IMM-529 for the prevention and treatment of Clostridium difficile - - PowerPoint PPT Presentation

IMM-529 for the prevention and treatment of Clostridium difficile infections

Clostridium difficile

http://www.nlm.nih.gov/medlineplus/images/clostridiumdifficile.jpg

• Gram positive, spore forming anaerobe
• Major nosocomial pathogen
• Pathogenic C. difficile strains produce toxins that

cause diarrhoea and mediate gut damage

• Resistant to most clinical antibiotics

Clostridium difficile

• Leading cause of infectious antibiotic-associated

diarrhoea in hospitals worldwide

• C. difficile only colonises the gut if the normal

microbiota is disrupted

• People at greatest risk include those on antibiotics,

the elderly and immunocompromised patients

• High rate of relapse, >25%

• Causesa spectrum of diseasescollectively knownas CDI:
• Mild self-limiting diarrhoea
• Pseudomembranouscolitis(PMC)
• May progressto toxic megacolon, sepsis, death
• S. Kirov, U Tas
• C. difficile infection (CDI)

normal gut PMC toxic megacolon

• C. difficile infection (CDI)

Centersfor Disease Control andPrevention(CDC):ANTIBIOTIC RESIST ANCETHREATS inthe UnitedStates, 2013

• C. difficile is listed as the number one antibiotic resistance

threat to the US healthcare system.

• C. difficile spores
• Spore-forming ability and spore

persistence are major problems

• Infectious particle
• Survival mechanism
• Heat, ethanol and UV resistant –

allowsfor persistence in hospitals

Ingestion of spores Germination of spores in the colon Toxin-mediated damage Colonisation by vegetative cells in the colon T

• xin production by

vegetative cells

• T
• xin A
• T
• xin B

Disruption to the gut microbiota by antibiotics

The infectious cycle of C. difficile

1 4 3 2 5

• T
• xin A (308 kDa) & T
• xin B (269.6 kDa)
• Monoglucosyltransferasesencoded on PaLoc (19.6 kb)

1 kb

tcdB tcdE tcdA tcdC tcdR PaLoc

T cdR = alternativesigmafactor T cdE = putative holin-like protein T cdC = anti-sigma factor(negativeregulator of toxin production)

• T
• xin B shown to be essential for disease (Lyras et al., 2009)
• Some strains also produce Binary toxin – may be involved in

colonisationand adherence

• C. difficile toxins

Pathogenesis of C. difficile toxins

Jank et al., 2007

InactivationofRho GTPases Depolymerisationofactin filaments Cytoskeleton disruption Cellroundingand death

The hypervirulent strains

• Emergence of hypervirulent strains associated with an

increase in disease incidence, severity and mortality

• Epidemicstrains (ribotype 027) have been isolated worldwide
• Higher relapse rates
• Increased virulence may be due to:

– A deletion in tcdC– loss of negative regulation of toxin production – More toxin produced = more bowel damage – Resistance to fluoroquinolones – Presenceof binary toxin

• C. difficile in hospitals
• 3% of healthy people and 20-40% hospitalised patients

colonised with C. difficile

• associated with short-stay hospitals
• poor hospital practices
• antibiotic stewardship
• ability to form spores

– major problem – resistantto many cleaning agents (notsporocidal) – persistin environmentfor6 months+

C.difficile spore image:JoanneWee

Current treatment of CDI

1) Mild disease - Discontinue use of antibiotics 2) Moderate/severe disease - Treat with metronidazole, vancomycin or fidaxomicin 3) A range of non-antibiotic treatments have also been tested:

• Intravenous IgG antibodies
• Monoclonal antibodies
• Probiotics
• Non-toxigenic strains of C. difficile
• Faecal transplant therapy

http://choices.studentlife.wfu.edu/files/2012/08/prescription-drug-addictions.jpg

Faecal transplant therapy

• Prepared by blending and filtering a fresh donor stool
• Is administered via the upper or lower gastrointestinaltract by

nasogastric/duodenaltube, colonoscopyor enema.

• Restore the diversity of the gut microbiota and reverse the

dysbiosisof CDI

• Concernsover the long-term unknown risks of the therapy:
• transmissionof unrecognised infectious agents
• potential associationof the gut microbiota with conditions such as irritable

bowel syndrome, metabolic syndromes, obesity and chronic fatigue

• Donorscreening protocols have not been established

New methods for the prevention and/or treatment of CDI are urgently required

• IMM-529 is a natural product which is intended to prevent

and treat C. difficile infections

• IMM-529 contains high concentrations of specific antibodies

that are predominantly IgG (86%), IgA (7%) and IgM (7%)

• IMM-529 does not destroy the gut microbiota like

antibiotictreatment

IMM-529 Production

Advantages of IMM-529

• Inexpensive
• Antibodiessurvive transit through the

stomachand remain functional in the large intestine (site of C. difficile infection and toxin production)

• Technology platform already used for the prevention

and treatment of other gastrointestinaldiseases

(Cryptosporidium,Rotavirus,EnterotoxigenicEscherichiacoli)

Evaluation of IMM-529 for passive immunotherapy in the prevention and treatment of

• C. difficile infections

Different markets for IMM-529

• Preventionof initial disease
• Treatment
• Preventionof disease relapse

Ingestionof spores Germinationofspores Colonisationby vegetativecells T

• xinproduction
• T
• xinA
• T
• xinB

Infection Disruptionto the gut microbiotaby antibiotics

Vaccine targets

IMM-529 Products

• IMM-529B contains high levels of specific antibodies which have

been generated against recombinant Toxin B

• IMM-529S contains high levels of specific antibodies which target

the very infectious C. difficle spores

• IMM-529V contains high levels of specific antibodies which target

cell surface antigens present on vegetative cells

• These are produced by vaccinating pregnant cows with specific

antigens and harvesting colostrum upon calving.

In vitro characterisation of C. difficile IMM-529 antibodies (spore, vegetative cell and toxin B)

IMM-529S1 antibodiesare cross-reactive with the exosporiumlayer from C. difficile spores

exosporium

250 kDa 250 kDa 250 kDa

Non-immune IMM-529S1 #1 IMM-529S1 #2

1 2 3 4 5 6 7 8

1-KI (A+B+) 2-M7404 (A+B+) 3-VPI10463 (A+B+) 4-GE (A+B+) 5-MDU2992 (A-B+) 6-JGS6133 (A+B+) 7-AI35 (A-B+) 8-1470 (A-B+)

IMM-529S1 contains antibodies that are cross-reactive with the exosporium layer of spores from a variety of isolates (human and animal)

Strain used to generate product

50 kDa 37 kDa 25 kDa 75 kDa

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Human Animal

IMM-529V1 antibodiesare cross-reactive with cell lysates from C. difficile vegetative cells

IMM-529V1 contains antibodies that are cross-reactive with vegetative cell whole cell lysates from a variety of human and animal C. difficileisolates

Strain used to generate product

IMM-529B1 antibodiesare cross-reactive with Toxin B from different C. difficile strains

IMM-529B1 contains antibodies specific to Toxin B from a variety

• f human and animal isolates

1 2 3 4 5 6 7 8 9 10 11

Non-immune IMM-529B1 #1 IMM-529B1 #2

250 kDa

1-KI (A+B+) 2-M7404 (A+B+) 3-VPI10463 (A+B+) 4-GE (A+B+) 5-MDU2992 (A-B+) 6-JGS6133 (A+B+) 7-AI35 (A-B+) 8-1470 (A-B+) 9-CD37 (A-B-) 10-Purified Toxin B (commercial)

250 kDa 250 kDa

N o N o n -im m u n e IM M -5 2 9 B 1 2 5 5 0 7 5 1 0 0

A n tib o d y % c e ll d e a th

**** ****

C o m m e rcia l p u rifie d T o xin B (stra in V P I1 0 4 6 3 )

N o N o n -im m u n e IM M -5 2 9 B 1 2 5 5 0 7 5 1 0 0

A n tib o d y % c e ll d e a th

*** ***

H isto rica l T o xin B (stra in 6 3 0 )

N o N o n -im m u n e IM M -5 2 9 B 1 2 5 5 0 7 5 1 0 0

A n tib o d y % c e ll d e a th

**** ****

H y p e rv iru le n t T o xin B (strain K I)

IMM-529B1 antibodies neutralise T

• xin B

from historical and hypervirulent strains

IMM-529B1 antibodies neutralise T

• xin

B from a historical strain

Vero + Toxin B-Hist Vero + Toxin B-Hist + anti-Toxin B antibody Vero + Toxin B-Hist + IMM-529B1 Vero + Toxin B-Hist + Non-immune

T

• xin neutralisationassays

50 µm 50 µm 50 µm 50 µm 50 µm

• Antibodies in IMM-529B1 neutralise historicalT
• xinB from the historical (Hist) strain 630

Vero cells only

IMM-529B antibodies neutralise T

• xin B

from a hypervirulent strain

Vero cells only Vero + Toxin B-HV Vero + Toxin B-HV + Toxin B antibody Vero + Toxin B-HV + Non-immune Vero + Toxin B-HV + IMM-529B1

T

• xin neutralisationassays

50 µm 50 µm 50 µm 50 µm 50 µm

• Antibodies in IMM-529B1 cross-neutraliseT
• xin B from a hypervirulent(HV) strain

In vivo characterisation of IMM-529

Monitor:

• Weight loss
• Physiological appearance
• Activity
• Diarrhoea
• 10
• 3

Day Antibiotics in drinking water to induce susceptibility to C. difficile

• C. difficile challenge

(103spores) IMM-529 administration C57BL/6 mice 6–7 weeks

• 2

The C. difficile mouse model

• 1

IMM-529S1 protects 40% of mice from C. difficile disease

Non-immunecontrol IgG IMM-529S1

% survival % weight loss

***

x N = 10 mice/group *** p = 0.0005 SEM x = mice culled

12 2 4 3 6 4 8 6 0 7 2 T im e (h o u rs ) 84 9 6 90 80 70 10 0

% survival % weight loss

11 0 % W e igh t (re lative to d a y 0 )

IMM-529B1 protects 80% of mice from C. difficile disease

Uninfected (N=11) No IgG(N=10) Non-immune IgG(N=20) IMM-529B1 (N=20)

*** p < 0.0001 SEM

***

IMM-529B1 protects mice from

• C. difficile disease (prophylaxis)

Uninfected Non-immuneIgG IMM-529B1

• Mouse colonic tissue from uninfected mice or mice that were pre-treated with Non-

immune or IMM-259B1 prior to C. difficile infection. Tissue was stained with PeriodicAcid Schiffs(P AS)/AlcianBlue stainingto detect glycoproteinsandmucopolysaccharides.

• Mice that received IMM-529B1 display colonic architecture similar to that observed in

uninfected mice. Mice that received non-immune IgG show extensive toxin-mediated colonic damage and inflammation

A combinationIMM-529 product protects 80% of mice from C. difficiledisease (prevention)

Non-immuneIgG IMM-529B1/IMM529S1/IMM-529V1 (1:1:1)

% survival % weight loss

**

x N = 5 mice/group ** p = 0.0027 SEM x = mice culled

1 2 2 4 3 6 4 8 6 0 7 2 8 4 9 6 2 0 4 0 6 0 8 0 1 0 0 1 2 0

h o u rs p o s t in fe c tio n P e rc e n t s u rv iv a l U n in fe c te d (n = 1 5 ) N o Ig G (n = 1 0 ) N o n -im m u n e Ig G (n = 1 5 ) IM M -5 2 9 B 1 (N = 1 4 ) V a n c o m y c in (n = 1 0 )

1 2 2 4 3 6 4 8 6 0 7 2 8 4 9 6 7 0 8 0 9 0 1 0 0 1 1 0

T im e (h o u rs ) W e ig h t (% re la tiv e to D 0 ) U n in fe c te d (n = 1 5 ) N o Ig G (n = 1 0 ) N o n -im m u n e Ig G (n = 1 5 ) IM M -5 2 9 B 1 (n = 1 4 ) V a n c o m y c in (n = 1 0 )

Treatment with IMM-529B1 post-infection protects 80% of mice from disease

x x = mice culled N=3, **** p < 0.0001 ****

Summary

1) IMM-529S1 (prophylaxis)

• Protected 40% of mice

2) IMM-529B 1(prophylaxis)

• Protected 80% of mice

3) IMM-529 product combination (prophylaxis)

• Mixture of IMM-529B1, IMM-529S1 and IMM-529V1

protected 80% of mice 4) IMM-529B1 (treatment)

• Protected 80% of mice when administered 6 hours post-

infection

IMP Manufacture & Process Development

Pilot Scale Production

• Immuron has partnered with Diary Innovations Australia Limited

and CSIRO to develop a scalable method of manufacture for IMM- 529 for a Clinical Development Program

• Three pilot scale production batched of IMM-529B2, IMM-529S2

and IMM-529V2 have been produced as well as a normal control batch

• This material has been made available to Monash University for

characterisation testing and preclinical proof of principle animal efficacy trials.

Specific-ELISAs for Pilot Scale Products

0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5

IMM-529B2

A n ti b o d y ti te r A b s o r b a n c e (4 5 0 n m ) IMM-529B N o n -i m m u n e IgG 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5

IMM-529V2

A n ti b o d y ti te r A b s o r b a n c e (4 5 0 n m ) IMM-529V N o n -i m m u n e 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0

IMM-529S2

A n ti b o d y ti te r A b s o r b a n c e (4 5 0 n m ) IMM-529E N o n -i m m u n e IgG

Endpointtiters: IMM529B - 16,000-64,000 IMM529V - 4000-16,000 IMM529S - 4000-16,000

IMM-529B2 protects 60% of mice from C. difficile disease

1 2 3 4 7 0 8 0 9 0 1 0 0 1 1 0

D a y s p o s t in fe c tio n % w e ig h tlo s s (re la tiv e to D 0 ) U n in fe c te d (n = 5 ) N o Ig G (n = 5 ) N o n -im m u n e Ig G (n = 5 ) IM M 5 2 9 B 2 (n = 1 0 ) IM M 5 2 9 B 2 1 :3 (n = 1 0 ) IM M 5 2 9 B 2 1 :1 0 (n = 1 0 ) IM M 5 2 9 B 2 1 :3 0 (n = 1 0 ) V a n c o m y c in (n = 1 0 )

0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 2 0 4 0 6 0 8 0 1 0 0

T im e (d a y s ) P e rc e n t s u rv iv a l U n in fe c te d (n = 5 ) N o Ig G (n = 5 ) N o n -im m u n e Ig G (n = 5 ) IM M 5 2 9 B 2 (n = 1 0 ) IM M 5 2 9 B 2 1 :3 (n = 1 0 ) IM M 5 2 9 B 2 1 :1 0 (n = 1 0 ) IM M 5 2 9 B 2 1 :3 0 (n = 1 0 ) V a n c o m y c in (n = 1 0 )

Datasummary(% survival):

• Uninfected-100%
• No IgG-0%
• Non-immuneIgG (neat)-20%
• IMM529B2(neat)-60%
• IMM529B2(1:3)- 20%
• IMM529B2(1:10)- 10%
• IMM529B2(1:30)- 10%
• Vancomycin-100%

0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 2 0 4 0 6 0 8 0 1 0 0

D a y s p o s t-in fe c tio n P e rc e n t s u rv iv a l U n in fe c te d (n = 1 0 ) N o Ig G (n = 1 0 ) N o n -im m u n e Ig G (n = 1 5 ) IM M 5 2 9 V 2 (n = 5 ) IM M 5 2 9 S 2 (n = 1 0 ) IM M 5 2 9 B 2 (n = 2 0 ) IM M 5 2 9 (n = 5 ) V a n c o m y c in (n = 1 0 )

0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 8 0 9 0 1 0 0 1 1 0

D a y s p o s t-in fe c tio n % w e ig h tlo s s (re la tiv e to D 0 ) U n in fe c te d (n = 1 0 ) N o Ig G (n = 1 0 ) N o n -im m u n e Ig G (n = 1 5 ) IM M 5 2 9 V 2 (n = 5 ) IM M 5 2 9 S 2 (n = 1 0 ) IM M 5 2 9 B 2 (n = 2 0 ) IM M 5 2 9 (n = 5 ) V a n c o m y c in (n = 1 0 )

IMM-529 protects 80% of mice from

• C. difficile disease

Datasummary(% survival):

• Uninfected-100%
• No IgG- 0%
• Non-immune IgG- 20%
• IMM529V2- 20%
• IMM529S2- 0%
• IMM529B2- 55%
• IMM529-80%
• Vancomycin-100%

IMM-529 is a combination of IMM-529B2, IMM- 529V2 and IMM-529S2 (1:1:1)

• 10

Day Antibiotic administration to induce susceptibility to C. difficile Oral gavage (C. difficile) Administration of antibiotic alone or both antibiotic and IMM-529

• 1

50 Monitor mice 20 Oral gavage antibiotic (12hr post-infection) Antibiotic treatment ceases 1 Administration

• f water or

IMM-529

Proposed model to test efficacy of IMM-529 against disease relapse

• Disease relapse model already established at Monash University
• Disease relapses occurs in all mice treated with vancomycin
• No disease relapse is observed in mice treated with fidaxomicin
• Will use this model to test the efficacy of IMM-529 against disease relapse

(compared to standard of care and published results from Merck trial)

IMM529: STUDY DESIGN

41

A Phase I/II study of IMM529 in patients with chronically-relapsing Clostridium difficile infection

• Part A:

A Phase I/II, dose evaluation safety study in combination with SOC in

• C. difficile patients
• Part B:

A Phase I/II, dose evaluation study investigating the activity of IMM529 in combination with SOC in patients with chronically-relapsing C. difficile infection

MAJOR INCLUSION/EXCLUSION CRITERIA

42

Inclusion criteria:

• In-patients
• 18-75 years;
• More than [TBC] episodes of diarrhoea bacteriologically confirmed C. difficile infection

within the preceding month

• Ability to provide written informed consent

Exclusion criteria:

• Receiving (or have received within 5 half lives, whichever is longer) experimental therapies
• Oral immunosuppressive therapy
• Known intolerance of milk or milk products
• Pregnancy or lactation

IMM529-1 STUDY: PART A

43

A Phase I/II study of IMM529 in patients with chronically-relapsing Clostridium difficile infection Part A: A Phase I/II safety assessment in combination with SOC in C. difficile patients

Objectives: (i) To determine the safety of IMM529 tid for 14 days in combination with SOC (ii) To select a dose for further clinical studies (iii) To preliminarily assess markers of response

Design

• Three escalating dose levels (TBC) in combination with SOC, starting at the lowest dose

level

• 14 day assessment per patient
• 6 patients per cohort

IMM529-1 STUDY: PART B

44

A Phase I/II study of IMM529 in patients with chronically-relapsing Clostridium difficile infection Part B: A Phase I/II study investigating the activity of IMM529 in combination with SOC in patients with chronically-relapsing C. difficile infection

Objectives: (i) To determine whether IMM529 can decrease the rate of relapse vs the historical relapse rate in each patient (ii) To determine whether IMM529 therapy ameliorates diarrhoeal frequency and severity (iii) To determine whether IMM529 therapy alters the gut microbiome

Parameters

• IMM529 dosing to commence with SOC and continue for 28 days
• At least two dose levels to be evaluated
• Symptomology measured daily
• Stool bacteriology at Baseline and D28
• Capacity to continue therapy beyond D28, should significant efficacy be observed

Acknowledgements

A/ProfDena Lyras Dr Melanie Hutton BlissCunningham Jerry Kanellos NickyKonstantopoulos AshleyTurner RezaMoussakhani Grant Rawlin