muc1 glycopeptide based anti- cancer vaccines...o oh ho oh ho o o oh oh ho o oh ho o ho h 4 α-gal...

1

MUC1 glycopeptide based anti-cancer vaccines

Philadelphia, USA

August 10, 2015

Sourav Sarkar

Glycobiology World Congress

TF(T)-Antigen

O

HOOH

AcHN

OO

HOOH

HO

HO O Ser/Thr

Tn-Antigen

O

HOOH

AcHN

HO

O Ser/Thr

STn-Antigen

O

HO

AcHN

HO

O Ser/Thr

O O

CO2OH OH

HOAcHN

HO

Agrawal et al. Mol. Med. Today 1998, 4, 397-402

MUC1 on Tumor CellsTumor Associated Carbohydrate Antigens (TACAs)

The Helper T-epitope

3

KLH

STn

STn

STn

STn

STn

STn

STn

T-cell help is required to obtain high antibody titers against TACAs.

Early Vaccines

Immunoresponse specific to the protein which suppresses the response for the cancer antigen

Modern Vaccines

O S NH

O

14

NHO

O

YAFKYARHANVGRNAFELFLG NH

NH

NHAc

O

3

O

O

O

NHAc

OH

HO OH

O

O

14

14

20 Aminoacid sequence derived from the outer membrane protein of Neisseria meningitides activate helper T-cells.

Peptide required to induce T cell dependant immunoresponse with the production of IgG against Tn.

(1) Kuberan et al. Curr. Org. Chem. 2000,4,653-677; (2) Boons et al. Angewandte Chemie 2005,44,5985-5988

O

OH

HO

OH

HOO

O

OHOH

HO

O

OH

HO

OHO

OH

4

α-Gal Conjugate Vaccines

• Immunogenicity against HIV gp120 was increased >100 fold by

conjugation with α-gal

– Galili et al. J Virol 2006, 6943.

• Vaccination with MUC1 expressing α-gal epitopes elicits effective

antibody production and induces tumor-specific T-cell responses.

– Deguchi, et al. Cancer Res. 2010, 70, 5259.

• But this only works in a subset of humans and still needs

improvement

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Anti-α-Gal Mediated Targeting of Vaccines to APCs

Motal UA et al. 2007. J. Virology 81, 9131-9141

L-Rhamnose: A Promising Xenoantigen

6Oyelaran, O.; McShane, L. M.; Dodd, L.; Gildersleeve, J. C. J. Proteome Res. 2009, 8, 4301-4310

7

APC

Helper T-Epitope(YAF)

CancerAntigen

(Tn)Rha

Free Anti-Rha Antibodies

Bound Anti-Rha Antibodies

Fc portionOf AntibodyFc Receptors

Fc-FcInteraction

MHC II

The Three component Rha-Vaccine

8Sarkar, S.; Lombardo, S. A.; Herner, D. N.; Talan, R. S.; Wall, K. A.; Sucheck, S. J. J. Am. Chem. Soc., 2010, 132, 17236–17246

The Three component Rha-Vaccine

Rhamnose-vaccine Control-vaccine

Outcomes:

• Anti-Tn antibody titers was significantly higher in groups of mice previously immunized with Rha-OVA and later challenged with Rha-YAF-Tn.

• Antibodies specific to Tn and evidence of IgG production was provided by the competitive binding experiments.

• T-cell proliferation assay showed that the peptide YAF was better displayed by the MHC in the presence of anti-Rha antibodies.

Incorporating Multivalency into

Vaccine Design

• Multiple epitope vaccine facilitates better vaccine internalization.

• High antibody titers have been reported against antigen clustered vaccines and multiple antigenic peptides (MAP).

9

NH

NH

HN

O

G

CH3O

O

H

ROOR

RO

AcHN

HO

CH3O

ORO

ROOR

AcHN

H

H

NH

HN

OCH3O

O

ORRO

RO

AcHN H

HO

O

OH

O

LFLEFANRGVNAHRAYKFAYNH

NH

HN

NH

H2N

OO

O

O

O

OH3C

ORRORO

H

O

O

ORRORO

H3C

O

O

ORRO

H3C

RO

H

Multivalent Vaccine Strategies

LiposomeEpitope Clustered Vaccine

MUC1 VNTR as Helper T Epitope

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MUC1 : Large polymorphic transmembrane glycoprotein containing numerous

20-amino acid long variable number tandem repeats (VNTR)

MUC1 VNTR = PDTRPAPGSTAPPAHGVTSA

Recent discovery :

TACA

MUC1 VNTR >> MUC1 VNTRImmune response

Our Hypothesis :

MUC1 VNTR Instead of YAF TACA

TACA

Helper T Epitope Helper T Epitope

(1) Patton et. al. Biochim. Biophys. Acta 1995, 1241, 407-423. (2) Finn et. al. Biol. Chem. 2009, 390, 611-618.

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Liposome Design

(1) Sarkar et. al. Bioconjugate Chem. 2013, 24, 363-375.

(2) PCT Int. Appl. (2013), WO 2013123282 A1 20130822, Inventors: Sucheck, S. J.; Wall, K. A.; Sarkar, S.

Synthesis of L-Rha Cholesterol

Conjugate

12

HO TsO

O

O

OAc

AcO

AcO

20 21 22

2324

TsCl / Pytetraethyleneglycol, 1,4-dioxane

, CH2Cl2, BF3.OEt2

NaOMe / MeOH

16 h ; 73 % 4 h ; 89 %

1 h ; 85 %

HOO

OO

OCholesterol

O

OAc

OAc

AcO

1

AcO

18 h ; 32 %

OO

OO

CholesterolO

O

OH

HO

HO

OO

OO

Cholesterol



Ot-BuFmocHN

O

SPamO

PamO

HN

FmocHN

O

SPamO

PamO

HN

PamHN

O

SPamO

PamO

28

2930

t-BuO SS Ot-Bu

O

NHR

NHR

O

Ot-BuFmocHN

O

SHO

HO

27

25 R = H

26 R = Fmoc

25

Fmoc-OSu,

N-ethyl-morpholine, THF

3 h, 87%

(i) Zn, MeOH-HCl-H2SO4

(100:7:1), 15 min

(ii) (S)-(-)-glycidol, 5 h,

40 oC

95 % (2 steps)

PamOH, DIC, DMAP, THF

R. T., 2 h, 88%

(i) TFA, R.T., 1 h

(ii) propargyl amine, PyBOP,

HOBt, DIPEA, CH2Cl2,

4 MS, R.T., 4 h

66 % (2 steps)

(i) ACN-CH2Cl2-Et2NH

(2:2:1), R.T., 2 h

(ii) PamOH, PyBOP, HOBt,

DIPEA, CH2Cl2, 4 MS,

R.T., 4 h

80 % (2 steps)

[Pam = CH3(CH2)14CO]

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Synthesis of alkyne functionalizedPam3Cys



14

Synthesis of Pam3Cys-MUC1 VNTR-TACA

O

HN

Fmoc

O

OAPPAHGVTSA

O

HN

NH

O

CH3OH

H

O

OAcAcO

AcO

AcHN

PDTRPAPGSNH

N3 OHAPPAHGVTSA

O

HN

NH

O

CH3OH

H

O

OHHO

HO

AcHN

PDTRPAPGSNH

N3

OHAPPAHGVTSA

O

HN

NH

O

CH3OH

H

O

ORRO

RO

AcHN

PDTRPAPGSNH

N

NN

O S

O

HN

NH

O

O

OO

SPPS i. resin cleavage

ii. NaOMe, MeOH, R.T., 2 h, 100%

31 32 33

34

HN

PamHN

O

SPamO

PamO

CuSO4.5H2O, Na-ascorbate, TBTA

H2O-MeOH-THF (1:1:2), R.T., 40 h,

100 %

O O

O



Liposome Preparation Procedure

• Lipid stock solutions prepared in chloroform and mixed in glass vials (total lipid conc. 30 mM).

• Chloroform layer evaporated and lipid film dried overnight under vacuum.

• Hydrated with HEPES buffer (pH 7.4).

• Agitated at 43 °C for 40 mins.

• Subjected to 10 freeze thaw cycles.

• Extruded 21 times through lipofast basic fitted with 100 nM polycarbonate membrane.

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Characterization of Liposomes

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A. Stability, Homogeneity and Size characterization

i. Dynamic Light Scattering (DLS) measurements.

ii. Scanning Electron Microscope (SEM) imaging.

B. Surface Display of MUC1 VNTR and L-Rha epitopes

i. Human anti-MUC1 antibody binding experiment.

ii. Anti-L-Rha antibody binding experiment.

Dynamic Light Scattering

Measurements

17

A B

DLS Measurements at 1/1000 Dilution (A) Buffer (HEPES pH = 7.4) (B) liposomes



SEM Images of L-Rha Displaying

Liposomes

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SEM images at 5 kV acceleration voltage (A) liposomes under 50000 X magnification, (B) liposomes under 250000 X magnification.



Anti-L-Rha and Anti-Human MUC1

antibody binding with Liposomes

19

A.

B.

C.

Fluorescence microscope images under 60 X magnification (A) Images with control antibodies (antibodies isolated from pre-immunization serum) (B) Images with anti-rhamnose antibodies (C) Images with anti-MUC1 antibodies [1st , 2nd and 3rd images: brightfield, FITC and overlay]



Immunization Plan

Group

A1

Group

A2

Group

B1

Group

B2

Non-

Immunized

Rha-Ficoll

Immunized

Non-

Immunized

Rha-Ficoll

Immunized

Pam3Cys-MUC1-Tn liposomes

Pam3Cys-MUC1-Tn + Rha liposomes



Anti-Rha Ab titers after 4th boost with Rha-Ficoll

0 1 2 30.0

0.5

1.0

1.5

2.0Group A1(non-immunized)

Group A2(Rha-Ficoll immunized)

Group B1(non-immunized)

Group B2(Rha-Ficoll immunized)

No Antigen

Log [1/Serum Dilution]

Ab

so

rban

ce



Anti-MUC1-Tn Antibody Titers after 1st boost with Pam3Cys-

MUC1-Tn or Pam3Cys-MUC1-Tn + Rha Liposomes

1.0 1.5 2.0 2.50.0

0.5

1.0

1.5





No Antigen

No 1o Antibody


Ab

so

rban

ce



Anti-Tn Antibody Titer after 1st boost with Pam3Cys-MUC1-Tn or

Pam3Cys-MUC1-Tn + RhaLiposomes

1.0 1.5 2.0 2.50.0

0.5

1.0

1.5





No Antigen

No 1o Antibody


Ab

so

rban

ce



Competitive Binding of Anti-MUC1-Tn Antibodies with bound MUC1-Tn

in presence of free MUC1-Tn

2 3 4 50.0

0.2

0.4

0.6

0.8

1.0GroupA1(non-immunized)

GroupA2(Rha-Ficoll immunized)

GroupB1(non-immunized)

GroupB2(Rha-Ficoll immunized)

No Antigen

No 1o Antibody

No free MUC1-Tn

Log [1/Free MUC1-Tn Conc. (M)]

Ab

so

rban

ce



Binding of Anti-MUC1-Tn Antibodies to Human Leukemia

U266 cells

(A) 2nd Antibody alone,....; with mouse Anti-Human MUC1 Antibodies,____

(B) with 1/5 dilution of non-immunized mouse serum,….; with 1/5 dilution of group B2 mice serum,____



Conclusions

• A fully synthetic two component vaccine construct containing the lipopeptide adjuvant Pam3Cys, 20-amino acid MUC1 VNTR conjugated cancer antigen GalNAc-O-Thr (Tn) was synthesized.

• The synthesized two component lipo-glycopeptide was successfully incorporated into L-Rha displaying liposomes.

• The formulated liposomes were homogenous in size and were stable at 4 °C for two days.

• Binding studies with both anti-rhamnose and mouse anti-human MUC1 antibodies revealed that the rhamnose and the MUC1 VNTR glycopeptide epitopes were surface displayed on the liposomes.

• Generation of glycosylated anti-MUC1 and anti-Tn antibodies and the anti sera bound human leukemia U266 cells.

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Acknowledgements

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Advisor : Dr. Steven J. Sucheck

Committee Members : Dr. Katherine A. Wall, Dr. Kana Yamamoto, Dr. Viranga Tillekeratne

Past And Present Group Members:

Dr. Aditya Kumar Sanki, Dr. Rommel Talan, Dr. Francis Umesiri, Parijat Srivastava, Vishwanath Gaitonde, Partha Karmakar, Kevin Trabbic, Amanda Lodzinski, Steven Lombardo, Paul Brown, Danielle Herner

Friends And Family The Department of Chemistry

Elsa U. Pardee

Foundation

Funding

muc1 glycopeptide based anti- cancer vaccines...o oh ho oh ho o o oh oh ho o oh ho o ho h 4 α-gal...

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