ1-BSA 17 -12 17 -26 0 -31 3 0 -28 0 non-human 752 PNAG 18 (10010) GlcNAcb1-6GlcNb1-6GlcNb1-6GlcNAcb1-6GlcNb1- 17 3 0 -11 3 -3 3 6 -9 -3 PNAG non-human 755 Xylb2 - 05 Xylb1-4Xylb1-BSA 17 -14 5 -42 0 261 0 0 -37 0 Blood Group 193 6'Neu5Ac-LNF V - Fuca1-2Galb1-3(Neu5Aca2-6)GlcNAcb1-3Galb1-APD- 14 85 54 5 39 -77 0 0 -45 -2 H 12 HSA glycolipid 522 GM2 tri (Ac) - 06 Neu5Aca2-3(GalNAcb1-4)Galb- 14 -14 3 -45 0 -128 12 11 -43 14 glycolipid 304 GQ2-Sp - 03 Neu5Aca2-8Neu5Aca2-8Neu5Aca2-8Neu5Aca2- 14 -14 3 -37 0 -59 0 6 -40 0 3[GalNAcb1-4]Galb1-4Glcb-Sp-BSA carb-type 2 311 LacNAc (dimeric)- (Galb1-4GlcNAcb1-3)2b-Sp-BSA 14 -17 237 -45 0 -57 -3 0 -37 -3 Sp - 06 Blood Group 92 2'F-B type 2-Sp - 07 Gala1-3[Fuca1-2]Galb1-4[Fuca1-3]GlcNAcb-Sp-BSA 11 -11 5 -34 0 -43 0 3 -31 5 B Blood Group 267 2'F-B type 2-Sp - 15 Gala1-3[Fuca1-2]Galb1-4[Fuca1-3]GlcNAcb-Sp-BSA 11 -11 0 -23 0 -54 0 3 -28 0 B carb-Sia 292 3'Neu5Ac-LacNAc- Neu5Aca2-3Galb1-4GlcNAcb-Sp-BSA 11 -6 3 -28 0 -54 0 3 -29 0 Sp - 10 peptide-Gal 410 Ac-S-S(Gala)-S-G - Ac-S-S(Gal a)-S-G-Hex-OH 11 -9 25 -14 31 -9 6 46 0 22 16 non-human 638 Forssman Di - 05-HSAGalNAca1-3GalNAcb1-HSA 11 -9 0 -17 0 -8 0 0 -14 -3 glycolipid 295 GT2-Sp - 03 Neu5Aca2-8Neu5Aca2-8Neu5Aca2-3[GalNAcb1- 11 -16 5 -48 0 -80 3 6 -54 0 4]Galb1-4Glcb-Sp-BSA y- 368 hsp90 Heat Shock Protein 90 11 33 20 51 5 62 6 8 43 17 glycoprotein carb-type 2 307 LacNAc (dimeric)- (Galb1-4GlcNAcb1-3)2b-Sp-BSA 11 -12 0 -39 0 -67 -3 3 -34 -3 Sp - 16 carb-GalNAc 323 LDN-Sp - 05 GalNAcb1-4GlcNAcb-Sp-BSA 11 -3 5 -42 0 -51 0 6 -20 3 Lewis 239 LeC (dimeric)-Sp - Galb1-3GlcNAcb1-3Galb1-3GlcNAcb-Sp-BSA 11 -9 3 -28 0 -43 3 2 -23 3 06 carb-Man 275 Man-a - 05 Man-a - BSA 11 0 0 -32 0 -33 3 0 -20 0 106 Table 3.3 (cont’d) non-human 764 PNAG 14 (01110) GlcNb1-6GlcNAcb1-6GlcNAcb1-6GlcNAcb1-6GlcNb1- 11 -5 5 -17 0 -26 0 3 -13 -3 PNAG non-human 767 PNAG 3 (00011) GlcNb1-6GlcNb1-6GlcNb1-6GlcNAcb1-6GlcNAcb1- 11 -3 4 -11 17 -14 0 9 -12 3 PNAG non-human 758 PNAG 5 (00101) GlcNb1-6GlcNb1-6GlcNAcb1-6GlcNb1-6GlcNAcb1- 11 -3 0 -6 0 -17 -3 3 -6 0 PNAG peptide-TF 725 TSSA(S-TF)TGHA(T- TF-5,10-MUC4- TSSA(S-TF)TGHA(T-TF)PLPVTD-BSA 11 -3 0 -20 0 -46 -3 0 96 -5 TF)PLPVTD-BSA carb-Sia 327 3'Neu5Ac-LDN-Sp - Neu5Aca2-3GalNAcb1-4GlcNAcb-Sp-BSA 9 -10 5 -26 0 -48 0 0 -20 0 11 peptide-Glc 393 Ac-A-S(Glcb)-S-G- Ac-A-S(Glcb)-S-G-Hex-BSA 9 -6 82 -23 0 -54 42 60 -20 0 Hex-18 peptide-TF 616 APF (asialo) 9 14 30 45 14 25 14 14 22 14 Blood Group 64 BG-B2-03 Gala1-3(Fuca1-2)Galb1-4GlcNAcb1-linker-BSA 9 -17 14 -65 11 -78 3 14 -43 3 B carb-Sia 298 CT/Sda-Sp - 05 Neu5Aca2-3[GalNAcb1-4]Galb1-4GlcNAcb-Sp-BSA; like 9 -23 5 -43 0 -71 0 3 -37 2 GM2 but on glycoproteins GAG-Hep 570 Hep-Octa-GT24-02 Glc(6S, Nac)a1-4GlcAb1-4Glc(6S, 3S, NS)a1-4IdoA(2S)a1- 9 15 20 -15 0 -20 11 6 48 9 4Glc(6S, NS)a1-4IdoA(2S)a1-4Glc(6S, NS)a1-4GlcAb- Benzamide- carb-type 1 278 LNT-Sp - 06 Galb1-3GlcNAcb1-3Galb1-4GlcNAcb-Sp-BSA 9 -20 0 -43 0 -50 5 0 -34 0 non-human 761 Manb1-4Glcb1 - 06 Manb1-4Glcb1 -BSA 9 -17 8 -32 0 28 3 3 -40 -3 non-human 10 Fuc-b - 22 Fuc-b - BSA 8 -12 60 -23 5 -46 3 15 -20 5 Blood Group 333 2'F-B type 2-Sp - 03 Gala1-3[Fuca1-2]Galb1-4[Fuca1-3]GlcNAcb-Sp-BSA 6 -20 5 -37 0 -52 0 0 -37 5 B carb-Sia 273 3'Neu5Ac-LacNAc- Neu5Aca2-3Galb1-4GlcNAcb-Sp-BSA 6 -20 2 -26 0 -78 0 0 -31 0 Sp - 05 carb-Sia 331 3'Neu5Ac-LDN-Sp - Neu5Aca2-3GalNAcb1-4GlcNAcb-Sp-BSA 6 -14 3 -34 0 -57 -3 0 -34 -3 05 Blood Group 334 A tetra type 2-Sp - GalNAca1-3[Fuca1-2]Galb1-4GlcNAcb-Sp-BSA 6 -14 2 -43 0 -54 3 3 -37 -6 A 05 peptide- 433 Ac-S-Core4(Thr)-S- Ac-Ser-Core4(Thr)-Ser-Gly-Hex-BSA 6 -17 22 -37 0 -71 3 0 -34 3 Core4 G-07 peptide-TF 464 Ac-S-TFa(Ser)- Ac-Ser-Ser(Galb1-3GalNAca-)-Ser(Galb1-3GalNAca-)- 6 -14 0 -31 0 -74 -1 3 -37 0 TFa(Ser)-G-03 Gly-Hex peptide-core 326 Ac-S-Thr(core 3)-S- Ac-Ser-(GlcNAcb1-3GalNAca)Thr-Ser-Gly-Hex-BSA 6 -6 5 -51 0 -48 0 0 -23 0 3 G - 05 peptide-F1a 341 Ac-S-Thr(F1a)-S-G - AcSer-(Galb1-4GlcNAcb1-6GalNAca)Thr-Ser-Gly-Hex- 6 -14 0 -32 0 -43 -3 0 -29 0 18 BSA y- 354 Alpha-fetoprotein alpha fetoprotein (AFP)-human, from cell culture 6 -3 5 -15 0 -31 -3 3 -8 0 glycoprotein peptide 449 AzHex-D-T-R-NH2- BSA--hexyl-D-T-R-amide 6 -6 0 -26 0 -48 0 6 -30 0 07 peptide- 531 AzHex-G-Y(GalNAc-b)-A-NH2 BSA-AzHex-G-Y(GalNAc-b)-A-amide - 04 6 -9 5 -17 0 -45 3 3 -29 2 GalNAc Tyr peptide 516 AzHex-G-Y-A-NH2 - BSA-AzHex-G-Y-A-amide 6 0 0 -9 0 -1 0 0 3 6 11 peptide- 527 AzHex-L-Y(GalNAc-a)-W-NH2 BSA-AzHex-L-Y(GalNAc-a)-W-amide - 03 6 -7 3 -10 0 -27 0 0 -11 8 GalNAc Tyr peptide- 520 AzHex-L-Y(GalNAc-a)-W-NH2 BSA-AzHex-L-Y(GalNAc-a)-W-amide - 11 6 -12 34 247 3 37 5 3 17 17 GalNAc Tyr peptide- 557 AzHex-L-Y(GalNAc-b)-W-NH2 BSA-AzHex-L-Y(GalNAc-b)-W-amide - 04 6 12 7 26 0 0 0 0 3 25 GalNAc Tyr peptide- 535 AzHex-L-Y(GalNAc-b)-W-NH2 BSA-AzHex-L-Y(GalNAc-b)-W-amide - 08 6 12 3 52 0 8 5 6 12 5 GalNAc Tyr peptide-Tn 440 AzHex-PD-Tn(Thr)- BSA--hexyl-PD-T(GalNAc-a)-RP-amide 6 -17 13 -40 0 -65 0 6 -45 0 RP-NH2-07 peptide-Tn- 425 AzHex-S-Tn(Ser)-V- BSA-AzHex-S-Tn(Ser)-V-G-HexOH 6 6 3 -3 0 3 3 0 3 0 Ser G-HexOH-15 peptide 452 AzHex-V-T-S-A-P-D- BSA--hexyl-VTSAPDTRPAPGS-amide 6 -6 0 -13 0 -37 -3 6 -20 0 T-R-P-A-P-G-S-NH2- 07 Blood Group 276 B tetra type 1-Sp - Gala1-3[Fuca1-2]Galb1-3GlcNAcb-Sp-BSA 6 -11 0 -29 0 -55 0 0 -34 -3 B 04 Blood Group 335 B tetra type 2-Sp - Gala1-3[Fuca1-2]Galb1-4GlcNAcb-Sp-BSA 6 -8 19 -33 0 -54 0 5 -29 0 B 05 non-human- 20 Bdi -23 Gala1-3Gal– BSA 6 -6 8 -34 5 -51 3 9 -28 3 aGal non-human- 242 Bdi-g - 16 Gala1-3Galb– BSA 6 -5 3 -23 0 -34 0 0 -17 0 aGal Blood Group 67 BG-A1-04 GalNAca1-3(Fuca1-2)Galb1-3GlcNAcb1-linker-BSA 6 -9 8 -29 3 -46 0 12 -22 0 A Blood Group 69 BG-A2-04 GalNAca1-3(Fuca1-2)Galb1-4GlcNAcb1-linker-BSA 6 -20 8 -42 14 -71 -3 9 -43 0 A Blood Group 101 BG-B (Dextra) - 13 Gala1-3(Fuca1-2)Galb-BSA [BG-B] from Dextra 6 -6 5 -26 0 -47 3 6 -28 6 B Blood Group 339 BG-B5-04 Gala1-3(Fuca1-2)Galb1-3Galb1-linker-BSA 6 -17 3 -26 0 -40 0 0 -24 -3 B 107 Table 3.3 (cont’d) Blood Group 342 BG-H1-10 Fuca1-2Galb1-3GlcNAcb1-linker-BSA 6 -17 5 -37 0 -62 0 0 -43 -3 H y- 357 BSM (deacetylated) Deacetylated-Bovine submaxillary mucin 6 3 3 12 0 3 3 0 0 0 glycoprotein y- 356 BSM (ox) periodate oxidized bovine submaxillary mucin 6 9 0 -13 0 -1 0 3 6 0 glycoprotein non-human 28 Cellobiose -13 Glcb1-4Glcb-BSA 6 -12 11 -45 4 -62 20 74 -39 3 non-human 618 Chitotriose - 07 GlcNAcb1-4GlcNAcb1-4GlcNAcb- 6 -37 0 -88 0 -134 3 6 -80 3 non-human 75 Chitotriose - 08 GlcNAcb1-4GlcNAcb1-4GlcNAcb-BSA 6 -8 0 -26 5 -48 0 6 -31 3 Lewis 329 DFLNHc, Galb1-4GlcNAcb1-6[Fuca1-2Galb1-3(Fuca1- 6 -3 4 -37 0 -41 -3 6 -17 0 LacNAc/LeB - 08 4)GlcNAcb1-3]Galb1-BSA Lewis 317 DFLNnH, LeX/LeX - Galb1-4(Fuca1-3)GlcNAcb1-6[Galb1-4(Fuca1- 6 -20 78 -40 0 -51 3 3 -23 12 10 3)GlcNAcb1-3]Galb1-BSA glycolipid 90 DSLNT - 06 Neu5Aca2-3Galb1-3(Neu5Aca2-6)GlcNAcb1-3Galb1- 6 -17 3 -43 0 -54 0 6 -40 0 BSA y- 360 FABP Fatty Acid Binding Protein 6 9 3 -3 0 20 3 0 23 0 glycoprotein y- 362 fetuin (asialo) asialofetuin from calf serum- type I (Sigma A4781; 6 8 0 23 0 20 0 6 9 0 glycoprotein Galb1-4GlcNAc, Galb1-3GlcNAc, Galb1-3GalNAc; mostly NA2 and NA3) y- 365 Fetuin (ox) periodate oxidized fetuin 6 9 0 0 0 11 -1 0 3 0 glycoprotein glycolipid- 57 GA1 tri- 20 Galb1-3GalNAcb1-4Galb1-BSA (GA1tri or asialo-GM1) 6 -14 21 -34 17 -60 -3 0 -34 -3 neutral glycolipid- 22 GA1di -11 Galb1-3GalNAcb – HSA 6 -15 5 -40 3 -62 0 6 -31 3 neutral carb-Gal 14 Gal-a- 24 Gal-a - BSA 6 -5 54 -31 3 -39 5 14 -28 3 carb-GalNAc 17 GalNAca1-6Galb - GalNAca1-6Galb-BSA 6 0 5 -20 11 -29 5 12 0 22 carb-GalNAc 9 GalNAc-b - 21 GalNAc-b - BSA 6 -34 25 -36 5 -65 3 43 -46 8 glycolipid- 81 Gb4 - 09 GalNAcb1-3Gala1-4Galb1-BSA (aka: P antigen) 6 -20 0 -34 5 -37 0 6 -22 3 neutral glycolipid- 620 Gb4 - 10-HSA GalNAcβ1-3Galα1-4Galβ1-HSA (Gb4) 6 -3 3 -17 0 -26 0 0 -14 0 neutral glycolipid 314 GD2-Sp - 04 Neu5Aca2-8Neu5Aca2-3[GalNAcb1-4]Galb1-4Glcb-Sp- 6 -29 0 -51 0 -60 0 2 -34 0 BSA y- 366 glycophorin (asialo) asialo-glycophorin A (aGn) 6 -22 5 -36 5 -62 0 3 -46 0 glycoprotein glycolipid 507 GM2 tri (Gc) - 04 Neu5Gca2-3(GalNAcb1-4)Galb- 6 -26 0 -57 0 -139 5 6 -62 6 GAG-Hep 587 Hep-Hexa-GT23-04 Glc(6S, NS)a1-4GlcAb1-4Glc(6S, 3S, NS)a1-4IdoA(2S)a1- 6 3 8 -11 5 -23 6 6 9 0 4Glc(6S, NS)a1-4GlcAb-Benzamide- GAG-Hep 581 Hep-NAc-Hexa-05 GlcNAca1-4GlcAb1-4GlcNAca1-4GlcAb1-4GlcNAca1- 6 -28 0 -85 0 -116 3 -2 -82 3 4GlcAb-Benzamide- (Heparosan ) GAG-Hep 585 Hep-Nona-GT14- GlcAb1-4GlcNSa1-4GlcAb1-4GlcNSa1-4GlcAb1-4Glc(6S, 6 -20 0 -54 0 -85 5 0 -46 0 02 NS)a1-4GlcAb1-4Glc(6S, NS)a1-4GlcAb-Benzamide- GAG-Hep 536 Hep-Octa-GT21-03 Glc(6S, NS)a1-4GlcAb1-4Glc(6S, NS)a1-4IdoA(2S)a1- 6 0 11 -17 0 -12 0 3 20 8 4Glc(6S, NS)a1-4IdoA(2S)a1-4Glc(6S, NS)a1-4GlcAb- Benzamide- carb-type 2 21 LacNAc - 22 Galb1-4GlcNAc – BSA 6 -14 5 -37 2 -48 -1 3 -31 3 carb-GalNAc 234 LDN-Sp - 14 GalNAcb1-4GlcNAcb-Sp-BSA 6 -15 3 -20 0 -57 0 6 -25 3 Lewis 37 LeA-Lac -18 (LNFP Galb1-3[Fuca1-4)GlcNAcb1-3Galb1-4Glcb- BSA 6 -17 7 -43 0 -65 0 9 -43 3 II) Lewis 279 LeC-Sp - 07 Galb1-3GlcNAcb-Sp-BSA 6 -14 3 -26 0 -22 0 0 0 glycolipid- 623 LNT - 17-HSA Galβ1-3GlcNAcβ1-3Galβ-HSA (LNT) 6 -6 0 -26 0 -36 0 0 -17 0 neutral carb-Man 439 Ma2Ma2Ma3(Ma3Ma6)-03 aMan(1-2)aMan(1-2)aMan(1-3)[aMan(1-3)aMan(1-6)] 6 181 11 -190 5 9 3 carb-Man 431 Ma2Ma3(Ma6)-08 aMan(1-2)aMan(1-3)[aMan(1-6)] 6 -31 11 -68 0 -122 -3 3 -68 6 carb-Man 469 Ma2Ma6(Ma2Ma3)Ma6-04 aMan(1-2)aMan(1-6)[aMan(1-2)aMan(1-3)]aMan(1-6) 6 -32 3 -74 0 -142 0 0 -79 -3 carb-Man 494 Ma6(Ma3)Ma6-04 aMan(1-6)[aMan(1-3)]aMan(1-6) 6 -11 5 -49 0 -44 0 3 -17 0 N-linked 84 Man7D3 - 08 Mana1-2Mana1-6(Mana1-3)Mana1-6(Mana1-2Mana1- 6 -20 17 -42 2 -51 -3 6 -34 0 3)Manb1-4GlcNAc-BSA N-linked 167 NGA4 - 06 GlcNAcb1-2(GlcNAcb1-6)Mana1-6[GlcNAcb1- 6 -20 0 -46 0 -82 3 5 -46 -3 2(GlcNAcb1-4)Mana1-3]Manb1-4GlcNAc -BSA y- 374 ovalbumin ovalbumin (Sigma A5503; 56% Man5+Man6) 6 3 5 19 5 20 0 0 11 4 glycoprotein y- 375 Ovalbumin (ox) periodate oxidized ovalbumin 6 17 8 26 2 76 3 6 20 3 glycoprotein y- 376 PSA Prostate Specific Antigen (PSA); human seminal fluid 6 6 3 9 37 10 0 3 14 0 glycoprotein 108 Table 3.3 (cont’d) y- 364 PSM A neg porcine submaxillary mucin (A-) 6 0 0 -6 0 6 3 -3 4 0 glycoprotein glycolipid 614 SSEA-4-penta-05 Neu5Aca2-3Galb1-3GlcNAcb1-3Gala1-4Galb1- 6 -14 5 -83 0 -105 -3 0 -67 2 glycolipid 617 SSEA-4-penta-11 Neu5Aca2-3Galb1-3GlcNAcb1-3Gala1-4Galb1- 6 -12 0 -40 0 -73 -3 0 -51 -3 glycolipid 231 SSEA-4-Sp - 05 Neu5Aca2-3Galb1-3GalNAcb1-3Gala1-4Galb1-4Glcb-Sp- 6 -17 5 -37 5 -77 -3 3 -51 3 BSA y- 377 Tgl Thyroglobulin -human 6 0 0 26 5 17 6 6 3 5 glycoprotein glycolipid 559 3'Kdn-Gb5 - 04 Kdna2-3Galb1-3GalNAcb1-3Gala1-4Galb- 6 -28 0 -85 0 -142 0 3 -74 0 carb-Sia 272 3'KDN-LacNAc-Sp - KDNa2-3Galb1-4GlcNAcb-Sp-BSA 6 -5 3 -17 0 -29 0 0 -14 6 12 Blood Group 277 A tetra type 2-Sp - GalNAca1-3[Fuca1-2]Galb1-4GlcNAcb-Sp-BSA 6 -34 0 -59 0 -94 0 6 -48 0 A 07 y- 353 Alpha-1-acid alpha1 Acid Glycoprotein-purified from human serum 6 3 0 -11 0 -9 3 3 6 0 glycoprotein glycoprotein (Sigma G9985) glycolipid 330 GD2-Sp - 10 Neu5Aca2-8Neu5Aca2-3[GalNAcb1-4]Galb1-4Glcb-Sp- 6 -8 3 -14 0 -23 0 0 -14 0 BSA Lewis 532 3'Neu5Ac-LeX-Gal - Neu5Aca2-3Galb1-4(Fuca1-3)GlcNAcb1-3Galb- 5 -26 0 -57 0 -92 -3 0 -43 0 04 glycolipid- 612 Gb5 tetra - 07 Galb1-3GalNAcb1-3Gala1-4Galb- 5 -31 0 -71 0 -102 -5 0 -68 3 neutral GAG-Hep 594 Hep-Octa-GT19-03 GlcNAca1-4GlcAb1-4GlcNSa1-4IdoA(2S)a1-4GlcNSa1- 5 -9 0 -28 3 18 0 3 -12 6 4IdoA(2S)a1-4GlcNSa1-4GlcAb-Benzamide- glycolipid- 578 iGb4 tri - 05 GalNAcb1-3Gala1-3Galb- 5 -20 0 -57 0 -85 3 0 -46 0 neutral Blood Group 253 2'F-A type 2-Sp - 05 GalNAca1-3[Fuca1-2]Galb1-4[Fuca1-3]GlcNAcb-Sp-BSA 3 -9 3 -29 0 -60 0 6 -25 0 A glycolipid 528 3'Kdn-LNT - 04 Kdna2-3Galb1-3GlcNAcb1-3Galb- 3 -11 0 -37 0 -89 0 2 -25 6 carb-Sia 232 3'Neu5Ac(9Ac)- Neu5Ac(9Ac)a2-3Galb1-4GlcNAcb-Sp-BSA 3 -11 0 -34 0 -46 0 3 -28 0 LacNAc-Sp - 10 carb-Sia 562 3'Neu5Ac8Me- Neu5Ac8Mea2-3Galb- 3 -43 0 -99 0 -153 -3 3 -94 0 Galb- 04 glycolipid 493 3'-Neu5Ac-Gb3 - 04 Siaa2-3Gala1-4Galb1- 3 -6 0 -34 0 -54 3 0 -29 3 glycolipid 472 3'-Neu5Ac-Gb3-08 Siaa2-3Gala1-4Galb- 3 -34 0 -85 0 -142 0 -3 -65 0 glycolipid 654 3'Neu5Ac-Gb5 Neu5Aca2-3Galb1-3GlcNAcb1-3Gala1-4Galb1- 3 -8 0 -23 0 -45 2 0 -26 6 glycolipid 657 3'Neu5Gc-Gb5 Neu5Gca2-3Galb1-3GlcNAcb1-3Gala1-4Galb1- 3 -17 0 -22 0 -75 6 0 -28 0 carb-Sia 256 3'Neu5Gc-LacNAc- Neu5Gca2-3Galb1-4GlcNAcb-Sp-BSA 3 -16 3 -34 0 -52 0 6 -31 0 Sp - 06 glycolipid 540 3'Neu5Gc-LNT - 06 Neu5Gca2-3Galb1-3GlcNAcb1-3Galb- 3 -6 0 -25 0 -83 0 0 -37 0 carb-Sia 47 6'Neu5Ac-Lac Neu5Aca2-6Galb1-4Glc-APD-HSA 3 -12 5 -48 167 -68 0 9 -37 2 Lewis 211 6'-sulpho-LeA - 16 6-SO3-Galb1-3[Fuca1-4)GlcNAc-BSA 3 -17 3 -34 0 -45 0 0 -31 0 Blood Group 265 A tetra type 1-Sp - GalNAca1-3[Fuca1-2]Galb1-3GlcNAcb-Sp-BSA 3 -17 0 -31 0 -57 5 3 -26 0 A 05 Blood Group 243 A tetra type 1-Sp - GalNAca1-3[Fuca1-2]Galb1-3GlcNAcb-Sp-BSA 3 0 3 0 0 -9 0 6 6 -3 A 15 N-linked 588 A2 (a2-3) - 01.4 Neu5Aca2-3Galb1-4GlcNAcb1-2Mana1-6(Neu5Aca2- 3 -9 0 -27 0 -46 0 0 -9 0 3Galb1-4GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb1- peptide-Fuc 386 Ac-G-S(Fuca)-S-G- Ac-G-S(Fuca)-S-G-Hex-BSA 3 -9 0 -20 0 -25 0 3 -12 0 Hex-05 peptide-Xyl 389 Ac-G-S(Xylb)-G-G- Ac-Gly-Ser(Xylb)-Gly-Gly-Hex-BSA 3 -5 3 -23 0 -25 3 3 -12 0 Hex-21 peptide 417 Ac-GSTAP-G-05 Ac-G-S-T-A-P-G- 3 -17 0 -28 0 -51 0 3 -26 0 peptide-Man 408 Ac-P-T(Mana)-A-G - Ac-Pro-Thr(Man a)-Ala-Gly-Hex-BSA 3 -6 0 -15 0 -35 0 3 -11 0 07 Peptide-core- 383 Ac-Ser(core 5)-S-G- Ac-(GalNAca1-3GalNAca)Ser-Ser-Gly-Hex-BSA 3 -15 0 -28 0 -34 2 0 -23 0 5 05 peptide-Gal 626 Ac-S-S(Gala)-S-G - Ac-S-S(Gal a)-S-G-Hex-OH 3 -9 0 -41 0 -44 0 0 -34 0 06 peptide-TF 123 Ac-S-TF(Ser)-S-G - Ac-Ser-(Galb1-3GalNAca)Ser-Ser-Gly-Hex-BSA 3 -6 0 -31 0 -49 0 6 -20 3 04 peptide-core 379 Ac-S-Thr(core 2)-S- Ac-Ser-(Galb1-3(GlcNAcb1-6)GalNAca)Thr-Ser-Gly-Hex- 3 -17 5 -35 0 -68 0 -6 -39 0 2 G - 12 BSA peptide-Tn- 120 Ac-S-Tn(Ser)-S-G - AcSer-(GalNAca)Ser-Ser-Gly-Hex-BSA 3 -11 3 -28 0 -60 0 6 -29 0 Ser 04 peptide-Tn- 126 Ac-S-Tn(Ser)-S-G - Ac-Ser-(GalNAca)Ser-Ser-Gly-Hex-BSA 3 -15 11 -23 5 -40 0 12 -26 0 Ser 33 peptide-Tn 163 Ac-S-Tn(Thr)-A-G - Ac-Ser-(GalNAca)Thr-Ala-Gly-Hex-BSA (muc1) 3 -14 5 -40 2 -59 3 3 -31 6 04 peptide-Tn 152 Ac-S-Tn(Thr)-G-G - Ac-Ser-(GalNAca)Thr-Gly-Gly-Hex-BSA (muc4) 3 -6 5 -19 0 -28 0 6 -17 0 19 peptide-Tn 122 Ac-S-Tn(Thr)-S-G - Ac-Ser-(GalNAca)Thr-Ser-Gly-Hex-BSA 3 -3 3 -31 3 -40 -3 21 -20 3 04 peptide-Tn 16 Ac-Tn(Thr)-G - 21 Ac(GalNAca)Thr-Gly-Hex-BSA 3 -29 17 -42 5 -68 3 34 -47 3 peptide-Tn 160 Ac-T-Tn(Thr)-P-G - Ac-Thr-(GalNAca)Thr-Pro-Gly-Hex-BSA (muc2,6,7) 3 -11 5 -26 0 -34 5 0 -14 3 04 Blood Group 18 Adi - 17 GalNAca1-3Galb-BSA 3 -17 8 -34 5 -40 0 6 -26 0 A y- 589 alpha 1 acid glycoprotein Alpha-1-Acid (isosep) Glycoprotein, Oroscomucoid, from Human serum 3 -3 0 -9 0 -9 0 0 -6 0 glycoprotein non-human 750 Arafa1-3(Arafa1-2)Xylb1 Arafa1-3(Arafa1-2)Xylb1 - 05 - BSA 3 23 0 -102 6 0 9 peptide 513 AzHex-L-Y-W-NH2 - 05 BSA-AzHex-L-Y-W-amide 3 -6 0 -22 0 -40 5 3 -20 6 peptide-Tn- 427 AzHex-S-Tn(Ser)-V- BSA-AzHex-S-Tn(Ser)-V-G-HexNH2 3 -6 0 -11 0 -23 0 0 -12 0 Ser G-HexNH2-16 Blood Group 70 BG-A2-16 GalNAca1-3(Fuca1-2)Galb1-4GlcNAcb1-linker-BSA 3 -20 5 -51 0 -71 0 6 -48 0 A 109 Table 3.3 (cont’d) Blood Group 72 BG-B1-15 Gala1-3(Fuca1-2)Galb1-3GlcNAcb1-linker-BSA 3 -34 5 -54 5 -97 0 3 -79 0 B Blood Group 347 BG-B6- 03 Gala1-3(Fuca1-2)Galb1-4Glcb1-linker-BSA 3 -14 0 -40 0 -51 0 -3 -17 0 B Blood group 586 BG-H di Fuca1-2Galb-BSA 3 -9 0 -29 0 -40 0 6 -20 0 H Blood Group 100 BG-H3- 04 Fuca1-2Galb1-3GalNAcα1-linker-BSA 3 -17 5 -37 0 -48 0 6 -28 0 H Blood Group 140 BG-H3- 14 Fuca1-2Galb1-3GalNAcα1-linker-BSA 3 -12 3 -34 0 -56 0 10 -25 0 H Blood Group 345 BG-H5-04 Fuca1-2Galb1-3Galb1-linker-BSA 3 -11 3 -25 0 -34 3 0 -20 0 H Blood Group 346 BG-H5-19 Fuca1-2Galb1-3Galb1-linker-BSA 3 -17 5 -40 0 -51 0 6 -25 -6 H non-human 669 cellotetraose - 05 Glcb1-4Glcb1-4Glcb1-4Glcb1- 3 -17 5 -51 0 -60 5 3 -29 8 non-human 577 Chitotriose - 04 GlcNAcb1-4GlcNAcb1-4GlcNAcb- 3 -28 0 -76 0 -148 -3 0 -78 3 carb-Sia 247 CT/Sda-Sp - 13 Neu5Aca2-3[GalNAcb1-4]Galb1-4GlcNAcb-Sp-BSA; like 3 -17 0 -37 0 -59 0 3 -29 0 GM2 but on glycoproteins glycolipid 496 di-sialyl type 1 - 04 NeuAcα2-8Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galb- 3 -11 0 -37 0 -65 0 -3 -32 -3 y- 378 FABP-3 Fatty Acid Binding Protein (Heart type) 3 2 0 0 0 22 3 0 12 0 glycoprotein non-human 119 Forssman Di - 04 GalNAca1-3GalNAcb1-BSA 3 -9 3 -52 0 -77 0 6 -44 3 non-human 108 Forssman Di - 21 GalNAca1-3GalNAcb1-BSA 3 -14 0 -34 0 -54 0 3 -29 -3 non-human 639 Forssman Di - 22-HSAGalNAca1-3GalNAcb1-HSA 3 -6 0 -9 0 -22 3 0 -5 0 carb-Fuc 11 Fuc-a - 22 Fuc-a - BSA 3 -6 11 -31 5 -45 0 9 -26 0 non-human- 76 Gal3- 07 Gala1-3Galb1-4Gala-BSA 3 -3 6 -23 0 -36 5 0 -7 0 aGal non-human 208 Gala1-2Gal - 13 Gala1-2Gal-BSA 3 -17 6 -31 0 -46 5 9 -31 0 glycolipid- 510 Gala1-4Galb- Gala1-4Galb- 3 -23 0 -43 0 -88 5 -3 -31 0 neutral non-human 25 Galb1-6Man-a - 13 Galb1-6Man-a - BSA 3 0 8 -12 0 -30 0 9 3 3 non-human- 605 Galili Hexa-13 Gala1-3(Galb1-4GlcNAcb1-3)2Galb- 3 -15 0 -32 0 -51 0 0 -40 -6 aGal non-human- 192 Galilli - 21 Gala1-3Galb1-4Glc-BSA 3 -10 5 -34 3 -43 5 6 -15 0 aGal glycolipid- 42 Gb3- 13 Gala1-4Galb1-4Glc-HSA [aka: Pk or CD77] 3 -17 5 -43 0 -49 0 3 -23 0 neutral glycolipid- 87 Gb4 tetra (P1 tetra)- GalNAcb1-3Gala1-4Galb1-4GlcNAcb-Sp-BSA 3 -20 5 -40 3 -54 0 6 -37 3 neutral Sp - 06 glycolipid 600 Gb5 analogue type Galb1-4GlcNAcb1-3Gala1-4Galb- 3 -6 0 -11 0 -12 0 0 -3 0 related 2-04 glycolipid 229 GD1a-Sp - 05 Neu5Aca2-3[Neu5Aca2-3Galb1-3GalNAcb1-4]Galb1- 3 -14 3 -46 0 -74 0 3 -40 0 4Glcb-Sp-BSA glycolipid 537 GD2 tetra (Kdn/Ac) Kdna2-8Neu5Aca2-3(GalNAcb1-4)Galb- 3 -5 0 -23 0 -37 0 0 -14 0 - 03 glycolipid 455 GD3(9-OAc)-b-propyl-03Neu5Ac(9-OAc)a2-8Neu5Aca2-3Galb1-4Glcb-propyl- 3 -14 0 -22 0 -63 0 3 -31 0 BSA glycolipid 457 GD3(Gc-Ac)-b-propyl-03Neu5Gca2-8Neu5Aca2-3Galb1-4Glcb-propyl-BSA 3 -3 0 -37 0 -57 0 0 -31 0 carb-GlcNAc 132 GlcNAca1-4Galb - GlcNAca1-4Galb-BSA 3 -8 3 -35 0 -43 0 6 -23 0 03 N-linked 197 GlcNAc-Man3 - 02 Mana1-6(GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb-BSA 3 -3 9 -31 0 -51 5 6 -22 5 N-linked 199 GlcNAc-Man5 - 03 Mana1-6(Mana1-3)Mana1-6(GlcNAcb1-2Mana1- 3 -12 4 -34 0 -37 5 3 -17 3 3)Manb1-4GlcNAcb-BSA Blood Group 110 Globo H - 03 Fuca1-2Galb1-3GalNAcb1-3Gala1-4Galb1-BSA 3 -9 3 -29 0 -48 0 3 -20 0 H Blood Group 611 Globo H analogue Fuca1-2Galb1-4GlcNAcb1-3Gala1-4Galb- 3 -12 85 -57 0 -65 -3 0 -40 0 H related type 2-10 y- 367 Glycophorin A Glycophorin A (Gn; Sigma G6017) 3 0 0 -8 0 -26 0 0 -9 0 glycoprotein glycolipid 467 GM1b-13 Siaa2-3Galb1-3GalNAcb1-4Galb- 3 -17 0 -37 0 -63 0 5 -43 0 glycolipid 46 GM3 - 12 Neu5Aca2-3Galb1-4Glc-APD-HSA 3 -17 9 -47 0 -78 0 8 -45 3 glycolipid 250 GM3(Gc)-Sp - 14 Neu5Gca2-3Galb1-4Glcb-Sp-BSA 3 -17 0 -49 0 -63 0 3 -27 0 glycolipid 434 GT1a - 04 Neu5Aca2-8Neu5Aca2-3Galb1-3GalNAcb1-4(Neu5Aca2- 3 -20 0 -35 0 -65 -1 0 -45 6 3)Galb- peptide-TF 188 GTSSASTGHA- BSA-PEG7-Gly-Thr-Ser-Ser-Ala-Ser-Thr-Gly-His-Ala- 3 -6 5 -34 0 -35 3 -3 -17 0 TF(Thr)-PLPVTD (Galb1-3GalNAca)Thr-Pro-Leu-Pro-Val-Thr-Asp 110 Table 3.3 (cont’d) GAG-Hep 565 Hep-NAc-Nona-04 GlcAb1-4GlcNAca1-4GlcAb1-4GlcNAca1-4GlcAb1- 3 -23 0 -56 0 -65 0 0 -40 0 4GlcNAca1-4GlcAb1-4GlcNAca1-4GlcAb-Benzamide- (Heparosan ) GAG-Hep 584 Hep-NS-Hepta-03 GlcAb1-4GlcNSa1-4GlcAb1-4GlcNSa1-4GlcAb1- 3 -14 0 -37 0 -43 0 0 -32 -3 4GlcNSa1-4GlcAb-Benzamide- GAG-Hep 574 Hep-NS-Penta-05 GlcAb1-4GlcNSa1-4GlcAb1-4GlcNSa1-4GlcAb- 3 -17 0 -43 0 -55 -3 0 -28 2 Benzamide- GAG-Hep 582 Hep-Octa-GT17-02 GlcNSa1-4GlcAb1-4GlcNSa1-4GlcAb1-4GlcNSa1- 3 -11 0 -32 0 -40 0 0 -19 0 4IdoA(2S)a1-4GlcNSa1-4GlcAb-Benzamide- GAG-Hep 590 Hep-Octa-GT18-02 GlcNSa1-4GlcAb1-4GlcNSa1-4IdoA(2S)a1-4GlcNSa1- 3 -8 0 -27 0 -31 0 0 -21 3 4IdoA(2S)a1-4GlcNSa1-4GlcAb-Benzamide- Carb-HNK-1 598 HNK 1 precursor- GlcAcb1-3Galb- 3 -12 0 -26 0 -37 0 0 -26 0 05 Carb-HNK-1 606 HNK 1 precursor- GlcAcb1-3Galb- 3 -14 0 -31 0 -40 0 0 -26 6 18 N-linked 214 Hybrid-M5N4B - 03 GlcNAcb1-2Mana1-3[Mana1-3(Mana1-6)Mana1- 3 -17 0 -43 0 -57 3 3 -29 -3 6](GlcNAcb1-4)Manb1-4GlcNAcb1-BSA glycolipid- 420 iGb5-06 Galb1-3GalNAcb1-3Gala1-3Galb- 3 -20 0 -37 3 -71 0 3 -43 0 neutral glycolipid- 23 Lac - 33 Galb1-4Glcb – BSA 3 -14 8 -43 0 -57 -3 6 -37 0 neutral glycolipid- 240 Lac-C5 - 05 Galb1-4Glcb – BSA 3 -6 0 -20 0 -49 5 3 -23 0 neutral carb-type 2 238 LacNAc-Sp - 15 Galb1-4GlcNAcb-Sp-BSA 3 -3 5 -28 0 -43 4 5 -17 0 Lewis 40 LeX-Lac Galb1-4[Fuca1-3)GlcNAcb1-3Galb1-4Glc-APD-HSA ; 3 -15 5 -37 0 -17 0 5 -14 3 (monomeric, SSEA-1; CD-15; Glycotech LNFIII) Lewis 621 LeY -08 (repeat) Fuca1-2Galb1-4[Fuca1-3)GlcNAc –HSA 3 -20 5 -45 0 -74 0 0 -31 0 glycolipid- 579 LNT tri - 10 Galb1-3GlcNAcb1-3Galb- 3 -11 0 -34 0 -54 3 0 -31 0 neutral glycolipid 54 LSTa - 10 Neu5Aca2-3Galb1-3GlcNAcb1-3Galb1-BSA 3 6 11 -8 19 -35 0 0 -19 0 carb-Man 484 Ma2Ma2-06 aMan(1-2)aMan(1-2) 3 -9 3 -23 0 -26 5 0 -15 6 carb-Man 491 Ma2Ma2-11 aMan(1-2)aMan(1-2) 3 -17 0 -53 0 -88 0 5 -48 0 carb-Man 454 Ma2Ma2Ma3(Ma6Ma6)-06 aMan(1-2)aMan(1-2)aMan(1-3)[aMan(1-6)aMan(1-6)] 3 -34 3 -85 0 -142 0 0 -77 0 carb-Man 443 Ma2Ma3(Ma6)-04 aMan(1-2)aMan(1-3)[aMan(1-6)] 3 -12 0 -37 0 -71 0 6 -34 3 carb-Man 478 Ma2Ma3-04 aMan(1-2)aMan(1-3) 3 -11 0 -25 0 -60 -3 -3 -29 6 carb-Man 441 Ma2Ma3Ma6(Ma3)-04 aMan(1-2)aMan(1-3)aMan(1-6)[aMan(1-3)] 3 -14 0 -29 0 -51 0 -3 -27 3 carb-Man 500 Ma2Ma3Ma6-03 aMan(1-2)aMan(1-3)aMan(1-6) 3 -20 0 -54 0 -68 0 0 -45 0 carb-Man 460 Ma2Ma6(Ma3)Ma6-07 aMan(1-2)aMan(1-6)[aMan(1-3)]aMan(1-6) 3 -22 0 -71 0 -116 3 -3 -65 0 carb-Man 446 Ma2Ma6-16 aMan(1-2)aMan(1-6) 3 -28 3 -82 0 -94 5 6 -57 3 carb-Man 463 Ma2Ma6Ma6-06 aMan(1-2)aMan(1-6)aMan(1-6) 3 -22 0 -54 0 -96 0 -3 -54 6 carb-Man 495 Ma3(Ma6)-07 aMan(1-3)[aMan(1-6)] 3 -20 0 -54 0 -153 0 3 -74 3 carb-Man 492 Ma3Ma6(Ma3)-04 aMan(1-3)aMan(1-6)[aMan(1-3)] 3 -15 6 -34 0 -60 0 -3 -29 3 carb-Man 497 Ma3Ma6(Ma3)-11 aMan(1-3)aMan(1-6)[aMan(1-3)] 3 -12 0 -15 0 -29 0 0 -17 0 carb-Glc 687 maltohexaose - 04 Glca1-4Glca1-4Glca1-4Glca1-4Glca1-4Glca1- 3 -8 0 -20 0 -26 0 -17 carb-Glc 104 Maltopentaose - Glca1-4Glca1-4Glca1-4Glca1-4Glca-BSA 3 -6 16 -29 0 -57 8 3 -26 6 11 N-linked 78 Man3 - 06 Mana1-6(Mana1-3)Manb1-4GlcNAc -BSA 3 -17 0 -45 3 -73 -5 6 -34 5 N-linked 508 Man3a - 07 Mana1-2Mana1-3Manb1-4GlcNAcb- 3 -22 5 -54 0 -130 0 0 -56 -6 N-linked 86 Man5 - 06 Mana1-6(Mana1-3)Mana1-6(Mana1-3)Manb1- 3 -6 8 -68 0 -77 0 3 -34 3 4GlcNAcb- N-linked 88 Man6 - II - 05 Mana1-2Mana1-3Mana1-6(Mana1-2Mana1-3)Manb1- 3 -23 5 -68 0 -79 3 3 -32 0 BSA N-linked 95 Man7D1 - 10 Mana1-6(Mana1-3)Mana1-6(Mana1-2Mana1-2Mana1- 3 3 3 -26 0 -40 0 3 -20 5 3)Manb1-4GlcNAc-BSA N-linked 506 Man7D2 - 05 Mana1-6(Mana1-2Mana1-3)Mana1-6(Mana1-2Mana1- 3 -17 0 -40 0 -43 -3 0 -31 -3 3)Manb1-4GlcNAcb- N-linked 615 Man7D3 - 06 Mana1-2Mana1-6(Mana1-3)Mana1-6(Mana1-2Mana1- 3 -26 0 -52 0 -94 0 3 -57 -3 3)Manb1-4GlcNAcb- carb-Man 26 Mana1-6Man-a - Mana1-6Man-a - BSA 3 9 4 -15 3 -14 0 3 -3 0 15 peptide-Tn 396 Muc1-Tn8 BSA--hexyl-G-V-T-S-A-P-D-T(GalNAc-a)-R-P-A-P-G-S-T- 3 0 3 -23 0 -43 5 3 -20 0 A-P-P-A-amide 111 Table 3.3 (cont’d) N-linked 170 NA4 - 05 Galb1-4GlcNAcb1-2(Galb1-4GlcNAcb1-6)Mana1- 3 -19 3 -54 0 -61 3 -3 -37 0 6[Galb1-4GlcNAcb1-2(Galb1-4GlcNAcb1-4)Mana1- 3]Manb1-4GlcNAc -BSA N-linked 168 NGA4(B)2 - 04 GlcNAcb1-2(GlcNAcb1-4)(GlcNAcb1-6)Mana1- 3 -22 5 -39 5 -60 3 6 -34 14 6[GlcNAcb1-2Mana1-3](GlcNAcb1-4)Manb1-4GlcNAc - BSA N-linked 169 NGA5B - 02 GlcNAcb1-2(GlcNAcb1-4)(GlcNAcb1-6)Mana1- 3 -15 5 -28 0 -34 3 6 -23 0 6[GlcNAcb1-2(GlcNAcb1-4)Mana1-3](GlcNAcb1- 4)Manb1-4GlcNAc -BSA non-human 744 PNAG 22 (10110) GlcNAcb1-6GlcNb1-6GlcNAcb1-6GlcNAcb1-6GlcNb1- 3 -14 3 -40 0 -80 3 3 -51 5 PNAG glycolipid 241 SSEA-4-Sp - 12 Neu5Aca2-3Galb1-3GalNAcb1-3Gala1-4Galb1-4Glcb-Sp- 3 0 0 -14 5 -33 0 6 -17 0 BSA glycolipid 301 GD3-Sp - 08 Neu5Aca2-8Neu5Aca2-3Galb1-4Glcb-Sp-BSA 3 -25 3 -31 0 -49 0 0 -26 0 GAG-Hep 526 Hep-NS-Hexa-04 GlcNSa1-4GlcAb1-4GlcNSa1-4GlcAb1-4GlcNSa1-4GlcAb- 3 -11 0 -39 0 -62 3 6 -32 3 Benzamide- Lewis 552 3'Neu5Ac8Me-LeX- Neu5Ac8Mea2-3Galb1-4(Fuca1-3)GlcNAcb1-3Galb- 2 -25 0 -66 0 -99 -1 0 -62 -3 Gal - 04 glycolipid 564 3'Neu5Ac8Me-LNT - Neu5Ac8Mea2-3Galb1-3GlcNAcb1-3Galb- 2 -28 0 -57 0 -82 0 0 -63 0 04 carb-Sia 249 6'Neu5Ac-LacNAc Neu5Aca2-6[Galb1-4GlcNAcb1-3)2b-Sp-BSA 2 -14 0 -29 0 -48 0 6 -22 -3 (dimeric)-Sp - 13 peptide-Glc 392 Ac-A-S(Glcb)-S-G- Ac-A-S(Glcb)-S-G-Hex-BSA 2 -6 3 -15 0 -29 5 6 -11 0 Hex-05 peptide-Tn- 79 Ac-S-Tn(Ser)-S-G - Ac-Ser-(GalNAca)Ser-Ser-Gly-Hex-BSA 2 -20 17 -40 5 -57 3 23 -37 0 Ser 22 peptide-Tn 161 Ac-S-Tn(Thr)-G-G - Ac-Ser-(GalNAca)Thr-Gly-Gly-Hex-BSA (muc4) 2 -9 3 -29 0 -17 3 6 -8 0 03 Blood Group 348 BG-B6-15 Gala1-3(Fuca1-2)Galb1-4Glcb1-linker-BSA 2 -23 14 -37 0 -71 0 3 -31 0 B Blood Group 98 BG-H4- 04 Fuca1-2Galb1-3GalNAcb1-linker-BSA 2 -6 0 -37 0 -37 -1 3 -17 3 H glycolipid 597 Gb5 analogue type Galb1-3GlcNAcb1-3Gala1-4Galb- 2 -3 0 -10 0 -15 0 0 -9 0 related 1-06 glycolipid 459 GD3(Gc-Gc)-b-propyl-03 Neu5Gca2-8Neu5Gca2-3Galb1-4Glcb-propyl-BSA 2 -15 0 -37 0 -62 3 0 -26 0 carb-Glc 6 Glc-a - 22 Glc-a - BSA 2 -20 4 -33 0 0 0 -38 0 glycolipid 461 GM1b-05 Siaa2-3Galb1-3GalNAcb1-4Galb- 2 -17 0 -63 0 1167 -85 0 0 -57 3 GAG-Hep 530 Hep-Nona-GT15- GlcAb1-4GlcNSa1-4GlcAb1-4Glc(6S, NS)a1-4GlcAb1- 2 -12 5 -23 0 -54 3 0 -12 3 04 4Glc(6S, NS)a1-4GlcAb1-4Glc(6S, NS)a1-4GlcAb- Benzamide- carb-type 2 227 LacNAc-Sp - 06 Galb1-4GlcNAcb-Sp-BSA 2 -11 5 -28 0 -44 0 -2 -28 3 N-linked 166 NGA3 - 01 GlcNAcb1-2Mana1-6[GlcNAcb1-2(GlcNAcb1-4)Mana1- 2 -17 5 -37 0 -45 5 6 -34 0 3]Manb1-4GlcNAc -BSA y- 371 OSM (ox) periodate oxidized ovine submaxillary mucin 2 0 0 -11 0 -11 -3 0 -6 6 glycoprotein non-human 810 Xylb5 - 17 Xylb1-4Xylb1-4Xylb1-4Xylb1-4Xylb1-BSA 2 -3 22 -3 0 621 0 3 -12 6 non-human 538 KDOa2-4KDOa - 09 KDOa2-4KDOa-APTE-BSA 0 -14 0 -25 0 -51 -3 0 -24 0 carb-Sia 517 3'Kdn-Galb- 03 Kdna2-3Galb- 0 -12 3 -34 0 -65 0 -3 -30 6 glycolipid 555 3'Kdn-iGb5 - 04 Kdna2-3Galb1-3GalNAcb1-3Gala1-3Galb- 0 -20 0 -47 0 -68 0 0 -34 0 carb-Sia 251 3'KDN-LacNAc-Sp - KDNa2-3Galb1-4GlcNAcb-Sp-BSA 0 -20 5 -23 0 -27 5 6 -23 6 05 Lewis 533 3'Kdn-LeX-Gal - 03 Kdna2-3Galb1-4(Fuca1-3)GlcNAcb1-3Galb- 0 -34 0 -85 0 -88 0 0 -51 0 glycolipid 551 3'Kdn-LNnT - 05 Kdna2-3Galb1-4GlcNAcb1-3Galb- 0 -20 0 -56 0 -74 0 0 -49 0 carb-Sia 222 3'Neu5Ac(9Ac)- Neu5Ac(9Ac)a2-3Galb1-4GlcNAcb-Sp-BSA 0 -23 5 -40 0 -51 -3 3 -34 0 LacNAc-Sp - 04 carb-Sia 195 3'Neu5Ac-3-FL - 07 Neu5Aca2-3Galb1-4(Fuca1-3)Glc -BSA 0 -17 5 -32 0 -48 0 -2 -29 -2 glycolipid 660 3'Neu5Ac-iGb5 Neu5Aca2-3Galb1-3GlcNAcb1-3Gala1-3Galb1- 0 -6 2 -14 0 -30 0 0 -15 0 carb-Sia 33 3'Neu5Ac-LacNAc - Neu5Aca2-3Galb1-4GlcNAc – BSA 0 -17 5 -43 3 -51 -3 6 -37 0 19 Lewis 127 3'Neu5Ac-LeX Neu5Aca2-3Galb1-4[Fuca1-3)GlcNAc – BSA 0 -23 4 -54 0 -88 0 0 -49 0 (Sialyl LeX) - ?? Lewis 41 3'Neu5Ac-LeX Neu5Aca2-3Galb1-4[Fuca1-3)GlcNAc – BSA 0 -9 4 -37 0 -39 0 6 -11 -3 (Sialyl LeX) - 09 Lewis 643 3'Neu5Ac-LeX Neu5Aca2-3Galb1-4[Fuca1-3)GlcNAc –APD-HSA 0 -8 2 -20 0 -51 0 0 -26 3 (Sialyl LeX) -APD- HAS carb-Sia 201 3'Neu5Ac-LNnT - Neu5Aca2-3Galb1-4GlcNAcb1-3Galb1-APD-HSA 0 -15 3 -43 0 -46 0 0 -32 3 09 glycolipid 561 3'Neu5Ac-LNT - 08 Neu5Aca2-3Galb1-3GlcNAcb1-3Galb- 0 -26 3 -71 0 -128 0 -2 -74 0 carb-Sia 563 3'Neu5Gc-Galb- 06 Neu5Gca2-3Galb- 0 -34 0 -60 0 -119 0 0 -74 -3 112 Table 3.3 (cont’d) glycolipid 663 3'Neu5Gc-iGb5 Neu5Gca2-3Galb1-3GlcNAcb1-3Gala1-3Galb1- 0 -6 0 -12 0 -28 0 0 -17 0 Lewis 556 3'Neu5Gc-LeX-Gal - Neu5Gca2-3Galb1-4(Fuca1-3)GlcNAcb1-3Galb- 0 -34 0 -94 0 -145 0 0 -80 -3 04 glycolipid 519 3'Neu5Gc-LNnT - Neu5Gca2-3Galb1-4GlcNAcb1-3Galb- 0 -37 0 -90 2 -165 -3 3 -85 3 03 Lewis 207 3'-sulpho-LeA - 15 3-SO3-Galb1-3[Fuca1-4)GlcNAc-BSA 0 -14 5 -29 3 -37 0 3 -20 0 Lewis 205 3'-sulpho-LeX - 15 3-SO3-Galb1-4[Fuca1-3)GlcNAc-BSA 0 -17 3 -40 0 -54 0 2 -32 0 non-human 748 4-Me-GlcAa1-2Xylb14-Me-GlcAa1-2Xylb1 - 07 -BSA 0 -26 0 -65 0 -63 -3 0 -46 0 non-human 805 4-Me-GlcAa1-2Xylb14-Me-GlcAa1-2Xylb1 - 25 -BSA 0 -8 0 -30 0 -32 0 0 -16 0 carb-Sia 261 6'Neu5Ac-LacNAc- Neu5Aca2-6Galb1-4GlcNAcb-Sp-BSA 0 -17 4 -42 0 -71 0 0 -40 5 Sp - 05 Lewis 209 6'-sulpho-LeX - 08 6-SO3-Galb1-4[Fuca1-3)GlcNAc-BSA 0 -6 0 -22 0 -40 3 3 -11 0 N-linked 593 A2 (a2-6) - 01.5 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-6(Neu5Aca2- 0 0 0 -34 0 -49 0 3 -31 -3 6Galb1-4GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb1- peptide 413 Ac-APGSTAPPA-G- Ac-A-P-G-S-T-A-P-P-A-G- 0 -28 4 -71 17 -100 5 12 -56 8 05 peptide 416 Ac-APGSTAPPA-G- Ac-A-P-G-S-T-A-P-P-A-G- 0 -20 0 -49 5 -71 3 12 -48 0 14 peptide-Tn 415 Ac-APGS-Tn(Thr)- Ac-A-P-G-S-Tn(Thr)-A-P-P-A-G- 0 -38 0 -88 0 -144 0 3 -91 -3 APPA-G-03 peptide-Tn 414 Ac-APGS-Tn(Thr)- Ac-A-P-G-S-Tn(Thr)-A-P-P-A-G- 0 0 0 -6 0 -9 0 3 -6 0 APPA-G-11 peptide-Tn 147 Ac-A-Tn(Thr)-S-G - Ac-Ala-(GalNAca)Thr-Ser-Gly-Hex-BSA (muc4) 0 -12 5 -28 0 -40 -3 6 -17 0 05 peptide-Tn 148 Ac-A-Tn(Thr)-S-G - Ac-Ala-(GalNAca)Thr-Ser-Gly-Hex-BSA (muc4) 0 -14 22 -29 0 -42 0 23 -33 0 08 peptide-N- 380 Ac-F-N(GlcNAcb1)- AcPhe-(GlcNAcb1)Asn-Ser-Gly-Hex-12 0 -6 3 3 0 -37 0 0 -11 0 GlcNAc S-G-Hex-12 peptide-Fuc 387 Ac-G-S(Fuca)-S-G- Ac-G-S(Fuca)-S-G-Hex-BSA 0 -10 3 -20 0 -40 -3 0 -12 0 Hex-15 peptide-Xyl 388 Ac-G-S(Xylb)-G-G- Ac-Gly-Ser(Xylb)-Gly-Gly-Hex-BSA 0 -11 3 -31 0 -66 3 0 -29 3 Hex-04 peptide-Tn 390 Ac-G-S-T(Tna)A-P- Ac-G-S-T(GalNAca)A-P-G-Hex-BSA 0 -6 0 -23 0 -35 -3 0 -20 0 G-Hex-06 peptide-Tn 391 Ac-G-S-T(Tna)A-P- Ac-G-S-T(GalNAca)A-P-G-Hex-BSA 0 -9 4 -28 0 -47 0 0 -32 0 G-Hex-19 peptide 419 Ac-GSTAP-G-15 Ac-G-S-T-A-P-G- 0 -17 5 -34 5 -48 3 14 -26 5 peptide-Tn 184 Ac-G-V-Tn(Thr)-S-A- Ac-Gly-Val-(GalNAca)Thr-Ser-Ala-Gly-Hex-BSA (muc1) 0 -14 5 -34 0 -43 0 6 -31 0 G - 04 peptide-Tn 185 Ac-G-V-Tn(Thr)-S-A- Ac-Gly-Val-(GalNAca)Thr-Ser-Ala-Gly-Hex-BSA (muc1) 0 -14 3 -45 0 -31 5 29 -20 0 G - 21 peptide-Man 411 Ac-P-T(Mana)-A-G - Ac-Pro-Thr(Man a)-Ala-Gly-Hex-BSA 0 -28 0 -51 0 -94 -3 0 -68 -6 22 peptide-Tn 171 Ac-P-Tn(Thr)-T-G - Ac-Pro-(GalNAca)Thr-Thr-Gly-Hex-BSA (muc2) 0 -22 14 -34 0 -37 0 17 -37 3 05 peptide-Tn 172 Ac-P-Tn(Thr)-T-G - Ac-Pro-(GalNAca)Thr-Thr-Gly-Hex-BSA (muc2) 0 -15 63 -31 0 -35 4 43 -32 0 08 peptide- 470 Ac-S-Core4(Thr)-S- Ac-Ser-Core4(Thr)-Ser-Gly-Hex-BSA 0 -20 43 -54 0 -94 0 0 -48 3 Core4 G-03 peptide- 435 Ac-S-Core4(Thr)-S- Ac-Ser-Core4(Thr)-Ser-Gly-Hex-BSA 0 -13 5 -31 0 -68 0 0 -34 0 Core4 G-10 Peptide-core- 384 Ac-Ser(core 5)-S-G- Ac-(GalNAca1-3GalNAca)Ser-Ser-Gly-Hex-BSA 0 -17 3 -32 0 -63 0 3 -23 -3 5 08 peptide-Man 625 Ac-S-S(Mana)-S-G - Ac-S-S(Man a)-S-G-Hex-OH 0 -17 0 -40 0 -60 2 0 -34 0 06 peptide-Man 409 Ac-S-S(Mana)-S-G - Ac-S-S(Man a)-S-G-Hex-OH 0 -20 13 -54 0 -92 0 0 -54 -3 20 Peptide-core- 381 Ac-S-Ser(core 5)-S- Ac-Ser-(GalNAca1-3GalNAca)Ser-Ser-Gly-Hex-BSA 0 -13 2 -14 0 -60 0 6 -28 -3 5 G-04 Peptide-core- 382 Ac-S-Ser(core 5)-S- Ac-Ser-(GalNAca1-3GalNAca)Ser-Ser-Gly-Hex-BSA 0 -17 0 -39 0 -73 0 6 -37 0 5 G-18 peptide 77 Ac-S-S-S-G - 24 Ac-Ser-Ser-Ser-Gly-Hex-BSA 0 -9 0 -34 0 -49 -5 3 -26 3 peptide-TF 103 Ac-S-TF(Ser)-S-G AcSer-(Galβ1-3GalNAcα)Thr-Ser-Gly-Hex-HSA - 04-HSA (S- 0 -9 5 -37 0 -63 0 0 -37 -3 TF-S) peptide-TF 109 Ac-S-TF(Ser)-S-G - Ac-Ser-(Galb1-3GalNAca)Ser-Ser-Gly-Hex-BSA 0 -14 6 -37 0 -54 3 0 -34 0 16 peptide-TF 128 Ac-S-TF(Ser)-S-G - Ac-Ser-(Galb1-3GalNAca)Ser-Ser-Gly-Hex-BSA 0 -9 0 -25 0 -48 -3 9 -31 0 28 peptide-TF 430 Ac-S-TFa(Ser)- Ac-Ser-Ser(Galb1-3GalNAca-)-Ser(Galb1-3GalNAca-)- 0 -17 0 -40 0 -68 -3 0 -46 -3 TFa(Ser)-G-15 Gly-Hex peptide-F1a 27 Ac-S-Thr(F1a)-S-G - Ac-Ser-(Galb1-4GlcNAcb1-6GalNAca)Thr-Ser-Gly-Hex- 0 -15 8 -40 0 -52 3 9 -31 -3 04 BSA 113 Table 3.3 (cont’d) peptide 196 Ac-S-Thr-S-G - 18 Ac-Ser-Thr-Ser-Gly-Hex-BSA 0 -20 230 -50 0 -85 0 0 -63 0 peptide-Tn- 429 Ac-S-Tn(Ser)-V-G- Ac-Ser-Ser(GalNAca)-V-G- 0 -12 0 -26 0 -49 3 0 -22 3 Ser 03 peptide-Tn- 499 Ac-S-Tn(Ser)-V-G- Ac-Ser-Ser(GalNAca)-V-G- 0 -14 0 -25 0 -45 6 0 -26 3 Ser 13 peptide-Tn 142 Ac-S-Tn(Thr)-A-G - Ac-Ser-(GalNAca)Thr-Ala-Gly-Hex-BSA (muc1) 0 -12 6 -37 0 -41 -3 15 -28 0 08 peptide-Tn 143 Ac-S-Tn(Thr)-A-G - Ac-Ser-(GalNAca)Thr-Ala-Gly-Hex-BSA (muc1) 0 -20 34 -28 0 -34 0 40 -23 3 22 peptide-Tn 151 Ac-S-Tn(Thr)-G-G - Ac-Ser-(GalNAca)Thr-Gly-Gly-Hex-BSA (muc4) 0 -12 5 -28 2 -48 3 6 -34 0 07 peptide-Tn 157 Ac-S-Tn(Thr)-S-G Ac-Ser-(GalNAca)Thr-Ser-Gly-Hex-HSA 0 -5 7 -31 0 -54 0 0 -25 0 HSA-04 peptide-Tn 156 Ac-S-Tn(Thr)-S-G Ac-Ser-(GalNAca)Thr-Ser-Gly-Hex-HSA 0 -12 0 -26 0 -34 0 3 -20 3 HSA-23 peptide-Tn 177 Ac-S-Tn(Thr)- Ac-Ser-(GalNAca)Thr-(GalNAca)Thr-Gly-Hex-BSA 0 -15 4 -26 0 -37 0 -3 -29 0 Tn(Thr)-G - 05 (muc2) peptide-Tn 178 Ac-S-Tn(Thr)- Ac-Ser-(GalNAca)Thr-(GalNAca)Thr-Gly-Hex-BSA 0 -20 5 -42 0 -43 -3 6 -28 3 Tn(Thr)-G - 09 (muc2) peptide-Tn 179 Ac-S-Tn(Thr)- Ac-Ser-(GalNAca)Thr-(GalNAca)Thr-Gly-Hex-BSA 0 -6 3 -26 0 -22 -3 0 -7 0 Tn(Thr)-G - 22 (muc2) peptide-Tn 182 Ac-S-Tn(Thr)-V-G - Ac-Ser-(GalNAca)Thr-Val-Gly-Hex-BSA 0 -12 11 -32 0 -37 5 9 -29 0 04 peptide-Tn 183 Ac-S-Tn(Thr)-V-G - Ac-Ser-(GalNAca)Thr-Val-Gly-Hex-BSA 0 -29 47 -31 0 -32 5 26 -45 3 22 peptide-TF 158 Ac-TF(Ser)-G - 04 Ac-(Galb1-3GalNAca)Ser-Gly-Hex-BSA 0 -9 3 -22 0 -34 3 6 -22 0 peptide-TF 159 Ac-TF(Ser)-G - 24 Ac-(Galb1-3GalNAca)Ser-Gly-Hex-BSA 0 -15 0 -32 0 -35 0 2 -26 0 peptide-Tn- 121 Ac- Ac-(GalNAca)Ser-(GalNAca)Ser-(GalNAca)Ser-Gly-Hex- 0 -17 5 -46 0 -74 5 0 -34 -3 Ser Tn(Ser)Tn(Ser)Tn(S BSA er)-G - 03 peptide-Tn 174 Ac-Tn(Thr)-Tn(Thr)- Ac-(GalNAca)Thr-(GalNAca)Thr-(GalNAca)Thr-Gly-Hex- 0 -17 5 -26 0 -51 5 0 -35 3 Tn(Thr)-G - 05 BSA (muc2) peptide-Tn 175 Ac-Tn(Thr)-Tn(Thr)- Ac-(GalNAca)Thr-(GalNAca)Thr-(GalNAca)Thr-Gly-Hex- 0 -12 0 -34 0 -42 3 6 -37 -3 Tn(Thr)-G - 08 BSA (muc2) peptide-Tn 154 Ac-T-Tn(Thr)-P-G - Ac-Thr-(GalNAca)Thr-Pro-Gly-Hex-BSA (muc2,6,7) 0 -11 5 -26 0 -31 3 3 -17 0 08 peptide-Tn 155 Ac-T-Tn(Thr)-P-G - Ac-Thr-(GalNAca)Thr-Pro-Gly-Hex-BSA (muc2,6,7) 0 0 5 -11 0 -11 0 13 -12 3 21 peptide-Tn 162 Ac-V-Tn(Thr)-S-G - Ac-Val-(GalNAca)Thr-Ser-Gly-Hex-BSA (muc1) 0 -12 5 -32 0 -23 3 3 -19 3 04 peptide-Tn 145 Ac-V-Tn(Thr)-S-G - Ac-Val-(GalNAca)Thr-Ser-Gly-Hex-BSA (muc1) 0 -12 5 -32 0 -48 0 6 -28 0 08 peptide-Tn 146 Ac-V-Tn(Thr)-S-G - Ac-Val-(GalNAca)Thr-Ser-Gly-Hex-BSA (muc1) 0 -3 8 -17 0 -14 0 6 -17 3 19 Blood Group 115 Adi - 04 GalNAca1-3Galb-BSA 0 -14 5 -39 0 -59 3 3 -29 0 A y- 352 AGE60 Advanced glycation endproducts day 60 0 -29 0 -57 0 -91 0 6 -51 -3 glycoprotein Blood Group 213 A-LeB hexa - 06 GalNAca1-3(Fuca1-2)Galb1-3(Fuca1-4)GlcNAcb1- 0 -9 5 -28 0 -48 0 3 -26 0 A 3Galb1-BSA y- 591 alpha 1 acid glycoprotein Asialo-alpha (asialo) 1-Acid Glycoprotein, Asialo- 0 -6 0 -8 0 -14 -3 0 0 0 glycoprotein Oroscomucoid, Human, from 75/54 treated with trifluoracetic acid non-human- 102 alphaGal- 08 Gala1-3Galb1-4GlcNAc-BSA 0 -5 0 -17 0 -40 0 9 -14 0 aGal non-human- 218 alphaGal-6-deoxy - Gala1-3Galb1-4(6deoxy-GlcNAc)-HSA (alphaGal)-HSA 0 -3 3 -32 0 -48 3 5 -24 3 aGal 11 peptide 631 APF peptide 0 -3 0 -5 0 9 3 0 -3 3 non-human 741 Ara2 - 08 Araa1-5Ara-BSA 0 -22 0 -48 0 -74 -5 0 -48 0 non-human 756 Ara4 - 08 Araa1-5Araa1-5Araa1-5Ara-BSA 0 -14 0 -32 0 -97 0 0 -62 0 non-human 798 Arafa1-3(Arafa1-2)Xylb1Arafa1-3(Arafa1-2)Xylb1 - 14 - BSA 0 -5 0 -3 0 -17 -3 0 -9 3 non-human 775 Arafa1-3(Arafa1-2)Xylb1-4Xylb Arafa1-3(Arafa1-2)Xylb1-4Xylb - 04 -BSA 0 -8 0 -27 0 -23 0 0 -11 3 Peptide 724 ATPLPVTD-BSA C-terminus MUC4- ATPLPVTD-BSA 0 -11 0 -28 0 -43 0 0 -33 0 non-human 509 AX2 - 04 3 0 -9 0 -48 0 -20 0 0 -17 0 Arafα1-2Xylβ1-4Xylβ-]; 2 -α-L-Arabinofuranosyl- xylotriose peptide-Tn 438 AzHex-D-Tn(Thr)-R- BSA--hexyl-D-T(GalNAc-a)-R-amide 0 -11 0 -26 0 -57 0 -3 -32 3 NH2-06 peptide- 524 AzHex-G-Y(GalNAc-b)-A-NH2 BSA-AzHex-G-Y(GalNAc-b)-A-amide - 12 0 0 0 4 0 3 0 0 0 3 GalNAc Tyr peptide 514 AzHex-G-Y-A-NH2 - BSA-AzHex-G-Y-A-amide 0 49 0 9 0 11 0 0 10 6 04 peptide 515 AzHex-L-Y-W-NH2 - 08BSA-AzHex-L-Y-W-amide 0 74 0 3 0 14 26 0 3 5 peptide 450 AzHex-P-D-T-R-P- BSA--hexyl-PDTRP-amide 0 -6 0 -12 0 -26 0 3 -18 3 NH2-07 peptide-Tn 442 AzHex-SAPD- BSA--hexyl-SAPD-T(GalNAc-a)-RPAP-amide 0 -14 0 -51 0 -91 0 5 -43 -3 Tn(Thr)-RPAP-NH2- 07 peptide 451 AzHex-S-A-P-D-T-R- BSA--hexyl-SAPDTRPAP-amide 0 -9 3 -40 0 -74 5 0 -31 3 P-A-P-NH2-07 peptide-Tn- 421 AzHex-S-Tn(Ser)-V- BSA-AzHex-S-Tn(Ser)-V-G-HexNH2 0 -29 3 -74 0 -122 0 0 -64 0 Ser G-HexNH2-06 peptide-Tn- 423 AzHex-S-Tn(Ser)-V- BSA-AzHex-S-Tn(Ser)-V-G-HexOH 0 -19 0 -42 0 -69 0 0 -51 0 Ser G-HexOH-06 114 Table 3.3 (cont’d) peptide-Tn 444 AzHex-VTSAPD- BSA--hexyl-VTSAPD-T(GalNAc-a)-RPAPGS-amide 0 -11 0 -28 0 -50 0 0 -14 0 Tn(Thr)-RPAPGS- NH2-06 Blood Group 226 B tetra type 1-Sp - Gala1-3[Fuca1-2]Galb1-3GlcNAcb-Sp-BSA 0 -6 3 -20 0 -34 3 0 -20 3 B 16 Blood Group 235 B tetra type 2-Sp - Gala1-3[Fuca1-2]Galb1-4GlcNAcb-Sp-BSA 0 -26 25 -26 0 -48 2 6 -25 0 B 07 non-human- 223 Bdi-g - 06 Gala1-3Galb– BSA 0 -8 5 -31 0 -54 0 6 -28 0 aGal Blood Group 44 BG-A -19 GalNAca1-3(Fuca1-2)GalbG -BSA 0 -11 5 -34 0 -45 0 6 -26 -3 A Blood Group 337 BG-A5-05 GalNAca1-3(Fuca1-2)Galb1-3Galb1-linker-BSA 0 -14 5 -37 0 -48 -1 -3 -26 -6 A Blood Group 343 BG-A6-04 GalNAca1-3(Fuca1-2)Galb1-4Glcb1-linker-BSA 0 -20 3 -34 0 -57 0 0 -28 -6 A Blood Group 344 BG-A6-23 GalNAca1-3(Fuca1-2)Galb1-4Glcb1-linker-BSA 0 -20 0 -57 0 -68 0 3 -29 0 A Blood Group 65 BG-B3-05 Gala1-3(Fuca1-2)Galb1-3GalNAcα1-linker-BSA 0 -23 68 -46 28 -85 -3 71 -57 0 B Blood Group 340 BG-B5-17 Gala1-3(Fuca1-2)Galb1-3Galb1-linker-BSA 0 -26 3 -31 0 -68 -3 0 -50 -6 B Blood Group 137 BG-H1- 06 Fuca1-2Galb1-3GlcNAcb1-linker-BSA 0 -9 4 -17 0 -17 0 3 -23 3 H Blood Group 138 BG-H2- 06 Fuca1-2Galb1-4GlcNAcb1-linker-BSA 0 -5 0 -29 0 -37 0 2 -14 3 H Blood Group 105 BG-H4- 15 Fuca1-2Galb1-3GalNAcb1-linker-BSA 0 -14 5 -40 0 -69 0 6 -45 0 H Blood Group 349 BG-H6-05 Fuca1-2Galb1-4Glcb1-linker-BSA 0 -28 5 -31 0 -43 0 3 -29 -3 H y- 355 BSM Bovine submaxillary mucin (Sigma M3895; STn, STF, S- 0 -6 0 -6 0 -9 0 3 -9 -3 glycoprotein GlcNAcb1-3, ~20% of Sia is acetylated at 7,8, or 9) y- 358 BSM (asialo) Asialo-Bovine submaxillary mucin (aBSM, Tn, TF, 0 -3 0 0 0 6 0 6 -3 -3 glycoprotein GlcNAcb1-3GalNAc) non-human 709 cellotetraose - 17 Glcb1-4Glcb1-4Glcb1-4Glcb1- 0 -5 0 -23 0 -12 -1 0 -14 0 non-human 684 chitobiose - 03 GlcNAcb1-4GlcNAcb1- 0 -23 0 -57 0 -77 0 0 -43 non-human 693 chitobiose - 12 GlcNAcb1-4GlcNAcb1- 0 -11 0 -23 0 -17 0 -17 non-human 688 chitopentaose - 06 GlcNAcb1-4GlcNAcb1-4GlcNAcb1-4bGlcNAcb1- 0 -11 0 -37 0 -57 0 6 -25 3 4GlcNAcb1- non-human 697 chitotetraose - 11 GlcNAcb1-4GlcNAcb1-4GlcNAcb1-4bGlcNAcb1- 0 -6 0 -32 0 -40 0 3 -26 3 non-human 718 chitotriose - 16 GlcNAcb1-4GlcNAcb1-4GlcNAcb1- 0 -8 0 -37 0 -48 0 3 -28 0 Lewis 89 DFpLNH I, LeB - 09 Fuca1-2Galb1-3(Fuca1-4)GlcNAcb1-3Galb1-4GlcNAcb1- 0 -27 0 -77 0 -111 0 6 -79 8 3Galb-BSA y-protein 784 EGFR EGFR Protein, Human, Recombinant (His Tag) 0 0 0 -6 0 -17 -3 0 -12 -3 y- 361 fetuin fetuin from calf serum (Sigma #F2379; most abundant 0 -3 0 -14 0 -20 0 3 -20 0 glycoprotein glycans are Neu5Ac2-3LacNAc, Neu5Ac2-6LacNAc, SiaLeC, STF) y- 412 fetuin (human) a2-HS-glycoprotein (aka human fetuin) 0 3 3 3 0 9 3 0 0 20 glycoprotein non-human 244 Forssman Tetra- GalNAca1-3GalNAcb1-3Gala1-4Galb-BSA 0 -3 3 -17 0 -17 0 3 -3 0 BSA - 05 Lewis+Sia 194 Fuc, 6'Neu5Ac- Galb1-4[Fuca1-3]GlcNAcb1-6[Neu5Aca2-6Galb1- 0 -9 3 -37 0 -51 3 3 -36 3 LNnH-APD-HSA - 4GlcNAcb1-3]Galb1-APD-HSA 12 carb-Fuc 130 Fuc-a - 04 Fuc-a - BSA 0 -17 5 16 0 -74 5 6 -37 3 non-human 131 Fuc-b - 04 Fuc-b - BSA 0 -12 8 -37 0 -45 5 9 -20 3 glycolipid 262 Fuc-GM1a - 08 Fuca1-2Galb1-3GalNAcb1-4(Neu5Aca2-3)Galb1-4-BSA 0 -17 0 -51 0 -85 0 6 -43 -3 glycolipid- 118 GA1 tri - 06 Galb1-3GalNAcb1-4Galb1-BSA (GA1tri or asialo-GM1) 0 -17 5 -51 0 -82 0 6 -46 -3 neutral glycolipid- 116 GA2di - 05 GalNAcb1-4Galb - BSA (aka: asialo-GM2) 0 -15 0 -32 0 -37 -3 6 -26 0 neutral non-human- 180 Gala3-type1 - 09 Gala1-3Galb1-3GlcNAc-BSA 0 -12 5 -28 0 -40 0 -6 -20 -5 aGal carb-Gal 675 Galb1-3Galb1 - 07 Galb1-3Galb1- 0 -11 0 -34 0 -105 3 0 -40 0 carb-Gal 673 Gal-BSA-21 Gal-BSA-21 (dorothy) 0 -3 0 -14 0 -15 5 0 -6 6 non-human- 596 Galili Hexa-05 Gala1-3(Galb1-4GlcNAcb1-3)2Galb- 0 -23 0 -37 0 -85 0 0 -51 -3 aGal non-human- 608 Galili Hexa-13-HSA Gala1-3(Galb1-4GlcNAcb1-3)2Galb- 0 -11 0 -34 0 -40 0 0 -28 0 aGal non-human- 602 Galili Octa-05 Gala1-3(Galb1-4GlcNAcb1-3)3Galb- 0 -26 3 -74 0 -113 0 0 -74 -3 aGal non-human- 610 Galili Octa-13 Gala1-3(Galb1-4GlcNAcb1-3)3Galb- 0 -39 0 -74 0 -119 -3 0 -82 0 aGal carb-GalNAc 113 GalNAc-a - 04 GalNAc-a - BSA 0 -17 3 -26 6 -46 0 6 -15 3 carb-GalNAc 114 GalNAca1-6Galb - GalNAca1-6Galb-BSA 0 -11 4 -28 0 -40 0 6 -20 0 04 carb-GalNAc 717 GalNAca-phenyl GalNAca-O-phenyl-amide linked 0 -28 0 -58 0 -94 0 0 -54 6 amide- low carb-GalNAc 729 GalNAca-phenyl Dz- GalNAca-O-phenyl-diazirine linker 0 -21 0 -17 0 -34 0 0 -20 0 high carb-GalNAc 732 GalNAcb-phenyl GalNAcb-O-phenyl-amide linked 0 -3 0 -12 0 -18 0 0 -9 0 amide- high 115 Table 3.3 (cont’d) carb-GalNAc 715 GalNAcb-phenyl Dz- GalNAcb-O-phenyl-diazirine linker 0 -6 0 -26 0 -32 0 3 -20 3 low glycolipid- 254 Gb4 tetra (P1 tetra)- GalNAcb1-3Gala1-4Galb1-4GlcNAcb-Sp-BSA 0 -6 3 -20 0 -23 5 3 -6 -3 neutral Sp - 15 glycolipid- 546 Gb4 tri - 07 GalNAcb1-3Gala1-4Galb- 0 -14 0 -37 0 -49 3 0 -34 -3 neutral glycolipid 603 Gb5 analogue type Galb1-3GlcNAcb1-3Gala1-4Galb- 0 -9 0 -26 0 -29 3 0 -15 0 related 1-14 glycolipid 607 Gb5 analogue type Galb1-4GlcNAcb1-3Gala1-4Galb- 0 -11 0 -26 0 -29 -3 0 -20 0 related 2-12 glycolipid 658 GD1a (Ac, Ac) Neu5Aca2-8Neu5Aca2-3(Galb1-3GlcNAcb1-4)Galb1- 0 -23 3 -68 0 -126 2 0 -68 6 glycolipid 655 GD1a (Kdn,Kdn) Kdna2-8Kdna2-3(Galb1-3GlcNAcb1-4)Galb1- 0 -11 0 -23 0 -31 -3 0 -17 0 glycolipid 549 GD2 tetra (Ac/Ac) - Neu5Aca2-8Neu5Aca2-3(GalNAcb1-4)Galb- 0 -20 0 -40 0 -68 3 -3 -45 -3 04 glycolipid 646 GD2 tetra (Ac-Gc) Neu5Aca2-8Neu5Gca2-3(GlcNAcb1-4)Galb1- 0 -7 3 -29 0 -37 0 0 -17 3 glycolipid 521 GD2 tetra (Gc/Ac) - Neu5Gca2-8Neu5Aca2-3(GalNAcb1-4)Galb- 0 -7 0 -20 0 -29 0 0 -23 0 04 glycolipid 505 GD2 tetra (Gc/Gc) - Neu5Gca2-8Neu5Gca2-3(GalNAcb1-4)Galb- 0 -22 4 -62 0 -119 -3 0 -57 -3 04 glycolipid 566 GD2 Tetra-06 GalNAcb1-4(Neu5Aca2-8Neu5Aca2-3)Galb- 0 -17 0 -46 0 -57 0 0 -38 0 glycolipid 569 GD2 Tetra-12 GalNAcb1-4(Neu5Aca2-8Neu5Aca2-3)Galb- 0 -28 0 -66 0 -109 -5 0 -70 -3 glycolipid 453 GD2-b-propyl-03 Neu5Aca2-8Neu5Aca2-3[GalNAcb1-4]Galb1-4Glcb- 0 -17 3 -43 0 -60 5 0 -32 3 propyl-BSA glycolipid 529 GD3 tri (Ac/Ac) - 04 Neu5Aca2-8Neu5Aca2-3Galb- 0 -38 0 -85 0 -161 -3 0 -85 -5 glycolipid 553 GD3 tri (Gc/Ac) - 04 Neu5Gca2-8Neu5Aca2-3Galb- 0 -25 0 -70 0 -114 0 0 -67 0 glycolipid 652 GD3 tri (Gc-Gc) Neu5Gca2-8Neu5Gca2-3Galb1- 0 -6 0 -25 0 -40 -3 0 -21 3 glycolipid 543 GD3 tri (Kdn/Ac) - Kdna2-8Neu5Aca2-3Galb- 0 -37 3 -80 0 -130 3 0 -91 0 05 carb-Glc 258 Glc-a - 05 Glc-a - BSA 0 -12 0 -37 0 -57 3 3 -23 0 Carb-Glc 759 Glca1-6Glca -05 Glca1-6Glca -BSA 0 -11 0 -28 0 -29 -1 0 -15 0 Carb-GlcA 448 GlcA-LNT-05 GlcAb1-3Galb1-3GlcNAcb1-3Galb- 0 -23 0 -65 0 -96 3 0 -54 3 Carb-GlcA 475 GlcA-LNT-16 GlcAb1-3Galb1-3GlcNAcb1-3Galb- 0 -28 0 -65 0 -108 -3 -3 -60 0 non-human 558 Glcb1-3Glcb- 06 Glcb1-3Glcb- 0 37 5 153 141 655 3 11 -26 6 non-human 781 Glcb1-3Glcb1-3Glcb Glcb1-3Glcb1-3Glcb -03 BSA 0 -11 3 -30 0 -31 0 6 -17 0 carb-Glc 720 Glcb1-4Glcb1-3Glc - Glcb1-4Glcb1-3Glcb1- 0 -17 0 -60 0 -49 0 6 -48 6 06 carb-Glc 691 Glcb1-4Glcb1-3Glc - Glcb1-4Glcb1-3Glcb1- 0 -9 5 -47 0 -10 3 11 -42 0 17 carb-Glc 708 Glcb1-4Glcb1-4Glc - Glcb1-4Glcb1-4Glcb1- 0 -14 0 -40 0 -3 0 3 -37 3 13 Carb-Glc 735 Glcb1-4Manb1 - 05 Glcb1-4Manb1 - BSA 0 -23 3 -40 0 -51 0 0 -37 0 Carb-GlcNAc 745 GlcNAcb-ITC - 06 from Beat Ernst 0 -9 0 -20 0 -23 -3 3 -20 3 Blood Group 106 Globo B - 05 Gala1-3(Fuca1-2)Galb1-3GalNAcb1-3Gala1-4Galb1-BSA 0 -17 12 -38 0 -48 0 9 -25 3 B Blood Group 609 Globo H analogue Fuca1-2Galb1-3GlcNAcb1-3Gala1-4Galb- 0 -8 0 -23 0 -17 -4 0 -15 3 H related type 1-04 Blood Group 604 Globo H analogue Fuca1-2Galb1-3GlcNAcb1-3Gala1-4Galb- 0 -11 0 -17 0 -34 0 0 -22 0 H related type 1-10 Blood Group 599 Globo H analogue Fuca1-2Galb1-4GlcNAcb1-3Gala1-4Galb- 0 -9 2 -37 0 -47 0 0 -26 -3 H related type 2-03 glycolipid 554 GM1 (Ac) - 06 Galb1-3GalNAcb1-4(Neu5Aca2-3)Galb 0 -8 0 -26 0 -43 0 -3 -32 -2 glycolipid 645 GM1 (Gc) Neu5Gca2-3(Galb1-3GlcNAcb1-4)Galb1- 0 -3 0 -20 0 -49 0 0 -11 2 glycolipid 648 GM1 (Kdn) Kdna2-3(Galb1-3GlcNAcb1-4)Galb1- 0 -3 0 -12 0 -46 -3 0 -14 3 glycolipid 82 GM1a - 29 Galb1-3GalNAcb1-4(Neu5Aca2-3)Galb 0 -15 5 -48 0 -74 0 6 -42 0 glycolipid 649 GM2 tri (Kdn) Kdna2-3(GlcNAcb1-4)Galb1- 0 -26 3 -65 0 -102 0 0 -49 3 glycolipid 336 GM2-Sp - 04 Neu5Aca2-3[GalNAcb1-4]Galb1-4Glcb-Sp-BSA 0 -25 5 -31 0 -37 3 0 -29 0 glycolipid 96 GM2-Sp - 14 Neu5Aca2-3[GalNAcb1-4]Galb1-4Glcb-Sp-BSA 0 -12 0 -17 0 -51 5 6 -31 0 glycolipid 282 GM3-Sp - 11 Neu5Aca2-3Galb1-4Glcb-Sp-BSA 0 -11 3 -15 0 -51 0 0 -17 0 carb-GlcNAc 252 GNLacNAc-Sp - 06 GlcNAcb1-3Galb1-4GlcNAcb-Sp-BSA 0 -20 110 -39 0 -71 3 3 -40 5 y- 370 gp120 gp120 from HIV; expressed in HEK293T cells; 0 -20 0 -22 0 -68 0 6 -31 0 glycoprotein y-protein 790 gp120 Clade B gp120 from HIV BAL-120; expressed in HEK293T cells; 0 -6 0 -17 0 -40 -3 -3 -14 0 glycolipid 257 GT3-Sp - 03 Neu5Aca2-8Neu5Aca2-8Neu5Aca2-3Galb1-4Glcb-Sp- 0 -14 3 -37 0 -57 -3 3 -31 0 BSA peptide-TF 186 GTSSAS-TF(Thr)- BSA-PEG7-Gly-Thr-Ser-Ser-Ala-Ser-(Galb1- 0 -9 5 -27 0 -41 -3 0 -17 3 GHATPLPVTD 3GalNAca)Thr-Gly-His-Ala-Thr-Pro-Leu-Pro-Val-Thr-Asp peptide 190 GTSSASTGHATPLP BSA-PEG7-Gly-Thr-Ser-Ser-Ala-Ser-Thr-Gly-His-Ala-Thr- 0 -14 3 -34 0 -43 0 6 -23 0 VTD Pro-Leu-Pro-Val-Thr-Asp peptide-TF 189 GTSSA-TF(Ser)- BSA-PEG7-Gly-Thr-Ser-Ser-Ala-(Galb1-3GalNAca)Ser- 0 -11 3 -37 0 -37 0 0 -20 3 TF(Thr)- (Galb1-3GalNAca)Thr-Gly-His-Ala-Thr-Pro-Leu-Pro-Val- GHATPLPVTD Thr-Asp peptide-TF 187 GTSSA-TF(Ser)- BSA-PEG7-Gly-Thr-Ser-Ser-Ala-(Galb1-3GalNAca)Ser- 0 -17 8 -28 0 -43 3 6 -25 0 TGHATPLPVTD Thr-Gly-His-Ala-Thr-Pro-Leu-Pro-Val-Thr-Asp (MUC4) 116 Table 3.3 (cont’d) GAG 203 Hep-5000 - 01 heparin polysaccharide (MW ~5000) 0 -8 5 -31 0 -37 3 6 -17 0 GAG 202 Hep-N-acetylated fully N-acetylated heparin polysaccharide 0 -12 3 -34 0 -37 3 6 -23 0 GAG-Hep 571 Hep-NAc-Hepta-05 GlcAb1-4GlcNAca1-4GlcAb1-4GlcNAca1-4GlcAb1- 0 -17 0 -34 0 -51 3 0 -34 0 4GlcNAca1-4GlcAb-Benzamide- (Heparosan ) GAG-Hep 568 Hep-NAc-Octa-03 GlcNAca1-4GlcAb1-4GlcNAca1-4GlcAb1-4GlcNAca1- 0 -6 0 -26 0 -43 -3 0 -29 0 4GlcAb1-4GlcNAca1-4GlcAb-Benzamide- (Heparosan ) GAG-Hep 542 Hep-NAc-Penta-04 GlcAb1-4GlcNAca1-4GlcAb1-4GlcNAca1-4GlcAb- 0 -17 0 -46 0 -79 -5 -3 -48 -3 Benzamide- (Heparosan ) GAG-Hep 511 Hep-NAc-Tetra-06 GlcNAca1-4GlcAb1-4GlcNAca1-4GlcAb-Benzamide- 0 -14 5 -34 0 -60 0 0 -20 -3 (Heparosan ) GAG-Hep 575 Hep-Nona-GT13- GlcAb1-4GlcNSa1-4GlcAb1-4GlcNSa1-4GlcAb1- 0 -20 0 -38 0 -54 0 0 -34 3 02 4GlcNSa1-4GlcAb1-4Glc(6S, NS)a1-4GlcAb-Benzamide- GAG-Hep 567 Hep-NS-Octa-03 GlcNSa1-4GlcAb1-4GlcNSa1-4GlcAb1-4GlcNSa1- 0 -9 0 -26 0 -40 0 3 -20 0 4GlcAb1-4GlcNSa1-4GlcAb-Benzamide- GAG-Hep 534 Hep-NS-Tetra-05 GlcNSa1-4GlcAb1-4GlcNSa1-4GlcAb-Benzamide- 0 -11 3 -31 0 -60 3 3 -31 0 GAG-Hep 738 Hep-Octa-GT24-C5- Glc(6S, Nac)a1-4GlcAb1-4Glc(6S, 3S, NS)a1-4IdoA(2S)a1- 0 3 4 -6 0 -20 3 0 11 3 03 4Glc(6S, NS)a1-4IdoA(2S)a1-4Glc(6S, NS)a1-4GlcAb- Benzamide- y-protein 787 Her2 Her2 / ERBB2 Protein, Human, Recombinant 0 0 0 -15 0 -17 -3 0 -11 3 (His Tag) GAG-Hya 576 Hya7-03 GlcAb1-3(GlcNAcb1-4GlcAb1-3)3- 0 -8 0 -31 0 -37 0 0 -32 0 GAG-Hya 206 Hya9 - 03 (GlcAb1-3GlcNAcb1-4)4b1-3GlcAb1-BSA 0 -6 5 -28 0 -51 5 6 -20 2 glycolipid- 432 iFs-05 GalNAca1-3GalNAcb1-3Gala1-3Galb- (iso-Forssman) 0 3 5 -17 0 -77 5 3 -37 3 neutral glycolipid- 436 iFs-11 GalNAca1-3GalNAcb1-3Gala1-3Galb- (iso-Forssman) 0 -38 0 -96 0 -156 3 0 -93 3 neutral glycolipid- 487 iGb4-19 GalNAcb1-3Gala1-3Galb- 0 -20 2 -57 0 -97 0 3 -51 -3 neutral glycolipid- 544 iGb5 tetra - 09 Galb1-3GalNAcb1-3Gala1-3Galb- 0 -11 0 -51 0 -82 0 0 -42 0 neutral glycolipid- 490 iGb5-15 Galb1-3GalNAcb1-3Gala1-3Galb- 0 -23 0 -77 0 -121 0 0 -66 3 neutral carb-type1 216 iLNO - 06 Galb1-3GlcNAcb1-3Galb1-4GlcNAcb1-6 (Galb1- 0 -6 5 -34 0 -29 0 3 -23 0 3GlcNAcb1-3)Galb1-BSA carb-Glc 30 Isomaltose - 13 Glca1-6Glcb-BSA 0 -15 5 -39 3 -51 6 3 -28 0 non-human 550 KDOa2-8KDOa - 09 KDOa2-8KDOa-APTE-BSA 0 -17 3 -32 0 -54 0 3 -26 0 non-human 541 KDOa2-8KDOa2-4KDOa KDOa2-8KDOa2-4KDOa-APTE-BSA - 02 0 -13 0 -43 0 -82 -1 0 -40 0 y- 369 KLH Keyhole limpet hemocyanin (Sigma H7017) 0 3 0 0 0 16 0 -3 9 0 glycoprotein glycolipid- 224 Lac-C5 - 14 Galb1-4Glcb – BSA 0 -8 5 -26 0 -29 0 6 -17 5 neutral carb-type 2 107 LacNAc (trimeric) - Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1- 0 -12 0 -37 0 -74 -5 6 -37 0 08 4GlcNAcb-APE-HSA N-linked 198 LacNAc-Man5 - 02 Mana1-6(Mana1-3)Mana1-6(Galb1-4GlcNAcb1- 0 -6 5 -23 0 -23 0 3 -17 0 2Mana1-3)Manb1-4GlcNAcb-BSA non-human 690 laminaritriose - 06 Glcb1-3Glcb1-3Glcb1- 0 -5 3 -26 0 -20 0 -12 glycolipid- 525 Lc3 di - 11 GlcNAcb1-3Galb- 0 -17 0 -32 0 -77 0 0 -37 2 neutral Lewis 700 LeA tri Galb1-3[Fuca1-4)GlcNAcb1; LeA trisacch from Dextra (NGP0704) 0 -6 0 -17 0 -11 0 0 -9 2 Lewis 38 LeB-Lac - 09 Fuca1-2Galb1-3(Fuca1-4)GlcNAcb1-3Galb1-4Glcb-BSA 0 -6 0 -34 3 -54 -3 6 -25 0 (LNDFH I) Lewis 221 LeC (dimeric)-Sp - Galb1-3GlcNAcb1-3Galb1-3GlcNAcb-Sp-BSA 0 -12 4 -34 0 -54 5 6 -26 0 16 lewis 111 LeC-Sp - 06 Galb1-3GlcNAcb-Sp-BSA 0 -16 -1 -37 0 -68 0 3 -39 0 Lewis 259 LeC-Sp - 15 Galb1-3GlcNAcb-Sp-BSA 0 -10 3 -28 0 -54 0 3 -20 0 Lewis 730 LeX trisaccharide Galb1-4[Fuca1-3)GlcNAc-; SSEA-1; CD-15; Dextra 0 -18 0 -17 0 -34 -1 0 -26 -6 Lewis 39 LeY -08 Fuca1-2Galb1-4[Fuca1-3)GlcNAc –HSA 0 -11 37 -34 0 -69 0 3 -20 0 Lewis 666 LeY tetra-HSA -05 Fuca1-2Galb1-4[Fuca1-3)GlcNAcb1- 0 -12 0 -22 0 -51 -3 0 -20 0 Lewis 667 LeY tetra-HSA -10 Fuca1-2Galb1-4[Fuca1-3)GlcNAcb1- 0 -6 0 -23 0 -43 0 0 -32 0 carb-type 133 LNH - 13 Galb1-4GlcNAcb1-6(Galb1-3GlcNAcb1-3)Galb1-BSA 0 -23 11 -63 0 -88 -3 0 -51 0 1+2 carb-type 640 LNH - 13-HSA Galb1-4GlcNAcb1-6(Galb1-3GlcNAcb1-3)Galb1- 0 -5 0 -14 0 -32 0 3 -14 -3 1+2 carb-type 2 134 LNnH - 11 Galb1-4GlcNAcb1-6(Galb1-4GlcNAcb1-3)Galb1-BSA 0 -15 5 -20 0 -31 -3 0 -17 6 carb-type 2 642 LNnH - 12-HSA Galb1-4GlcNAcb1-6(Galb1-4GlcNAcb1-3)Galb1- 0 -11 0 -17 0 -20 -4 0 -17 3 carb-type 2 595 LNnO-05 Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1- 0 -23 0 -57 0 -85 0 0 -51 0 4GlcNAcb1-3Galb- carb-type 2 601 LNnO-16 Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1- 0 -17 0 -31 0 -60 0 3 -34 0 4GlcNAcb1-3Galb- glycolipid- 135 LNnT - 04 Galb1-4GlcNAcb1-3Galb1-BSA 0 -11 3 -17 0 -71 3 -3 -28 0 neutral glycolipid- 580 LNnT - 04 Galb1-4GlcNAcb1-3Galb- 0 -28 0 -71 0 -122 0 0 -65 3 neutral glycolipid- 622 LNnT - 13-HSA Galβ1-4GlcNAcβ1-3Galβ1-HSA (LNnT) 0 -17 0 -32 0 -40 0 3 -22 0 neutral glycolipid- 85 LNnT - 14 Galb1-4GlcNAcb1-3Galb1-BSA 0 -17 5 -42 0 -59 3 6 -40 0 neutral glycolipid- 117 LNT - 05 Galb1-3GlcNAcb1-3Galb-BSA 0 -14 5 -37 0 -68 0 0 -34 0 neutral glycolipid- 53 LNT - 21 Galb1-3GlcNAcb1-3Galb-BSA 0 -26 5 -77 0 -121 0 9 -71 3 neutral glycolipid 56 LSTc - 07 Neu5Aca2-6Galb1-3GlcNAcb1-3Galb1-BSA 0 -17 0 -46 3 -51 0 6 -34 3 glycolipid 483 LSTd-03 Siaa2-3Galb1-4GalNAcb1-3Galb- 0 -15 0 -26 0 -52 3 3 -20 3 117 Table 3.3 (cont’d) glycolipid 483 LSTd-03 Siaa2-3Galb1-4GalNAcb1-3Galb- 0 -15 0 -26 0 -52 3 3 -20 3 carb-Man 489 Ma2Ma2Ma2-04 aMan(1-2)aMan(1-2)aMan(1-2) 0 -32 0 -74 0 -110 -3 -6 -51 0 carb-Man 486 Ma2Ma2Ma3(Ma3Ma6)-09 aMan(1-2)aMan(1-2)aMan(1-3)[aMan(1-3)aMan(1-6)] 0 -26 0 -71 0 -127 -3 -3 -74 6 carb-Man 473 Ma2Ma2Ma3(Ma6)-07 aMan(1-2)aMan(1-2)aMan(1-3)[aMan(1-6)] 0 -23 0 -68 0 -94 0 3 -51 -3 carb-Man 424 Ma2Ma2Ma3-04 aMan(1-2)aMan(1-2)aMan(1-3) 0 -12 0 -51 0 -108 3 3 -60 -2 carb-Man 477 Ma2Ma2Ma3-12 aMan(1-2)aMan(1-2)aMan(1-3) 0 -28 0 -80 0 -131 3 0 -76 0 carb-Man 456 Ma2Ma3(Ma6)Ma6-09 aMan(1-2)aMan(1-3)[aMan(1-6)]aMan(1-6) 0 -37 0 -85 0 -145 -3 -6 -82 3 carb-Man 466 Ma2Ma3Ma6(Ma3)-10 aMan(1-2)aMan(1-3)aMan(1-6)[aMan(1-3)] 0 -23 0 -45 0 -77 0 0 -43 -1 carb-Man 479 Ma2Ma3Ma6-15 aMan(1-2)aMan(1-3)aMan(1-6) 0 -23 0 -63 0 -99 -4 2 -53 -3 carb-Man 422 Ma2Ma6(Ma2Ma3)-03 aMan(1-2)aMan(1-6)[aMan(1-2)aMan(1-3)] 0 -17 5 -43 0 -85 3 9 -37 5 carb-Man 498 Ma2Ma6(Ma2Ma3)-07 aMan(1-2)aMan(1-6)[aMan(1-2)aMan(1-3)] 0 -17 0 -40 0 -117 0 -2 -49 0 carb-Man 501 Ma2Ma6(Ma3)-03 aMan(1-2)aMan(1-6)[aMan(1-3)] 0 -25 0 -48 0 -147 3 6 -55 3 carb-Man 447 Ma2Ma6(Ma3)-07 aMan(1-2)aMan(1-6)[aMan(1-3)] 0 -17 0 -37 0 -54 0 0 -40 3 carb-Man 474 Ma3(Ma6)Ma6(Ma2Ma3)-06 aMan(1-3)[aMan(1-6)]aMan(1-6)[aMan(1-2)aMan(1-3)] 0 -17 7 -43 0 -94 0 0 -45 0 carb-Man 480 Ma3Ma6(Ma2Ma3)-03 aMan(1-3)aMan(1-6)[aMan(1-2)aMan(1-3)] 0 -9 0 -32 0 -63 5 0 -29 3 carb-Man 485 Ma3Ma6(Ma2Ma3)-10 aMan(1-3)aMan(1-6)[aMan(1-2)aMan(1-3)] 0 -19 0 -51 0 -74 0 3 -48 -2 carb-Man 445 Ma3Ma6-05 aMan(1-3)aMan(1-6) 0 -25 0 -65 0 -100 0 -6 -60 -3 carb-Man 471 Ma3Ma6-13 aMan(1-3)aMan(1-6) 0 -8 0 -20 0 -44 0 -3 -29 0 carb-Man 465 Ma4(Ma6)-07 aMan(1-4)[aMan(1-6)] 0 -12 0 -28 0 -60 0 6 -36 0 carb-Man 468 Ma6Ma4(Ma6Ma6)-06 aMan(1-6)aMan(1-4)[aMan(1-6)aMan(1-6)] 0 -20 0 -51 0 -91 0 0 -49 3 carb-Man 462 Ma6Ma6(Ma2Ma3)-06 aMan(1-6)aMan(1-6)[aMan(1-2)aMan(1-3)] 0 -15 3 -49 0 -108 0 3 -54 0 carb-Man 482 Ma6Ma6-05 aMan(1-6)aMan(1-6) 0 -9 0 -26 0 -28 0 0 -17 -3 carb-Man 488 Ma6Ma6-09 aMan(1-6)aMan(1-6) 0 -14 0 -48 0 -66 0 3 -46 0 carb-Glc 705 maltoheptaose - 04 Glca1-4Glca1-4Glca1-4Glca1-4Glca1-4Glca1-4Glca1- 0 -8 0 -23 0 -34 -3 0 -9 0 carb-Glc 721 maltononaose - 04 Glca1-4Glca1-4Glca1-4Glca1-4Glca1-4Glca1-4Glca1- 0 3 0 -26 0 -45 0 6 -22 3 4Glca1-4Glca1- carb-Glc 678 maltononaose - 14 Glca1-4Glca1-4Glca1-4Glca1-4Glca1-4Glca1-4Glca1- 0 -8 0 -17 0 -32 0 0 -17 2 4Glca1-4Glca1- carb-Glc 712 maltooctaose - 04 Glca1-4Glca1-4Glca1-4Glca1-4Glca1-4Glca1-4Glca1- 0 -20 2 -42 0 -74 3 0 -39 3 4Glca1- carb-Glc 696 maltooctaose - 20 Glca1-4Glca1-4Glca1-4Glca1-4Glca1-4Glca1-4Glca1- 0 -6 0 -20 0 -26 3 -20 4Glca1- carb-Glc 706 maltopentaose - 06 Glca1-4Glca1-4Glca1-4Glca1-4Glca1- 0 -11 0 -20 0 -28 5 0 -15 0 carb-Glc 703 maltose - 05 Glca1-4Glca1- 0 -6 0 -31 0 -43 0 0 -23 -3 carb-Glc 679 maltose - 20 Glca1-4Glca1- 0 -17 0 -29 0 -36 3 0 -20 6 N-linked 264 Man1 - 04 Manβ1-4GlcNAcβ1-4GlcNAcβ1-BSA 0 -17 0 -40 0 -48 0 3 -28 0 N-linked 204 Man6 - I - 04 Mana1-6(Mana1-3)Mana1-6(Mana1-2Mana1-3)Manb1- 0 -14 5 -37 0 -51 0 3 -23 3 BSA N-linked 613 Man6 I - 03 Mana1-6(Mana1-3)Mana1-6(Mana1-2Mana1-3)Manb- 0 -22 3 -59 0 -102 5 0 -54 3 N-linked 394 Man8D1D3 #2 Mana1-2Mana1-6(Mana1-3)Mana1-6(Mana1-2Mana1- 0 -11 0 -30 0 -51 0 6 -32 3 2Mana1-3)Manb1-4GlcNAc-BSA N-linked 91 Man9 - 05 #2 Mana1-2Mana1-6(Mana1-2Mana1-3)Mana1-6(Mana1- 0 -10 8 -46 0 -85 3 3 -37 0 2Mana1-2Mana1-3)Manb1-4GlcNAc-BSA N-linked 503 Man9 - 08 Mana1-2Mana1-6(Mana1-2Mana1-3)Mana1-6(Mana1- 0 -29 0 -54 0 -75 -3 6 -61 0 2Mana1-2Mana1-3)Manb1-4GlcNAcb- carb-Man 504 Man-a - 20 Man-a - BSA 0 -3 0 -20 0 -33 0 3 -12 0 Carb-Man 681 Mana1-2Man Mana1-2Man-BSA 0 -9 0 -23 0 -28 0 0 -17 5 Carb-Man 694 Mana1-3Man Mana1-3Man-BSA 0 -9 0 -14 0 -23 2 0 -17 2 carb-Man 225 Mana1-6Man-a - Mana1-6Man-a - BSA 0 -12 0 -39 2 -46 0 3 -26 0 04 non-human 747 Manb2 - 04 Manb1-4Manb1-BSA 0 21 0 -63 3 0 0 non-human 753 Manb3 - 04 Manb1-4Manb1-4Manb1-BSA 0 -14 0 -43 0 -85 0 0 -48 3 non-human 804 Manb3 - 10 Manb1-4Manb1-4Man 0 3 8 -11 0 1 0 0 -8 0 non-human 733 Manb3 - 16 Manb1-4Manb1-4Manb1-BSA 0 13 20 -60 0 128 0 2 -43 -6 carb-Man 32 ManT -26 Mana1-6[Mana1-3]ManbG-BSA 0 -9 5 -48 3 -62 0 3 -37 0 Blood Group 217 MFLNH I - 11 Galb1-4GlcNAcb1-6 (Fuca1-2Galb1-3GlcNAcb1-3)Galb1- 0 -12 0 -32 0 -50 3 0 -23 0 H BSA Lewis 219 MFLNH III - 14 Galb1-4(Fuca1-3)GlcNAcb1-6 (Galb1-3GlcNAcb1- 0 -16 5 -43 0 -33 0 6 -29 0 3)Galb1-BSA Blood Group 212 MSMFLNH I - 11 Neu5Aca2-6Galb1-4GlcNAcb1-6 (Fuca1-2Galb1- 0 -8 0 -45 0 -57 3 3 -29 0 H 3GlcNAcb1-3)Galb1-BSA Lewis 215 MSMFLnNH - 09 Galb1-4(Fuca1-3)GlcNAcb1-6 (Neu5Aca2-6Galb1- 0 -8 3 -32 0 -34 0 0 -29 0 4GlcNAcb1-3)Galb1-BSA peptide 395 Muc1 BSA--hexyl-G-V-T-S-A-P-D-T-R-P-A-P-G-S-T-A-P-P-A- 0 -3 0 -20 0 -23 3 3 -11 0 amide peptide-Tn 397 Muc1-Tn15 BSA--hexyl-G-V-T-S-A-P-D-T-R-P-A-P-G-S-T(GalNAc-a)- 0 -11 2 -31 0 -40 3 3 -20 0 A-P-P-A-amide N-linked 153 NA2 - 08 Galb1-4GlcNAcb1-2Mana1-6[Galb1-4GlcNAcb1- 0 -9 3 -31 0 -54 0 3 -25 -3 2Mana1-3]Manb1-4GlcNAc -BSA N-linked 150 NA3 - 05 Galb1-4GlcNAcb1-2Mana1-6[Galb1-4GlcNAcb1- 0 -23 5 -48 0 -60 -4 5 -48 3 2(Galb1-4GlcNAcb1-4)Mana1-3]Manb1-4GlcNAc -BSA N-linked 144 NGA2 - 07 GlcNAcb1-2Mana1-6(GlcNAcb1-2Mana1-3)Manb1- 0 -15 3 -40 0 -69 -5 -6 -34 -3 4GlcNAc -BSA N-linked 141 NGA2B - 05 GlcNAcb1-2Mana1-6(GlcNAcb1-2Mana1-3)(GlcNAcb1- 0 -17 0 -35 0 -63 0 0 -31 0 4)Manb1-4GlcNAc -BSA N-linked 165 NGA3B - 06 GlcNAcb1-2Mana1-6[GlcNAcb1-2(GlcNAcb1-4)Mana1- 0 -15 3 -37 0 -54 -3 3 -26 0 3](GlcNAcb1-4)Manb1-4GlcNAc -BSA y- 385 OSM Ovine submaxillary mucin (94% STn, 4% TF, 2% Fuca1- 0 11 2 -37 0 -71 0 3 203 0 glycoprotein 2Galb1-3GalNAc) y- 372 OSM (asialo) asialo-Ovine submaxillary mucin 0 6 0 3 0 3 3 3 8 0 glycoprotein y-protein 793 PD-L1 Human PD-L1/B7-H1 Protein, His Tag 0 12 0 8 0 17 3 3 11 0 carb-type 1 136 pLNH - 07 Galb1-3GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-BSA 0 -17 5 -43 0 -40 2 3 -46 0 118 Table 3.3 (cont’d) carb-type 1 83 pLNH - 21 Galb1-3GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-BSA 0 -23 5 -69 0 -116 -3 0 -68 6 non-human 739 PNAG 10 (01010) GlcNb1-6GlcNAcb1-6GlcNb1-6GlcNAcb1-6GlcNb1- 0 -14 0 -22 0 -29 0 -2 -14 0 PNAG non-human 742 PNAG 11 (01011) GlcNb1-6GlcNAcb1-6GlcNb1-6GlcNAcb1-6GlcNAcb1- 0 -11 0 -17 0 -32 0 0 -17 3 PNAG non-human 751 PNAG 12 (01100) GlcNb1-6GlcNAcb1-6GlcNAcb1-6GlcNb1-6GlcNb1- 0 3 0 -14 0 -28 5 0 -17 0 PNAG non-human 757 PNAG 13 (01101) GlcNb1-6GlcNAcb1-6GlcNAcb1-6GlcNb1-6GlcNAcb1- 0 -6 5 0 -34 5 3 -34 0 PNAG non-human 771 PNAG 15 (01111) GlcNb1-6GlcNAcb1-6GlcNAcb1-6GlcNAcb1-6GlcNAcb1- 0 -3 0 -8 0 -31 -1 3 -14 3 PNAG non-human 762 PNAG 2 (00010) GlcNb1-6GlcNb1-6GlcNb1-6GlcNAcb1-6GlcNb1- 0 -8 0 -21 0 -16 0 0 -3 3 PNAG non-human 777 PNAG 21 (10101) GlcNAcb1-6GlcNb1-6GlcNAcb1-6GlcNb1-6GlcNAcb1- 0 -6 0 -6 0 -8 0 0 -3 3 PNAG non-human 780 PNAG 23 (10111) GlcNAcb1-6GlcNb1-6GlcNAcb1-6GlcNAcb1-6GlcNAcb1- 0 -20 0 -43 0 -148 0 3 -57 -6 PNAG non-human 783 PNAG 24 (11000) GlcNAcb1-6GlcNAcb1-6GlcNb1-6GlcNb1-6GlcNb1- 0 -17 0 -40 0 -119 0 0 -43 0 PNAG non-human 765 PNAG 26 (11010) GlcNAcb1-6GlcNAcb1-6GlcNb1-6GlcNAcb1-6GlcNb1- 0 -15 0 -12 0 -26 -3 0 -20 0 PNAG non-human 786 PNAG 28 (11100) GlcNAcb1-6GlcNAcb1-6GlcNAcb1-6GlcNb1-6GlcNb1- 0 0 0 -1 0 -28 0 2 -12 0 PNAG non-human 768 PNAG 29 (11101) GlcNAcb1-6GlcNAcb1-6GlcNAcb1-6GlcNb1-6GlcNAcb1- 0 -6 0 -11 0 -37 5 0 -15 0 PNAG non-human 789 PNAG 30 (11110) GlcNAcb1-6GlcNAcb1-6GlcNAcb1-6GlcNAcb1-6GlcNb1- 0 -6 0 -9 0 -25 0 3 -6 0 PNAG non-human 774 PNAG 6 (00110) GlcNb1-6GlcNb1-6GlcNAcb1-6GlcNAcb1-6GlcNb1- 0 -3 0 -6 0 -25 -3 0 -9 3 PNAG non-human 754 PNAG 9 (01001) GlcNb1-6GlcNAcb1-6GlcNb1-6GlcNb1-6GlcNAcb1- 0 -3 0 -20 0 -25 3 -2 -11 0 PNAG non-human 426 Rha-a - 05 Rha-a – BSA 0 -17 0 -48 0 -90 -3 6 -40 0 non-human 428 Rha-b - 05 Rha-b - BSA 0 23 8 -28 0 -46 5 3 -29 2 Blood Group 220 TFiLNO(1-2,1-2,1- Fuca1-2Galb1-3GlcNAcb1-3Galb1-4(Fuca1-3)GlcNAcb1- 0 -6 4 -25 0 -26 0 0 -8 0 H 3) - 04 6(Fuca1-2Galb1-3GlcNAcb1-3)Galb-BSA (mixed with other stuff by MS) peptide-TF 723 TSSA(S-TF)-TGH- TF-S5-N-terminus Muc4- TSSA(S-TF)-TGH-BSA 0 -15 0 -37 0 -63 0 0 -43 0 BSA peptide-TF 632 TSSA-(TF)S-TGHATPLPVTD BSA-Mal-PEG6-TSSA(Galb1-3GalNAca)STGHATPLPVTD 0 -19 0 -43 0 -66 -1 0 -54 0 peptide-TF 727 TSSASTGHA(T- TF-10-MUC4- TSSASTGHA(T-TF)PLPVTD-BSA 0 -17 0 -20 0 -48 0 0 -30 0 TF)PLPVTD-BSA peptide 629 TSSASTGHATPLPVTDBSA-Mal-PEG6-TSSASTGHATPLPVTD (MUC4 TR) 0 -23 0 -26 0 -67 0 3 -40 0 Blood Group 36 BG-H1-Lac- 20 (LNF Fuca1-2Galb1-3GlcNAcb1-3Galb1-4Glcb–APD-HSA -2 -34 4 -48 0 -110 0 0 -79 3 H I) non-human 702 chitotetraose - 03 GlcNAcb1-4GlcNAcb1-4GlcNAcb1-4bGlcNAcb1- -2 -12 5 -34 0 -57 3 3 -22 6 glycolipid 664 GD1a (Gc, Ac) Neu5Gca2-8Neu5Aca2-3(Galb1-3GlcNAcb1-4)Galb1- -2 -14 11 -48 0 -88 -1 0 -46 6 Blood Group 248 A tetra type 2-Sp - GalNAca1-3[Fuca1-2]Galb1-4GlcNAcb-Sp-BSA -3 -26 3 -34 0 -45 0 0 -29 -3 A 17 Blood Group 35 2'F-A type 2-Sp - 13 GalNAca1-3[Fuca1-2]Galb1-4[Fuca1-3]GlcNAcb-Sp-BSA -3 -12 2 -25 0 -26 3 6 -20 3 A carb-Sia 548 3'Neu5Ac-Galb- 07 Neu5Aca2-3Galb- -3 -42 5 -116 0 -158 -3 0 -110 0 non-human 763 4-Me-GlcAa1-2Xylb1-4Xylb- 4-Me-GlcAa1-2Xylb1-4Xylb- 07 BSA -3 -17 0 -23 0 -50 0 0 -26 0 carb-Sia 230 6'Neu5Gc-LacNAc- Neu5Gca2-6Galb1-4GlcNAcb-Sp-BSA -3 -20 3 -26 0 -48 0 6 -32 0 Sp - 05 Blood Group 68 BG-A1-12 GalNAca1-3(Fuca1-2)Galb1-3GlcNAcb1-linker-BSA -3 -15 3 -37 0 -66 0 0 -37 0 A y-protein 682 CRM197 ecoCRM197 from ______ -3 -6 0 6 0 13 3 0 3 3 glycolipid 661 GD1a (Kdn,Gc) Kdna2-8Neu5Gca2-3(Galb1-3GlcNAcb1-4)Galb1- -3 -22 0 -54 0 -119 -3 0 -66 0 GAG-Hep 572 Hep-Nona-GT16- GlcAb1-4Glc(6S, NS)a1-4GlcAb1-4Glc(6S, NS)a1- -3 -12 0 -37 0 -48 0 -23 0 03 4GlcAb1-4Glc(6S, NS)a1-4GlcAb1-4Glc(6S, NS)a1- 4GlcAb-Benzamide- glycolipid- 418 iGb4-07 GalNAcb1-3Gala1-3Galb- -3 -57 6 -128 0 -213 -3 3 -128 -3 neutral Lewis 200 LeA-LeX - 21 Galb1-3(Fuca1-4)GlcNAcb1-3Galb1-4(Fuca1- -3 -20 3 -31 0 -50 0 3 -34 -5 3)GlcNAcb1-3Galb1-APD-HSA Lewis 592 LeY-LeX Fuca1-2Galb1-4(Fuca1-3)GlcNAcb1-3Galb1-4(Fuca1- -3 -17 0 -40 0 -55 0 0 -32 0 3)GlcNAcb1-O-APE-HSA; Tri-fucosyl-Lewis y- heptasaccharide glycolipid 55 LSTb - 11 Galb1-3(Neu5Aca2-6)GlcNAcb1-3Galb1-BSA -3 -31 5 -65 3 -114 -3 0 -68 0 carb-Man 476 Ma2Ma3-11 aMan(1-2)aMan(1-3) -3 -17 0 -68 0 -110 0 5 -54 -3 119 Table 3.3 (cont’d) carb-Man 437 Ma3(Ma6Ma6Ma3)-05 aMan(1-3)[aMan(1-6)]aMan(1-6)[aMan(1-3)] -3 -34 8 -91 3 -139 3 6 -85 6 carb-Man 458 Ma6(Ma3)Ma6-10 aMan(1-6)[aMan(1-3)]aMan(1-6) -3 -17 0 -40 0 -59 0 0 -26 3 carb-Glc 711 maltotetraose - 18 Glca1-4Glca1-4Glca1-4Glca1- -3 -14 0 -28 0 -37 0 3 -26 0 carb-Glc 714 maltotriose - 05 Glca1-4Glca1-4Glca1- -3 -40 0 -83 0 -105 0 6 -49 0 Carb-Man 699 Mana1-4Man Mana1-4Man-BSA -3 -11 0 -23 0 -26 0 -12 non-human 799 Manb1-4Glcb1 - 14 Manb1-4Glcb1 -BSA -3 -20 0 -54 0 31 0 0 -43 -3 non-human 778 Manb5 - 04 Manb1-4Manb1-4Manb1-4Manb1-4Man -3 -3 0 -8 0 -15 -5 0 -9 -6 non-human 760 PNAG 17 (10001) GlcNAcb1-6GlcNb1-6GlcNb1-6GlcNb1-6GlcNAcb1- -3 -6 0 -14 0 -29 1 0 -14 0 PNAG non-human 772 PNAG 8 (01000) GlcNb1-6GlcNAcb1-6GlcNb1-6GlcNb1-6GlcNb1- -3 -3 0 -8 0 -13 0 3 -3 0 PNAG peptide-TF 726 TSSAS(T-TF)GHA(T- TF-6,10-MUC4- TSSAS(T-TF)GHA(T-TF)PLPVTD-BSA -3 -11 0 -23 0 -31 -1 0 -23 5 TF)PLPVTD-BSA non-human 796 Xylb2 - 13 Xylb1-4Xylb1-BSA -3 -6 0 -12 0 812 0 3 -11 0 z-control 1 Alexa Fluoro 647 Alexa Fluoro 647-BSA (25 ug/ml +100ug/mL BSA, 125 71 79 133 29 120 -15 54 151 23 79 ug/ml total) z-control 808 Alexa Fluoro 647 Alexa Fluoro 647-BSA (25 ug/ml +100ug/mL BSA, 125 11 -3 10 -14 11 -28 12 9 -22 14 end ug/ml total) z-control 634 Biotin-BSA Biotin-BSA 0 -8 0 -22 0 -29 0 0 -15 3 z-control 2 BSA Bovine serum albumin 6 -11 8 -32 8 -43 5 6 -26 0 z-control 245 BSA - C5 (Alkyne) - DF-168B-175-1 C5-alkyne-BSA 3 3 3 -8 0 -17 3 3 -6 0 10 z-control 332 BSA - C5 (Alkyne) - DF-168C-16-B5 C5-alkyne-BSA 6 6 7 -5 0 -23 2 3 -3 6 23 z-control 670 BSA (ozonyzed) bovine serum albumin treated with ozone 0 -11 3 -17 0 -40 -3 3 -12 3 z-control 813 Cy3 Cy3-BSA undoped z-control 815 Cy3 Cy3-BSA undoped 2572 2427 3871 2786 3060 2896 2465 2237 2422 2185 z-control 4 Cy3 Cy3-BSA undoped 2186 950 2094 3617 2549 3489 2192 2165 2291 1878 2053 z-control 814 Cy3 Cy3-BSA undoped 2151 1910 2529 2556 4861 1004 3022 3680 1245 z-control 29 HSA Human serum albumin (isolated from serum) 3377 3 3534 0 4196 11 4420 -40 2478 8 2294 -51 3018 2 17609 1865 -31 1609 0 z-control 689 HSA (ozonyzed) human serum albumin treated with ozone 444 -6 33 -28 0 -32 15 0 -22 -3 z-control 93 HSA (recomb) human serum albumin (recombinant) 6 -17 3 -37 0 -60 5 6 -29 0 z-control 399 human IgA use 50ug/mL + 75ug/mL BSA 3 -8 5 -12 0 -74 0 0 -23 3 z-control 400 human IgG use 50ug/mL + 75ug/mL BSA 130 15 145 9 215 3 119 125 20 215 z-control 624 Human IgG1 used 50ug/mL + 75 ug/mL BSA; next time up to 17 -3 112 -20 293 -34 12 48 -3 43 100ug/mL + 25 BSA z-control 630 Human IgG2 used 50ug/mL + 75 ug/mL BSA; next time up to 224 17 108 12 551 5 246 94 17 326 100ug/mL + 25 BSA z-control 633 Human IgG3 used 50ug/mL + 75 ug/mL BSA; next time up to 576 22 124 30 425 -9 432 62 3 381 100ug/mL + 25 BSA z-control 627 Human IgG4 use 50ug/mL + 75 ug/mL BSA 128 80 474 119 569 374 131 227 125 257 z-control 398 human IgM use 50ug/mL + 75ug/mL BSA; 14 -6 8 -11 5 -29 15 8 -6 8 z-control 404 mouse IgG use 50ug/mL + 75ug/mL BSA z-control 403 mouse IgM use 50ug/mL + 75ug/mL BSA; monoclonal IgM purified 11105 12 8260 -33 8650 3 9118 -74 22370 0 22144 -150 9452 5 195026 21579 -74 23503 14 from hybridoma z-control 405 No Data (BSA for Bovine serum albumin 3 -3 3 -17 0 -60 0 -3 -20 0 carry over) z-control 636 No Data (BSA for 29 -9 12 -20 0 -74 22 11 -29 34 carry over) z-control 407 No Data (BSA for 31 3 66 -1 82 -6 26 46 0 57 carry over) z-control 7 No Data (BSA for BSA 14 -12 34 -34 20 -40 12 20 -23 11 carry over) z-control 373 No Data (BSA for 0 -11 0 -28 0 -74 0 3 -29 -3 carry over) z-control 406 No Data (BSA for 3 -20 3 -57 0 -127 0 0 -57 -3 carry over) z-control 637 No Data (BSA for 0 -5 0 -17 0 -23 -1 0 -11 0 carry over) z-control 685 No Data (BSA for 0 -6 0 -25 0 -37 0 0 -22 0 carry over) z-control 811 No Data (BSA for BSA -3 -3 0 -13 0 -10 0 -3 -11 0 carry over) z-control 191 PEG-linker - 06 OH-(CH2)2-NH-Gly-CO-PEG7-NH-(CO)Hept-SH-Mal- 0 -6 0 -30 0 -15 0 6 -15 3 Cychex-CO-BSA z-control 402 rabbit IgG use 50ug/mL + 75ug/mL BSA 94 37 93 28 349 44 78 94 46 85 z-control 260 Triazole linker from BSA-linker-triazole from Xuefei 8 -3 5 -9 0 -8 5 3 -3 8 Xuefei - 43 z-control 776 XH Cys linker Cys-S-CH2CONHCH2CH2CONH-BSA 14 -5 5 -11 0 -20 0 0 -9 3 816 6 3 21 0 26 -1 6 14 5 11 120 REFERENCES 121 REFERENCES [1] Y. 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