Neue Möglichkeiten der Immuntherapie Martin Bachmann, Immunologie Bern. Use of virus-like particles for therapeutic vaccination

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1 Neue Möglichkeiten der Immuntherapie Martin Bachmann, Immunologie Bern Use of virus-like particles for therapeutic vaccination against Regulation addiction of anti-viral and B other cell responses chronic diseases 1

2 History of Vaccinology specificity and safety live virulent live attenuated/ inactivated recombinant Variolation (pre-1700) Vaccinia (1796) Europe: 1800 (India, China: 1000) 2 Rabies (1885) HBV subunit (1982) Cholera (1896) Conjugate (Hib) (1987) Allergy (1911) Genetically-modified pertussis (1995) Tuberculosis (1921) HPV Diphtheria (1923) Tetanus (1926) Yellow Fever (1935) Polio (1952) Measles (1958) Mumps (1967) Rubella (1970) year 1900 Source: Bachmann and Dyer, Nature Reviews Drug Discovery, 3, (2004), WHO 2000

3 Risk Factors in the 21 st Century Growing Burden of Ageing Societies 3

4 Risk Factors in the 21st Century Poor compliace is a general problem Hypertension Ø 40%-60% Patienten non-compliant within 1 year Hypercholesterinema Ø 45% non-compliant within 1 year Ø 60% non-compliant within 18 months Asthma Ø 50% non-compliant within year 4 Sources: Am J Med 1997; 102: Am J Man Care 1997; 3:s Ann Allergy Asthma Immunol 1997;79:

5 Risk Factors in the 21 st Century The Treatment of Risk Factors Drugs treating risk factors have to be: Conveniant, to maximize Compliance à Vaccines are conveniant since they have a longlasting effect 5

6 Hypertension Vaccine Modulate the Action of Angiotensin II (Renin inhibitors) ACE inhibitors AIIR blocker 6

7 Hypertension Remaining Problems Compliance: An estimated 50-80% do not take all of the prescribed medication Morning pressor surge: Pharmacokinetic profile of current inhibitors of the RAS may limit efficacy in early morning hours Vaccine targeting angiotensin II may address these two issues, due to long-lived antibody responses 7 Sources: NHS, (2004); AJH 17: (2004)

8 Antigen Organization drives B cell responses Repeptitivenes: a pathogen associated geometric pattern Organization: high low absent Antibody response Induction of auto-antibodies Science 262,

9 Role of epitope densitiy and organisation Why is epitope reptitiveness so important Our immune system exploits structural features for the discrimination of self and foreign. Viruses have small genomes and therefore consist of a small number of proteins. Viruses cannot help but have to express highly repetitive and highly ordered arrays of antigens on their surface 9 à Antigen Organisation is a geometric PAMP (Pathogen associated molecular pattern)

10 Active immunization: Mechanism Bachmann & Dyer Nat Rev Drug Discov. 2004

11 No IgG antibodies in the absence of T help T help provided by the carrier is required for antibody production à Hence no boosting of the response by endogenous cytokine Bachmann & Dyer Nat Rev Drug Discov. 2004

12 Carrier-specific help bypasses Th cell tolerance Bachmann & Dyer Nat Rev Drug Discov. 2004

13 Vaccine Design Antigen Linker (SMPH) Carrier Cys O N O O N O O O N O Lys 13 Bachmann&Jennnings Nature reviews Immunology 10: Non-replicating Contains RNA as natural TLR7/8 ligand Very stable Economic production in bacteria 2 g/l bacterial culture of GMP grade material

14 High Antibody Titers in Mice and Men Mouse Human Antibody titer (OD 50 ) Factor 100 Peptide Peptide Peptide- Peptide- Protein Carrier VLP Antibody titer (Endpoint) µg i.m. 50µg i.m. 10µg s.c. 50µg s.c. Adjuvants none Alum Alum none 14 J Allergy Clin Immunol.117:1470

15 Vaccine Design Vaccine Design CGGDRVYIHPF Angiotensin II 30 nm Qbeta virus-like particle CYT006-AngQb 15

16 Strong antibody responses against Angiotensin II Efficient Antibody Induction in SHR Rats ELISA titer (OD50%) 40'000 30'000 20'000 10'000 CYT006-AngQb 0 d7 d14 d21 d28 Days after immunization 16 J Hypertens 25:63-72

17 Reduction of blood pressure in rats SHR Rats: BP Reading by Telemetry Systolic BP (mm Hg) Titer CYT006-AngQb BP VLP BP CYT006-AngQb p < 0.05 * Anti Ang II titer (OD50%) Days 0 17 J Hypertens 25:63-72

18 Study Outline (1) A Placebo-controlled Phase IIa Clinical Trial Indication Mild to moderate hypertension (systolic mmHg; diastolic mmHg) Design Double-blind, randomized, placebo-controlled, sequential two-dose comparison study in 72 patients Study period: 4 months plus 8 months safety follow-up Endpoints Safety, tolerability, and exploratory efficacy (change from baseline blood pressure) 18

19 Study Outline (2) Two Dose Levels vs. Placebo months N=24 N=12 100µg AngQb placebo safety follow up 24h ambulatory blood pressure measurement N=24 N=12 300µg AngQb placebo safety follow up 19 Injection The Lancet, 371 (2008) The Lancet, 371 (2008)

20 Evidence for Affinity Maturation Antibody Responses in Study 01 ELISA titer µg AngQb 100 µg AngQb Placebo weeks 20

21 Day-time blood pressure Mean Change of ABP: 300 µg vs. Placebo Day-time Blood Pressure -9.0 / -4.0 mm Hg p=0.015 / p=0.064 at 8am -25 / -13 mm Hg p< / p= The Lancet, 371 (2008)

22 24 Hours Measurement Ambulatory blood pressure (mmhg) run-in Time (24-hour) 22 placebo (SBP/DBP), n=12 300µg AngQb (SBP/DBP), n=21 The Lancet, 371 (2008)

23 Conclusion Immunization against angiotensin II can significantly reduce blood-pressure in hypertensive individuals Regimen and dosing, however, needs optimization 23

24 Vaccination against Parkinson`s disease Martin Bachmann, Jenner Institute, University of Oxford, UK Use of virus-like particles for therapeutic vaccination against Regulation addiction of anti-viral and B other cell responses chronic diseases

25 Parkinson s Disease (PD) Neurodegenerative disease Loss of dopaminergic neurons Leading to motor control disorder Characterised by Lewy bodies = protein aggregates in the brain 25 Mis-folded / aggregated disease-specific proteins common feature of neurodegenerative diseases PrP, Kreuzfeld-Jakob disease, etc Amyloid-β (Aβ), tau - Alzheimer s disease (AD) α-syn Parkinson s disease (PD) Huntigtons, ALS

26 α-synuclein α-synuclein is expressed in various tissues and adopts a monomeric but largely unfolded structure (J Biol Chem May 4;287(19): ). α-synuclein undergoes heavy posttranslational modification, including phosphorylation, nitration and sometimes aggregation (Nat Rev Neurosci Jan;14(1):38-48) Mild overexpression (2-fold) but also single point mutations may cause familial Parkinson`s disease (Lancet 364(9440): ; Science 276: 2045) Aggregated α-synuclein is thought to be central for Parkinson`s disease development 26

27 Parkinson s Disease (PD) t c g M Jucker and LC Walker, (2013) Self-propagation of pathogenic protein aggregates in neurodegenerative diseases Nature; 501(7465):45-51 t 27

28 Antibodies may stop propagation Small aggregates / oligomers neutralised by antibodies Protection of degenerative pathology Hope for approach to stop progression and spread of proteo-pathological disorders 28

29 Antibodies protect against PD in mouse model Passive Immunization Reduces Behavioral and Neuropathological Deficits in an Alpha-Synuclein Transgenic Model of Lewy Body Disease Eliezer Masliah 1,2 *, Edward Rockenstein 1, Michael Mante 1, Leslie Crews 2, Brian Spencer 1, Anthony Adame 1, Christina Patrick 1, Margarita Trejo 1, Kiren Ubhi 1, Troy T. Rohn 3, Sarah Mueller-Steiner 4, Peter Seubert 4, Robin Barbour 4, Lisa McConlogue 4, Manuel Buttini 4, Dora Games 4, Dale Schenk 4 1 Department of Neurosciences, University of California San Diego, La Jolla, California, United States of America, 2 Department of Pathology, University of California San Diego, La Jolla, California, United States of America, 3 Department of Biology, Boise State University, Boise, Idaho, United States of America, 4 ELAN Pharmaceuticals, South San Francisco, California, United States of America PLoS ONE 1 April 2011 Volume 6 Issue 4 e

30 Active versus passive vaccination mab therapy is highly effective, but à cost-intensive à many patients develop anti-antibody responses which neutralize the therapeutic potential of mabs Increasingly older populations pose a pharmacoeconomic threat to health- care systems resulting in downward pressure on drug-costs Innovative and cost-effective therapies are needed Vaccination addresses these issues Drug Discov Today Nov;11(21-22): Nat Rev Drug Discov Jan;3(1):81-8 Vaccine Apr 3;31(14):

31 Goal 1) Induction of antibodies recognizing oligomeric and aggregated α-synuclein 2) Oligomers should be recognized preferentially over monomeric α-synuclein (à even though native α-synuclein is intracellular and not obviously accessible to antibodies). 3) Specific T cells should be avoided (see ELAN and their AD vaccine) à no strong adjuvants and peptide epitopes 31

32 Vaccine Design Antigen Linker (SMPH) Carrier Cys O N O O N O O O N O Lys 32 Bachmann&Jennnings Nature reviews Immunology 10: Non-replicating Contains RNA as natural TLR7/8 ligand Very stable Economic production in bacteria 2 g/l bacterial culture of GMP grade material

33 CAD106: A Vaccine for Alzheimer`s now in Phase III based on the same principle Aβ 1-42 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA Aβ 1-6 DAEFRHGGC AβQb Epitopes per Qβ monomer Qβ QβAβ

34 Vaccine-Design: Epitope N- C- terminal terminal 140 Amphipathic region NAC domain Acidic tail N-terminal mab target C-terminal - 3 peptides chosen for vaccine design: MDVFMKGL, KNEEGAPQ, EGYQDYEPEA - Sequence comprised between 8-10 AA à essentially no T cell epitopes - C-term and N-term of proteins generally accessible in aggregates

35 Good response with all peptides N- C- terminal terminal 140 Amphipathic region NAC domain Acidic tail Figure 2. Peptide-specific antibody responses induced by the three vaccines and a biosimilar of CAD106 (a vaccine against 35 Alzheimer s disease currently developed by Novartis) (n=4 mice/group). N-terminal mab target C-terminal

36 Recognition of aggregated α-synuclein (human brain tissue) IgG from immunised mice (VLP- pep6de 3) day 70 Figure 4. Immunohistochemistry on paraffin-embedded post-mortem brain tissue from a PD patient, Braak stage 4. Purified IgGs (1 mg/ml) used at 1:1000. Region sampled, substantia nigra. Scale bar, 50 µm. Lewy bodies (white arrows), Lewy neurites (black 36 arrows), Pale body (white arrowhead), intracellular aggregation (white dashed arrows).

37 Recognition of aggregated α-synuclein (human brain tissue) IgG from immunised mice (VLP- pep6de 1) day 70 IgG from immunised mice (VLP- pep6de 2) day 70 Figure 3. Immunohistochemistry on paraffin-embedded post-mortem brain tissue from a PD patient, Braak stage 4. Purified IgGs (1 mg/ml) used at 1:1000. Region sampled, substantia nigra. Scale bar, 50 µm. Lewy bodies (white arrows), intracellular aggregation 37 (white dashed arrows).

38 hα syn SNCS hα syn Snca / hα syn hα syn SNCS Snca / Snca / Snca / Figure 2 SNCA OVX hα syn SNCA S hα syn hα syn SNCA S hα syn Figure Figure 2 22 Figure SNpc +AC Figure 2 SNCA-OVX LB m 20 m 20 m 20 m B CA3 Snca-/-sg [DA] (% of Snca / ) CA3 CA3 sg SNCA-OVX E 18 months SNCA OVX SNCA OVX 0.5 s Syn 1 CPu F ' months' ** F H D LB509 PK NAc Snca-/- Snca-/-SNCA-OVX SNCA-OVXhα-syn hα-syn 18 months 18 months G G Snca ( / ) SNCA OVX h -syn 0.5!" F' 18 months 18 months 1818 months months G G **G *** SNCA- OVX Snca-/- H C C F + PK SNCA-OVX h -syn D H H SNpc SNpc Travers H H H spsnpc spspe' Snca ( / ) SNpc 18' SNCA OVX months' SNpc SNpr Snca / Snca / E NAc NAc D SNCA OVX CPu CPu SNCA OVX SNCA OVX G0.5 sd0.5cs D D G C57Bl6 E C' SNCA-OVXhα-syn Snca-/-SNCA-OVX SNCAhα-syn OVX Snca / Snca / 0.5!"0.5!" Snca ( / ) 3 months 3 months SNCA OVX 3 months Snca / 3 months 3 months Snca-/α syn E α syn ** SNCA OVX *** Snca-/- E' [DA] (% of Snca / ) hα syn 3 months B'B hα-syn B 20 m LB509hα-syn LB509 LB509 Snca / SNCA OVX hα syn C57Bl6 Snca / SNCA OVX hα syn C57Bl6 hα-syn Snca / E SNCA OVX hα syn SNCA-OVX C57Bl6 Snca-/Snca-/Syn 1 E F F Syn 1 Syn 1 F E F PK ++ PK Thioflavine S PKE LB509 PK PK SNCA-OVX + PK hα-syn F F LB509 F E 12 mth 18 mth C BC 18 mth D C D 20SNCA-OVX m 20 m hα-syn hα-syn 20 m SNCA-OVX Snca-/hα-syn Snca-/SNCA-OVX SNCA-OVX SNCA-OVX Snca-/hα-syn Snca-/Snca-/hα-syn Snca-/SNCA-OVX hα-syn Snca / SNCA OVX hα syn C57Bl6 Snca / SNCA OVX hα syn C57Bl6 Syn 1 ein/ THα syn Eα syn α synuclein TH α synuclein/ F THSyn 1F PK PK + PK + PK α syn E Snca ( / ) 3 months Snca-/- B FD BD D DF Snca / Snca / Agure 1EC A 3 C AFigure C C E 20 m sp SNpr Figure 2 AB B Figure 2 SNpc SNpctosp sptest the sp atients. BWeB will Mouse use thismodel state-of-the art model Figure 2 SNpr SNprdisease SNpr of Parkinson`s A B A CA3 sg C D spsnpr sp Figure 2 + PK SNpr vaccination approach targeting α-synuclein. CA3 CA3 CA3 D C sg sg sg sg SNpc SNpc sp SNpc SNpr SNpc SNpr SNpr F D Snca-/- Foot'slips' hα syn E F mice show expression of αf the SNCA-OVX mouse model. SNCA-OVX D C D F E C Cns of the SNc (A), have twice Dthe level of endogenous α-synuclein SNpr sg sg 40 spsnpr sp CA3 CA3 CA3 sg sg CA3 CA3 sg 18 months months 18 months months D Snca / 3 months months 3 months months EE'FF C at 18 FF %12 months of age E Fneurons Eofmth F F' compared SNpc 20SNc 20 F EtoEthe control transgenic F mth th SNCA-OVX Snca-/Snca-/SNCA-OVX SNCA-OVX0hα-syn hα-syn Snca-/Snca-/-SNCA-OVX hα-syn F Snca-/-SNCA-OVX SNCA-OVXhα-syn hα-syn hα-syn 0 Snca-/E F sp f 0E α-synuclein (hα syn) (C). (D) SNCA-OVX mice exhibit reduced evoked Snca ( / ) CPu NAc SNpr Snca ( / ) CPu NAc Snca-/CPu NAc SNCA-OVX Snca-/Snca-/Snca-/- Snca-/Snca-/SNCA-OVX hα-syn SNCA-OVXhα-syn hα-syn Snca-/SNCA-OVX hα-syn Snca-/Snca-/SNCA OVX SNCA OVX Regio( *** dorsal striatum. Regio( Regio( months of age in the (E) At 18 months of age SNCA-OVX 38 5!" 0.5!" Janezic etca3 al., PNAS, 2013 sg 0.5 to s 0.5 s ility remain on the rotarod. (F) SNCA-OVX at 18 human αlb509 5sH 3 mth mth immunofluorescence I accelerating erization of α-syn transgenic mice. (A and B) 18 Double labeling for α-syn mice and TH confirms 18 mth Snca ( / ) Syn 1 + PKin 3-mo-old SNCA-OVX and hα-syn animals. (Scale bars, 200 mmunoreactive dopamine neurons of the PK (A) SNc and (B) VTA

39 39 α-syn is recognized in tg-mouse model

40 Goal 1) Induction of antibodies recognizing oligomeric and aggregated α-synuclein 2) Oligomers should be recognized preferentially over monomeric α-synuclein (à even though native α-synuclein is intracellular and not obviously accessible to antibodies). 3) Specific T cells should be avoided (see ELAN and their AD vaccine) à no strong adjuvants and peptide epitopes 40

41 Soluble proteins versus aggregates Bivalent Binding à avidity Monovalent Binding à affinity à Low affinity antibodies recognize aggregates but not soluble proteins 41

42 α-synuclein specific Abs have low affinity Affinity of immune sera Day 14 Day 70 Peptide nm 190 nm Peptide 3 30 nm 35 nm 42

43 Efficacy Experiment Ongoing Vaccination of young mice (6 week-old) VLP-peptide (20 μg), s.c. administra0on (every 2 weeks for 1 month, then monthly) (1) (2) (3) (4) (5) (6) (12) (18) Experimental day (month) 6 wks 10 wks 14 wks (3.5 mo.) 12 mo. 18 mo. Age of animal Study 1 Study 2 1. Biochemistry: assess α-synuclein protein burden (ELISA, WB) 2. a. Dopamine neurotransmission (FCV) 2. b. Behavioural studies Controls: - SNCA-OVX mice: administration of non-coupled VLPs 43

44 Acknowledgements Cytos Biotechnology, Zürich Gunther Spohn Patrik Maurer Gary Jennings University Hospital Zürich Franziska Zabel Antonia Fettelschoss Thomas Kündig University of Oxford Aadil El-Turabi Marika Doucet Richard Wade-Martins BRSC Riga Paul Pumpens Andris Zeltins Andris Dishlers 44 Hypertension Robert Sabat, Berlin Frank Wagner, Berlin Jürg Nussberger, Lausanne