Altered Peptide Ligands in the immunotherapy of Multiple Sclerosis: the role of citrullination

Deraos Georgios MSc, PhD ELDRUG S.A.

Patras, Greece

Multiple Sclerosis

Epidemiology of MS

Age onset 20 – 50 years old

Women are 2 times more likely to develop MS

500,000 cases in US

Over 2.5 million people around the world

More prevalent whites of northern European ancestry

Genetic Influences

• Vision problems

• Numbness

• Difficulty walking

• Fatigue, Depression

• Coordination and Balance problems,

• Pain

• Spasticity

Symptoms of MS

http://www.chumsweb.org/types.htm

MS Types

Relapsing-remitting MS (RRMS)Attacks followed by partial or completerecovery Symptoms may be inactive for months or years

•Secondary-progressive MS (SPMS)Occasional relapses but symptoms remain constant, no remissionProgressive disability late in disease course

Primary-progressive MS (PPMS)Affects approximately 10% of MS population. Slow onset but continuous worsening condition

Benign MS. small attacks followed by complete recovery.

Immunopathogenesis of MS

Immunodominant epitopes in MS

MBP 83-99 ENPVVHFFKNIVTPRTP

MOG 35-55 MEVGWYRSPFSRVVHLYRNGK

PLP 139-151 HCLGKWLGHPDKF

HLA-DR2 – PEPTIDE COCMPLEX

Val 87

Phe 90

Lys 91

Crystal Structure of HLA-DR2 (DRA*0101, DRB1*1501) Complexed with a Peptide from Human Myelin Basic Protein kathine J. Smith, Jason Pyrdol, Laurent Gauthier, Don C Wiley, Kai W. Wucherpfenning.

Citrullination in Multiple Sclerosis

Medications and MS

Therapies Administration Class

Avonex IM 1x a week Interferon beta-1a

Betaseron SC, every other day Interferon beta-1b

Copaxone SC 1x a day Glatiramer acetate

Rebif SC 3x a week Interferon beta-1a

Gilenya Oral capsule 1x day Fingolimod

Tysabri IV Monthly at Center Natalizumab

1 Myelin Peptide Analogues2 MBP83-99(Tyr91)

3 MBP83-99(Phe91)

4 MBP83-99(Glu91)

5 Cyclo(83-99)MBP83-99(Ala91, 96)

6 MBP87-99(Cit91, 97)

7 CycloMBP87-99(Cit91, 97)

8 MBP87-99(Arg91Ala96)

9 CycloMBP87-99(Arg91Ala96)

10 MBP87-99(Ala91,96)

11 CycloMBP87-99(Ala91,96)

12 MOG35-55 rat mouse

13 MOG35-55 Human

14 CyclicMOG35-55 rat mouse

15 CyclicMOG35-55 human

Synthesis of peptide analogues Solid Phase peptide Synthesis

Conversion of the citrullinated analogues

HPLC analysis of the citrullinated analogues

Biological evaluation

Citrullinated analoguesMBP87-99(Cit91,97)cycloMBP87-99(Cit91,97)

PBMCs of MS patientsCytokine secretion

Mannan conjugated peptides• MBP83-99(Tyr91)• MBP83-99(Phe91)• MBP83-99(Arg91)• MBP83-99(Glu91)

EAE experimentIn SJL/J miceCytokine secretion Cross reaction antibodies assays

Department of Medical school, University of Patras,

Burnet Institute, Australia

Deraos et al. J. Med. Chem. 2008 Dec 25;51(24):7834-42. .

Citrullinated peptides in PBMCs from MS patients

Katsara et al , Mol Immiunol2008 Aug;45(13):3661-70.

Manosylated Peptideanalogs

IFN-γ IL-4 IL-10 Proliferation Abs Cross reaction MBP

MBP83-99 ++ --     +++ +++

MBP83-99(A91) -- ++     + +

MBP83-99(E91) -- ++     +++ +

MBP83-99(F91) ++ ++ ++ +++ +/- -

MBP83-99(Y91) -- ++ ++ +/- +++ -

MBP83-99(R91,A96) -- ++     +++ +++

Manosylation of MBP83-99 peptide analogues

Katsara et al , Mol Immiunol2008 Aug;45(13):3661-70.

Manosylated Peptideanalogs

IFN-γ IL-4 IL-10 Proliferation Abs Cross reaction MBP

MBP83-99 ++ --     +++ +++

MBP83-99(A91) -- ++     + +

MBP83-99(E91) -- ++     +++ +

MBP83-99(F91) ++ ++ ++ +++ +/- -

MBP83-99(Y91) -- ++ ++ +/- +++ -

MBP83-99(R91,A96) -- ++     +++ +++

Manosylation of MBP83-99 peptide analogues

Katsara et al , Mol Immiunol2008 Aug;45(13):3661-70.

Manosylated Peptideanalogs

IFN-γ IL-4 IL-10 Proliferation Abs Cross reaction MBP

MBP83-99 ++ --     +++ +++

MBP83-99(A91) -- ++     + +

MBP83-99(E91) -- ++     +++ +

MBP83-99(F91) ++ ++ ++ +++ +/- -

MBP83-99(Y91) -- ++ ++ +/- +++ -

MBP83-99(R91,A96) -- ++     +++ +++

Manosylation of MBP83-99 peptide analogues

Katsara et al , Mol Immiunol2008 Aug;45(13):3661-70.

Manosylated Peptideanalogs

IFN-γ IL-4 IL-10 Proliferation Abs Cross reaction MBP

MBP83-99 ++ --     +++ +++

MBP83-99(A91) -- ++     + +

MBP83-99(E91) -- ++     +++ +

MBP83-99(F91) ++ ++ ++ +++ +/- -

MBP83-99(Y91) -- ++ ++ +/- +++ -

MBP83-99(R91,A96) -- ++     +++ +++

Manosylation of MBP83-99 peptide analogues

Katsara et al , Mol Immiunol2008 Aug;45(13):3661-70.

Manosylated Peptideanalogs

IFN-γ IL-4 IL-10 Proliferation Abs Cross reaction MBP

MBP83-99 ++ --     +++ +++

MBP83-99(A91) -- ++     + +

MBP83-99(E91) -- ++     +++ +

MBP83-99(F91) ++ ++ ++ +++ +/- -

MBP83-99(Y91) -- ++ ++ +/- +++ -

MBP83-99(R91,A96) -- ++     +++ +++

Manosylation of MBP83-99 peptide analogues

Katsara et al , Mol Immiunol2008 Aug;45(13):3661-70.

Manosylated Peptideanalogs

IFN-γ IL-4 IL-10 Proliferation Abs Cross reaction MBP

MBP83-99 ++ --     +++ +++

MBP83-99(A91) -- ++     + +

MBP83-99(E91) -- ++     +++ +

MBP83-99(F91) ++ ++ ++ +++ +/- -

MBP83-99(Y91) -- ++ ++ +/- +++ -

MBP83-99(R91,A96) -- ++     +++ +++

Manosylation of MBP83-99 peptide analogues

Katsara et al , Mol Immiunol2008 Aug;45(13):3661-70.

Manosylated Peptideanalogs

IFN-γ IL-4 IL-10 Proliferation Abs Cross reaction MBP

MBP83-99 ++ --     +++ +++

MBP83-99(A91) -- ++     + +

MBP83-99(E91) -- ++     +++ +

MBP83-99(F91) ++ ++ ++ +++ +/- -

MBP83-99(Y91) -- ++ ++ +/- +++ -

MBP83-99(R91,A96) -- ++     +++ +++

Manosylation of MBP83-99 peptide analogues

Conclusions

➢Citrullination is essential for antigenic recognition of self epitopes by T cells and

may be a new strategy of citrullinated inhibitors like drugs in the future

➢Mutations to the native MBP83–99 peptide were able to induce IL-4 responses,

although IFN-γ responses were still present.

➢ However, conjugating MBP83–99 peptide analogs to mannan was able to further

divert the immune response from Th1 to Th2, demonstrating the importance of

mannan as conjugates.

➢ APLs linear or cyclic can be used as immunomodulator agents in the MS.

Although linear peptides have the disadvantage of being degradated easily from

proteolytic enzymes and can not be used as oral administered drugs.

ELDRUG S.A COMPANY

PATRAS GREECE