bovine colostrum antibodies for prevention and treatment

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 thatcause diarrhoea and mediate gut damage

• Resistant to most clinical antibiotics













































Clostridium difficile

• Leading cause of infectious antibiotic-associateddiarrhoea in hospitals worldwide

• C. difficile only colonises the gut if the normalmicrobiota is disrupted

• People at greatest risk include those on antibiotics,the elderly and immunocompromised patients

• High rate of relapse, >25%

• Causes a spectrum of diseases collectively known as CDI:

- Mild self-limiting diarrhoea- Pseudomembranous colitis (PMC)

• May progress to toxic megacolon, sepsis, death

S. Kirov, U Tas

C. difficile infection (CDI)

normal gut PMC toxic megacolon

C. difficile infection (CDI)

Centers for Disease Control and Prevention (CDC): ANTIBIOTIC RESISTANCE THREATS in the United States, 2013

C. difficile is listed as the number one antibiotic resistance threat to the US healthcare system.

C. difficile spores

• Spore-forming ability and sporepersistence are major problems

• Infectious particle

• Survival mechanism

• Heat, ethanol and UV resistant –allows for persistence in hospitals

Ingestion of spores

Germination of sporesin the colon

Toxin-mediated damageColonisation byvegetative cellsin the colon

Toxin production by vegetative cells

• Toxin A• Toxin B

Disruption to thegut microbiota by

antibiotics

The infectious cycle of C. difficile

1 4

3

2

5

• Toxin A (308 kDa) & Toxin B (269.6 kDa)

• Monoglucosyltransferases encoded on PaLoc (19.6 kb)

1 kb

tcdB tcdE tcdA tcdCtcdR

PaLoc

TcdR = alternative sigma factor

TcdE = putative holin-like protein

TcdC = anti-sigma factor (negative regulator of toxin production)

• Toxin B shown to be essential for disease (Lyras et al., 2009)

• Some strains also produce Binary toxin – may be involved in colonisation and adherence

C. difficile toxins

Pathogenesis of C. difficile toxins

Jank et al., 2007

Inactivation of Rho GTPases

Depolymerisation of actin filaments

Cytoskeleton disruption

Cell roundingand death

The hypervirulent strains

• Emergence of hypervirulent strains associated with anincrease in disease incidence, severity and mortality

• Epidemic strains (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

– Presence of binary toxin

C. difficile in hospitals

• 3% of healthy people and 20-40% hospitalised patientscolonised with C. difficile

• associated with short-stay hospitals

• poor hospital practices

• antibiotic stewardship

• ability to form spores– major problem

– resistant to many cleaning agents (not sporocidal)

– persist in environment for 6 months+

C.difficile sporeimage: 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 gastrointestinal tract bynasogastric/duodenal tube, colonoscopyor enema.

• Restore the diversity of the gut microbiota and reverse thedysbiosis of CDI

• Concerns over the long-term unknown risks of the therapy:

• transmission of unrecognised infectious agents• potential association of the gut microbiota with conditions such as irritable

bowel syndrome, metabolic syndromes, obesity and chronic fatigue• Donor screening 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 antibodiesthat are predominantly IgG (86%), IgA (7%) and IgM (7%)

• IMM-529 does not destroy the gut microbiota like

antibiotic treatment

IMM-529 Production

Advantages of IMM-529

• Inexpensive

• Antibodies survive transit through thestomach and remain functional in the large intestine(site of C. difficile infection and toxin production)

• Technology platform already used for the preventionand treatment of other gastrointestinal diseases

(Cryptosporidium, Rotavirus, Enterotoxigenic Escherichiacoli)

Evaluation of IMM-529 for passiveimmunotherapy in the prevention

and treatment of

C. difficile infections

Different markets for IMM-529

• Prevention of initial disease

• Treatment

• Prevention of disease relapse

Ingestion of spores

Germination of spores

Colonisation byvegetative cells

Toxin production• Toxin A• Toxin B

Infection

Disruption to the gutmicrobiota by

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. difficileIMM-529 antibodies

(spore, vegetative cell and toxin B)

IMM-529S1 antibodies are cross-reactive with the exosporium layer 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 exosporiumlayer 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

HumanAnimal

IMM-529V1 antibodies are 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. difficile isolates

Strain used to generate product

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

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

0

2 5

5 0

7 5

1 0 0

A n tib o d y

% c

ell

de

ath

****

****

C o m m e r c ia l p u r if ie d T o x in B

(s t r a in V P I1 0 4 6 3 )


0

2 5

5 0

7 5

1 0 0

A n tib o d y

% c

ell

de

ath

***

***

H is to r ic a l T o x in B

(s tr a in 6 3 0 )


0

2 5

5 0

7 5

1 0 0

A n tib o d y

% c

ell

de

ath

****

****

H y p e r v ir u le n t T o x in B

(s tra in K I)

IMM-529B1 antibodies neutralise Toxin Bfrom historical and hypervirulent strains

IMM-529B1 antibodies neutralise ToxinB from a historical strain

Vero + Toxin B-Hist Vero + Toxin B-Hist + anti-Toxin B antibody

Vero + Toxin B-Hist + IMM-529B1Vero + Toxin B-Hist + Non-immune

Toxin neutralisation assays

50 µm 50 µm 50 µm

50 µm50 µm

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

Vero cells only

IMM-529B antibodies neutralise Toxin Bfrom 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

Toxin neutralisation assays

50 µm 50 µm 50 µm

50 µm 50 µm

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

In vivo characterisation ofIMM-529

Monitor:•Weight loss•Physiological appearance•Activity •Diarrhoea

-10 -3 0 Day

Antibiotics in drinking water to induce susceptibility to C. difficile

C. difficile challenge (103 spores)

IMM-529 administration C57BL/6 mice 6–7 weeks

-2

The C. difficile mouse model

-1

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

Non-immune control IgGIMM-529S1

% survival % weight loss

***x

N = 10 mice/group*** p = 0.0005

SEMx = mice culled

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

T im e ( h o u rs )

8 4 9 6

9 0

8 0

7 0

1 0 0


1 1 0

%W

eig

ht

(re

lati

ve

to

da

y

0)

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

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

*** p < 0.0001SEM

***

IMM-529B1 protects mice fromC. 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 Periodic AcidSchiffs (PAS)/Alcian Blue stainingto detect glycoproteinsand mucopolysaccharides.

• 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 combination IMM-529 product protects80% of mice from C. difficile disease (prevention)

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


**

x

N = 5 mice/group** p = 0.0027

SEMx = mice culled

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

0

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 t io n

Pe

rc

en

t s

urv

iva

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 )

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 )

We

igh

t (%

re

lati

ve

to

D0

)

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

xx = 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 t i te r

Ab

so

rb

an

ce

(4

50

nm

)

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


Ab

so

rb

an

ce (4

50

nm

)

IMM-529V

N o n -i m m u n e

0 . 0

0 . 5

1 . 0

1 . 5

2 . 0

IMM-529S2


Ab

so

rb

an

ce (4

50

nm

)

IMM-529E

N o n -i m m u n e IgG

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

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

0 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

eig

htl

os

s (

re

lati

ve

to

D0

)

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

0

2 0

4 0

6 0

8 0

1 0 0

T im e (d a y s )

Pe

rc

en

t s

urv

iva

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 )

Data summary(% survival):• Uninfected-100%• No IgG-0%• Non-immune IgG (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

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

Pe

rc

en

t s

urv

iva

l


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

eig

htl

os

s (

re

lati

ve

to

D0

)


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 fromC. difficile disease

Data summary(% 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 0 Day

Antibiotic administration to induce susceptibility to C. difficile

Oral gavage(C. difficile)

Administration ofantibiotic alone or both

antibiotic and IMM-529

-1 50

Monitor mice

20

Oral gavageantibiotic (12hr post-infection)

Antibiotic treatment ceases

1

Administration of 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


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


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/Prof Dena LyrasDr Melanie HuttonBliss Cunningham

Jerry KanellosNickyKonstantopoulos AshleyTurner Reza Moussakhani Grant Rawlin

bovine colostrum antibodies for prevention and treatment

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