Immunotherapies for cancer and infectious diseases

MVA TECHNOLOGY IN THE DEVELOPMENT OF HIGHLY COMPLEXED TB VACCINE CANDIDATES

TBVI Symposium Les Diablerets, 3 February 2016

Stéphane Leung-Theung-Long Geneviève Inchauspé

2

Early results in clinical trials with a MVA-TB vaccine

Efficacy results from the phase 2b based on MVA-85A did not match expectations. Many factors may have played a role:

► vaccine target: new borns are a most difficult population

► vaccine make-up: based on a single antigen

► trial design: vaccine injection too close to BCG prime

► vaccine dose, schedule and routes of administration

► vaccine platform: MVA not potent enough, not generating the right response

MVA remains a competitive platform in the TB field and requires to be further explored

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► MVA belongs to the vaccinia viruses Large ds enveloped DNA viruses (approx. 170 Kbp, 230 genes)

► Highly attenuated strain

> 570 passages in chick embryo fibroblasts cells ± 15% of DNA lost compared with VV space for transgenes lysis of infected cells increased immunogenicity without production of infective particles

► No safety concerns

Developed in Germany, in the 70’s (Dr Anton Mayr) to specially vaccinate subjects at risk for smallpox vaccination (CNS

disorders, allergy, skin diseases, etc.) 150,000 subjects vaccinated against smallpox Many trials in prophylaxis (prime-boost), HIV, Malaria, Ebola, Flu, TB… Few hundreds of patients have received MVA-based therapeutic vaccines

(review by Boukhebza et al., Human Vaccines and Immunotherapeutics, 2012)

Poxvirus MVA (Modified Vaccinia Ankara)

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2: Adult vaccines prophylactic and post-exposure

1: Pediatric vaccine prophylactic

Vaccine approaches in the fight against tuberculosis

INFECTION PHASES AND DISEASE OCCURRENCE

3: Immunotherapeutic (P3) (combination with antibiotics)

3: Therapeutic vaccines in combination with antibiotics: Increase/acceleration of cure and/or prevention of rebound or re-infection

Transgene priority

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Therapeutic vaccines

• Definition : Manipulation of the immune system in an antigen specific fashion

Positive way: enhancement of immunity: cancers, infectious diseases

Negative: attenuation of an immune response: autoimmune diseases

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Add a mechanism of action not and/or poorly used by current therapies (antivirals, antibiotics) i.e. enroll the host’s immune system to participate in viral/bacterial…. clearance of an already (actively) infected carrier

These novel immunotherapeutics should try and capture major immune features found in resolvers/controllers

Knowledge on immune correlates of control/resolution

Avoid exacerbation of diseases

Add vaccine in already treated patients (early control of replication)

Aim of therapeutic vaccines targeting chronic infectious diseases

7

Therapeutic vaccines Technologies

and Marketed Vaccines

Anti-idiotype vaccines • Made of antibodies that see other

antibodies as the antigen and bind to it • They stimulate the body to produce

antibodies against tumour cells

Dendritic cell vaccines • Absorb and present antigen to

lymphocyte for immune system activation. Patient specific vaccines

• One marketed therapeutic vaccine in US: prostate cancer, “Provenge” (DC+ PAP/GM-CSF)

Whole-cell Tumour vaccines • Solve the problem of undiscovered

antigen as they expose a large range of tumour

• Autologous: derived from patient own tumour; allogenic: prepared for any patient

pDNA vaccines

• DNA is taken up by the APCs and instructs them to produce antigens continuously

A whole virus/antigen/adjuvant vaccines • Designed to stimulate the immune

system by using individual antigens • An adjuvant is combined with the

vaccine, which help boost immune response

• VZV vaccine for the elderly: prevention of reactivation/attenuation of zoster

Viral vectors • Utilize viral vector to transfer DNA of the

tumoral or viral antigen to produce antigen proteins in APCs (poxvirus, adenovirus, …)

• One therapeutic vaccine in China: AdenoP53 in Head and Neck cancers

Source: Arrowhead, Capgemini Life Sciences Team Analysis

A diversity of therapeutic vaccine technologies have emerged and it is yet unclear which platforms will prevail

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THE CHECK POINT MODEL FOR HOST IMMUNITY AGAINST TB Immunotherapy could restore key checkpoints Schön et al., J Internal Medicine, 2013

Loss of protective T cell

responses

ie loss of functional CD4/ CD8

responses, hampered

cytolytic functions,

hampered innate immunity,

increase T-regs, pD1, IL10,

Inflammation

MVA inducing cellular-based

immunity:

Priming de novo poly-functional and

multi-antigenic

CD4+ and CD8+ T cells capable to

exhert effector

functions at site of infection

Re-boost innate immunity

Ideally once inflammation is first (in

part) controlled by antibiotics

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Active

Latent

Resuscitation

High plasticity of the MVA has allowed to generate highly complexed candidates

MVA / ACT-LAT-RES Modified Vaccinia Ankara virus (MVA)

Multi-phase antigens covering

all phases of infection (active,

resuscitation, latent)

Phases of infection

17 Mtb antigens evaluated

Transgene: Development of multiphasic vectorized TB vaccines

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Antigens

Bioinformatic • Exhaustive data basis on known

epitopes • MHC Binding/epitope

predictions (class I and II)

Biochemistry • Known structure and homologs • Prediction of Stability and

difficulty of expression

Data mining • Immunogenicity • Protection • Biological properties

Design and selection of immunogenic sequences (fusions)

Construction and ranking of fusions Construction, in vitro (expression) and in vivo (DNA vaccine) testing

Construction and in vitro ranking of the vaccine candidates – genetic stability

Testing and ranking of the vaccine candidates in in vivo efficacy experiments

Lead vaccine candidate

General Approach

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Example of antigenic Fusion Design

All fusions are blocks1+2 or blocks1+3 or blocks1+2+3 Example: 3 fusions in MVATG18377: Fusion 11: RpfB-Dhyb*-Ag85B*-TB10.4-ESAT6 Fusion 13: SS-Rv2029*-Rv2626-Rv1733*-Rv0111* Fusion 5: SS-Rv0569-Rv1813*-Rv3407-Rv3478-Rv1807-TM

* Antigen mutated and/or truncated

Antigens with described fold :

Ag85B*, Rv2029*, Rv2626, Rv0569,

RpfB-Dhyb*

Fold unknown or problematic: Rv1813*,

Rv3407, Rv3478, Rv1807, ESAT6, TB10.4

Membrane anchorage (signal

seq. added): Rv1733*, Rv0111* or

added TM

Block 1 Block 2 (optional) Block 3 (optional)

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Active (3) / Resusc. (2) Latent (4) Latent (5)

Active (2) / Latent (2)

Latent (5) Active (1) / Resusc. (2)

/ Latent (2)

Active Active Active Latent Resusc.

Latent Active Active Active Active

Latent-2A- Active

Active-X-Active

Active-X-Active

Resusc. Latent-2A-

Latent

Latent-X- Active

Active-X-Active

Active-X-Active

Resusc. Latent-X-

Latent

2A cleavage peptides + Linker

Linker

Active (1)/ Resusc. (2) / Latent (2)

Large Fusions

Individual Ag or

Short fusions

Examples of derived MVA-TB vectors

Active (3)/ Resusc. (2) Latent (2)

Active (3)/ Resusc. (2)

Active (1) / Latent (1)

Large and short fusions

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●●●●●●●●● 13

D0

Immunization (s.c, 107 pfu/mouse)

D7 D9 D14 …………

• Mouse strains: • BALB/c (H-2d) • C57Bl/6 (H-2b) • C3H/HeN (H-2k) • HLA-A2 (I-Ab, HLA-A2)

• Readouts:

• ELISpot IFNg (production by splenocytes activated in vitro by peptides) • In vivo CTL • ICS CD4/CD8; polyfunctionality • Antibodies

Mouse

Typical early assessment of immunogenicity in mice (Leung-Theung-Long et al., PLoS One, Nov 2015 + unpublished)

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Immunogenicity of MVA candidate vaccines in BALB/c mice Illustration of IFN-γ responses specific of 14 antigens (3 expression cassettes)

Broad and significant responses observed (Rv1813, Rv3407, Rv3478 and Rv1807) with the MVA including SS and TM domains in fusion sequence.

0

100

200

300

400

500

600

700

sfc/

10

6 c

ells

** * **

*

**

MVATG18377 -Rv2029-Rv2626-Rv1733-Rv0111 + RpfB-Dhyb-Ag85B-TB10.4-ESAT6 + -Rv0569-Rv1813-Rv3407-Rv3478-Rv1807-MVATG18379 -Rv2029-Rv2626-Rv1733-Rv0111 + RpfB-Dhyb-Ag85B-TB10.4-ESAT6 + Rv0569-Rv1813-Rv3407-Rv3478-Rv1807 MVATGN33.1

Medium Irr pept P1

Rv1813

P2 P3

Rv3478

P4 P1 P2 P3 P4

Rv1807 Rv0569 Rv3407

Medians of each group U Mann Whitney test * : p < 0.05 ** : p < 0.01 ----- : cut-off P: peptide pool

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IMMUNOGENICITY OF MVA CANDIDATE VACCINES IN HLA-A2 MICE ILLUSTRATION OF IFN-g RESPONSES SPECIFIC OF 4 ANTIGENS (1 EXPRESSION CASSETTE)

0

400

800

1200

1600

2000

sfc/

10

6 c

ells

MVATGN33.1 MVATG18376 -Rv2029-Rv2626-Rv1733-Rv0111 + -RpfB-Dhyb-Ag85B-TB10.4-ESAT6- + -Rv0569-Rv1813-Rv3407-Rv3478-Rv1807-

Medium Irr pept

RpfB-Dhyb

P1 P2 P3 P4

Ag85B

P1 P2 P3

TB10.4 ESAT6

** **

**

** **

**

** *

Broad and significant responses observed with the MVA in HLA-A2 transgenic mice. Following CD4 T cell depletion, significant IFNγ response was still detected for antigens such as RpfB-RpfD fusion protein.

Medians of each group U Mann Whitney test * : p < 0.05 ** : p < 0.01 ----- : cut-off P: peptide pool

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Rv2029

% o

f sp

ecific

lysis

P1 P2 P3 P40

20

40

60

*

*

*

Rv1807

% o

f sp

ecific

lysis

P1 P2 P3 P40

20

40

60

*

**

ESAT-6

% o

f sp

ecific

lysis

0

20

40

60

*

TB10.4

% o

f sp

ecific

lysis

0

20

40

60

Ag85B

% o

f sp

ecific

lysis

P1 P2 P30

20

40

60

*

RpfB-Dhyb

% o

f sp

ecific

lysis

P1 P2 P3 P40

20

40

60

* *

Rv3478

% o

f sp

ecific

lysis

P1 P2 P3 P40

20

40

60

Rv1733

% o

f sp

ecific

lysis

P1 P20

20

40

60

*

Rv2626

% o

f sp

ecific

lysis

P1 P20

20

40

60

* *

Rv1813

% o

f sp

ecific

lysis

0

20

40

60

*

Rv3407

% o

f sp

ecific

lysis

0

20

40

60

Rv0569

% o

f sp

ecific

lysis

0

20

40

60

*

Rv0111

% o

f sp

ecific

lysis

P1 P2 P3 P40

20

40

60

*

*

MVA-TB immunization can trigger antigen-specific cytotoxic activity in mice

The 14 Ag MVATG18377 candidate induced cytotoxic activity specific of 11 out of 14 Mtb antigens following two injections in BALB/c mice.

in vivo CTL assay

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Immunogenicity studies in non-human primates (naïve animals)

PBMC

• Substantial MVA-TB immunogenicity demonstrated in primates. • Multiple antigens /multiple epitopes targeted.

MVATG18377 (14 Ag) 108 pfu, i.m.

MVATG18377

Weeks

0 8 18 2 10 20 27 29

MVATG18377 MVATG18377

31

†

sp

ots

/10

6 c

ell

s

0 2 10 18 20 27 29 31 0 2 10 18 20 27 29 31 0 2 10 18 20 27 29 31

200

400

600

800

1000

1200

1400

1600

1800

2000

ESAT-6

Rv3478

Rv3407

Rv2626

Rv2029

Ag85B

Rv1813

Rv1807

Rv1733

Rv0569

TB10.4

Rv0111

RpfB-Dhyb

R632 R634 R818

Weeks

Primate ID

†, data not available

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VACCINES ANTIGENS #

fusions #

Ag

MVATG18639 Rv2626/Ag85B - CFP10/ESAT6 - TB10.4/Rv0287 - RpfB/D - Rv1813/Rv3407 5 10

MVATG18598 Rv2626/2A/Ag85B - CFP10/ESAT6 - TB10.4/Rv0287 - RpfB/D - Rv1813/2A/Rv3407 5 10

MVATG18633 Ag85B - ESAT6 - RpfB/D - Rv2626 - Rv1813 5 6

MVATG18690 RpfB/D/Ag85B/TB10.4/ESAT6 - Rv2626/Rv3407 2 7

MVATG18692 RpfB/D/Ag85B/TB10.4/ESAT6 - Rv3478/2A/Rv1733 2 7

MVATG18827 Rv2029/TB10.4/ESAT6/Rv0111 - SS-RpfB/D 2 6

Active – Resuscitation – Latent Heterodimeric partners

SS: signal sequence 2A: auto-cleavage peptide

MVA-TB genetically stable ie fit for manufacturing

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Therapeutic efficacy studies in mice : Reduction/ Prevention of Rebound and/or Acceleration of control

2

4

6

8

10 20 5 15

CFU

per

lun

g (l

og 1

0)

25 Time after infection

(weeks)

Antibiotics

+ antibiotics

MVA n x injections

Antibiotics + MVA-TB vaccines

Mtb (H37Rv)

Initial experiment performed with one MVA-TB genetically stable candidate (10 antigens)

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-4

H37Rv challenge

2.5 log10 cfu

Mouse BALB/c

0

RHZ 5 days/week, oral gavage

Weeks 23 11

RHZ: Rifampin, Isoniazid, Pyrazinamide

• Mice: BALB/c • RHZ-treated group as control • MVATG18598 prototype (10 Ag, 107 pfu/s.c. injection)

MVA-TB therapeutic efficacy study in combination with drugs: control of relapse TB Alliance and Dr Eric Nuermberger (Johns Hopkins University) support

8

CFU evaluation in lung

MVA schedule 1

MVA schedule 3

MVA schedule 2

3

21

Median lung CFU count at Week 8 : shortening

• Safety : Bacterial burden and histology : MVA did not impair RHZ therapy efficacy (no negative interference). Multiple MVA injections did not worsen lung inflammation.

• Trend to reduced bacterial burden in the MVA-TB groups compared with RHZ group

(not statistically significant)

Week 8

log

10

M.

tub

erc

ulo

sis

CF

U

Untr

eate

dRHZ

RHZ +

MVA

3x

RHZ +

MVA

5x

RHZ +

MVA

7x

1.0

1.5

2.0

2.56.0

6.5

7.0

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Groups Mice relapsing CFU

[median (IQR)] Number Percentage

RHZ 10 / 12 83% 2.9 (0.5-3.7)

RHZ / MVA schedule 1 (3x) 6 / 12 50% 0.2 (0.0-3.3)

RHZ / MVA schedule 2 (5x) 8 / 12 67% 2.5 (0.0-3.8)

RHZ / MVA schedule 3 (7x) 7 / 12 58% 2.8 (0.0-3.6)

CFU count at Week 23: relapse

• MVA schedule 1 (3x) has the most robust impact on relapse – 50% of mice do not relapse while 83% do in the control group

• Bacterial burden is lowered (median 3 logs lower)

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Ongoing

● Therapeutic mouse efficacy studies with all 6 genetically stable candidates MVA-TB in mice

● Different kinetics; Different routes; Different positionings (during/post antibiotic treatment)

● Prophylactic heterologous prime-boost in non-human primates including a multi-antigen MVA-TB vaccine

● Collaboration with GSK and AERAS

● Supported by AERAS

● Organisation of potential therapeutic efficacy in Guinea Pig with MVA-TB candidates with Chinese National Institutes for Food and Drug Control (NIFDC)

● Pre-requisite for a development in China

● Development of a cell-line based manufacturing process for large scale production of MVAs-TB

● Transgene/Emergent BioSolutions + NIH grant

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Acknowledgements

TRANSGENE

Lyon, France

•Marie Gouanvic • Charles-Antoine Coupet •Aurélie Ray • Clément Levin •Audrey Glaize • Cécile Bény • Emmanuel Tupin • Stéphane Leung-Theung-Long •Geneviève Inchauspé

•Romain Micol •Valentina Ivanova-Segura • Ludovic Dendane

Strasbourg, France •Martine Marigliano

• Jean-Baptiste Marchand •Nathalie Silvestre • Thierry Menguy • Joan Foloppe •Doris Schmitt • Chantal Hoffmann •Murielle Klein •Véronique Koerper • Sophie Steinbach • Fabrice Le Pogam • Patricia Kleinpeter •Dominique Villeval • Sophie Jallat •Annick Hoh

•Anthony Cristillo •Maria Cecilia Huaman •Philip Seegren •Priyanka Dhopeshwarkar

NIH support through grant awarded to Emergent BioSolutions/Transgene subcontractor

• Eric Nuermberger • Paul Converse • Sandeep Tyagi

• Tom Evans •Barry Walker •Nathalie Cadieux

•William Reiley