Alopexx Pharmaceuticals Founded in 2006 Daniel Vlock, M.D. and - - PowerPoint PPT Presentation

A L O P E X X

Pharmaceuticals, LLC

Antibody Targeting of Poly N-Acetyl Glucosamine (PNAG)

A conserved surface polysaccharide on multiple microbial pathogens

Potential as a broad-spectrum biological therapeutic

PAHO, Washington DC November 2014

Alopexx Pharmaceuticals

• Founded in 2006

– Daniel Vlock, M.D. and Gerald Pier, Ph.D.

• Established to further development of an antibody

platform produced in Dr. Pier’s laboratory at Harvard Medical School

• Promising new alternative for the treatment

and prevention of bacterial infections

Desirable Characteristics for an Effective Antibody Against Bacterial Infections

• Broad distribution of target antigen

– Not limited to only a few serotypes

• Clinically relevant target

– Bacteria cannot simply avoid the immune therapy by mutating to not produce the target antigen

• Loss of the target antigen would cripple the bacterium’s ability to cause infection

– Binds to capsular polysaccharides

• With rare exception immunologic protection against bacterial infections are directed

against capsular sugars

• Induce immune-mediated bacterial killing

– Intact antibody required to induce phagocytic killing

• Single agent activity

– Simplifies clinical development – Provides a signal to justify combination therapy

PNAG: a surface polysaccharide common to multiple and diverse bacterial pathogens

• Poly N-acetyl glucosamine
• Conserved surface polysaccharide

produced by major bacterial, fungal and protozoal parasites

– Present as a capsular antigen surrounding the outside of the cell

• Capsules well known targets of effective vaccines and

passive therapies

• Employed by bacteria to

– Facilitate adherence to biomaterials – tissue, prostheses – Protect the bacterial cell from host defenses

• Critical virulence factor

PNAG-β-1-6-linked polymer

• f N-acetyl glucosamine

residues

O H O H H O H H H C H

O O H H O H O H H H C H

O O H H O H H O H H H O C H

O H H O H H O H H N H

H C H

O O N H N H N H C H

C O C H

C O C H

C O C H

C O

O-linked acetates and succinates

CH2 C O C O OH CH2

N-linked acetates

PNAG Expression Determination Bacterial Species

Genes for biosynthetic proteins identified and polysaccharide isolated § S. aureus including MRSA § S. epidermidis and other coagulase-negative staphylococci § E. coli including 0157 and other Shiga-toxin producers § Yersinia pestis § Aggregatibacter actinomycetemcomitans § Actinobacillus pleuropneumoniae § Acinetobacter baumannii § Vibrio parahemolyticus Genes for biosynthetic proteins identified and expression confirmed by immunochemical confirmation § Bordetella pertussis, § B. parapertussis § B. bronchiseptica § Y. entercolitica, § Y. pseudotuberculosis § Burkholderia (including B. mallei) § Stenotrophomonas § Klebsiella § Shigella § Group B streptococcus § S. pneumoniae § Vibrio cholerae § Enterococcus faecalis § Salmonella typhi Immunolochemical Confirmation Only § Bacteroides fragilis § Bacillus subtilis § Borrelia burgdorferi § Brucella abortus § Clostridium difficle § Campylobaccter jejunii § Candida albicans § Chlamydia trachomatis § Francisella tularensis § Fungal pathogens § Helicobacter pylori § Hemophilus ducreyi § Hemophilus influenzae § Listeria monocytogenes § Mycobacterium tuberculosis § Plasmodium species § Neisseria gonorrhoeae § Neisseria meningitides § Propionobacterium acnes

• Streptococcus pyogenes
• Trichmonas vaginalis/Tritrichomonas foetus

§ Rhodococcus equi § Streptococcus equi § Hemophilus parasuis § Salmonella cholerasuis § Streptococcus suis § Streptococcus uberis § Streptococcus dysgalactiae § Staphylococcus pseudintermedius

Wide range of bacteria, fungi and protozoa shown to produce PNAG but lack an identifiable genetic loci

• Proc. Natl Acad of Sci, June 2013

Pathogens that make PNAG

• Initially identified on a limited number of bacterial species
• 4-gene biosynthetic locus identified
• Polysaccharide isolated
• PNAG found to be chemically identical across species
• Small variations in acetylation levels of the amino groups
• Variations in the amount of O-linked acetates and succinates
• Recent large expansion of organisms known to express PNAG

PNAG Expression Determination Bacterial Species

Genes for biosynthetic proteins identified and polysaccharide isolated § S. aureus including MRSA § S. epidermidis and other coagulase-negative staphylococci § E. coli including 0157 and other Shiga-toxin producers § Yersinia pestis § Aggregatibacter actinomycetemcomitans § Actinobacillus pleuropneumoniae § Acinetobacter baumannii § Vibrio parahemolyticus Genes for biosynthetic proteins identified and expression confirmed by immunochemical confirmation § Bordetella pertussis, § B. parapertussis § B. bronchiseptica § Y. entercolitica, § Y. pseudotuberculosis § Burkholderia (including B. mallei) § Stenotrophomonas § Klebsiella § Shigella § Group B streptococcus § S. pneumoniae § Vibrio cholerae § Enterococcus faecalis § Salmonella typhi Immunolochemical Confirmation Only § Bacteroides fragilis § Bacillus subtilis § Borrelia burgdorferi § Brucella abortus § Clostridium difficle § Campylobaccter jejunii

§ Candida albicans

§ Chlamydia trachomatis § Francisella tularensis

§ Fungal pathogens

§ Helicobacter pylori § Hemophilus ducreyi § Hemophilus influenzae § Listeria monocytogenes

§ Mycobacterium tuberculosis § Plasmodium species

§ Neisseria gonorrhoeae § Neisseria meningitides § Propionobacterium acnes

• Streptococcus pyogenes
• Trichmonas vaginalis/Tritrichomonas foetus

§ Rhodococcus equi § Streptococcus equi § Hemophilus parasuis § Salmonella cholerasuis § Streptococcus suis § Streptococcus uberis § Streptococcus dysgalactiae § Staphylococcus pseudintermedius

Wide range of bacteria, fungi and protozoa shown to produce PNAG but lack an identifiable genetic loci

• Proc. Natl Acad of Sci, June 2013

Pathogens that make PNAG

Control' (F429)'

N.#gonorrhoeae#252'

Control' (F429)' An01PNAG' (F598)' An01PNAG' (F598)'

N.#gonorrhoeae#2399# Non1typable#H.#influenzae#75' N.#gonorrhoeae#FA1090#

Control'(F429)' An01PNAG'(F598)' Control'+'An01 serogroup'A'

An01PNAG'(F598)'+'an01serogroup'A'

N.#meningi1dis#serogroup'B# N.#gonorrhoeae#252'

N.#meningi1dis#serogroup'A#

Non1typable'H.#influenzae#200' Non1typable'H.#influenzae#140'

A B C D I J E F G H

An01PNAG'(F598)'+' an01S.#pneumoniae' serogroup'19A'

PNAG is intercalated on the surface with the classic capsular polysaccharides

• Demonstrated by

immunochemical confocal microscopy, electron microscopy

• n:
• H. influenzae
• N. gonorrhoeae
• Serogroups A & B N. meningitidis
• Serogroup 19A S. pneumoniae
• PNAG is intercalated on the surface with the

classic capsular polysaccharides

• Demonstrated by immunochemical confocal

microscopy, electron microscopy on:

• H. influenzae
• N. gonorrhoeae
• Serogroups A & B N. meningitidis
• Serogroup 19A S. pneumoniae
• PNAG molecules spatially located in the same

area as capsular antigens

• Co-staining with
• anti-serogroup A N. meningitidis
• anti-S.pneumoniae serogroup 19A

Detection of PNAG expression on bacterial surfaces.

In vivo expression of PNAG by microbial pathogens.

!Chi%nase!!!!!!Chi%nase!!!!Dispersin!B

Strain'070' S.'pneumoniae'samples'A)D'

Anti PNAG Control Anti S. pneumoniae Anti PNAG Control Anti S. pneumoniae Control Anti PNAG Control Anti S. pneumoniae Anti S. pneumoniae Anti PNAG Control Anti PNAG Anti H. influenzae Chi%nase!!!!!Chi%nase!!!!!!Dispersin!B!!!!!Periodate' Control Anti PNAG Anti H. influenzae

Human!MEF!samples! Chinchilla!NP!samples! Animal'1'

Chi%nase!!!!!!Chi%nase!!!!!Dispersin!B' Control Anti PNAG Anti S. pneumoniae 19A Anti PNAG Control Phase contrast DNA stain Anti-Mtb Anti-PNAG Composite Phase contrast DNA stain Anti-Mtb Anti-PNAG Composite

M.#tuberculosis#infected!human!lung!%ssue!

Phase contrast DNA stain Anti-Mtb Control Composite

C.#roden1um3GI!infec%on#

Control & DNA stain Anti PNAG & DNA stain

A B C D E F K L M G H I

C.#albicans?kera%%s#

DNA stain Control Overlay DNA stain Overlay Anti PNAG

J

Animal'2'

Chi%nase!!!!!!Chi%nase!!!!!Dispersin!B'

H.'influenzae'(non)typable)'samples'E'&'F'

Chi%nase!!!!!Chi%nase!!!!!!Dispersin!B!!!!!Periodate'

Human!MEF!samples!

Anti S. pneumoniae 19A !Chi%nase!!!!!!Chi%nase!!!!Dispersin!B !Chi%nase!!!!!!Chi%nase!!!!Dispersin!B !Chi%nase!!!!!!Chi%nase!!!!Dispersin!B Anti-PNAG

N

Anti-PNAG Anti-PNAG

• S. pneumoniae
• Infected middle ear fluid (MEF) from

humans (A-D)

• Otitis media chinchilla model (G-H)
• Non-typable H. influenzae
• Human otitis media (E-F)
• C. rodentium
• Colonic sections from mice ( I)
• closely related to pathogenic E. coli
• C. albicans
• Cornea of a mouse with keratitis (J)
• M. tuberculosis
• Human lung tissue (K-N)

Clinical Relevance of PNAG

Loss of Target Reduces Virulence of Bacteria

Andrea Kropec A , Infection and Immunity 73:6868–6876, 2005

• Loss of PNAG production decreases survival of S. aureus in

the blood

• Loss or mutation cripples the bacterium’s ability to cause

infection

101 102 103 Strain: WT Comp Δica WT Comp Δica Time: 2 hours 4 hours cfu/ml blood P<.05 P<.001

• S. aureus Mn8

Given the ubiquity of PNAG why don’t we all have protective antibodies?

10 9 8 7 6 5 4 3 2 1

100 1000 10000 Sample of normal human serum

Titer

• Everyone has antibody to PNAG
• But
• Natural antibody to PNAG is not opsonic nor protective in

animals

• Among adults examined to date
• nly 2 of 45 had detectable, PNAG-specific opsonic killing

antibody titers

• Opsonic antibody to PNAG is uncommon in spite of

the natural exposure to this antigen

• IVIgG from human donors with very high titers of natural

antibody to PNAG did not protect mice against S. aureus bacteremia or skin abscesses

• Humans with severe staphylococcal infections
• Make opsonic antibodies specific to PNAG,
• Intensity of exposure to Staphylococci dictates production of

these antibodies

O H H O H H O H H H C H

2

O O H H O H H O H H H C H

2

O O H H O H H O H H H O C H

2

O H H O H H O H H N H

2

H C H

2

O O

PNAG

N H C H

3

C O N H C H

3

C O

dPNAG

N H C H

3

C O NH2 NH2

Modification of PNAG

A More Effective Target

• dPNAG
• modified version of PNAG
• Deacetylated
• Well established means to produce

effective immune response

• Modification of PNAG permits identifying

and developing antibodies that effectively target bacteria

• Native PNAG induces antigenic but

NOT immunogenic antibody response

• Explains why bacterial infections can
• ccur more than once
• Human antibodies that can kill the bacteria

bind primarily to the non-acetylated form

• dPNAG
• F598 binds best to dPNAG
• Enhanced killing
• F628 binds best to native PNAG
• No enhanced killing

20 40 60 80 100 25 12 6 3 1.5

mAb concentration (µg/ml) Percent bacteria killed

F598 F628 F630

• Antibodies to dPNAG bind comparably to

both native and dPNAG

Protection against

• S. pneumoniae-induced lung infection

and Carbapenemase-producing K. pneumoniae

10 20 30 40 50 60 70 80 90 20 40 60 80 100

Control Anti-9GlcNH2(TT) F598-200µg F598-100µg F598-50µg F598-25µg F598-12.5µg Time (h)

Percent survival

• S. pneumoniae-induced lung infection model
• F598 administrated intravenously 4 hours before intranasal

bacterial challenge (108 cfu/mouse)

• Dose-dependently prevented the development of infection
• At 200 µg/mouse effect comparable to curative treatment

with 30 mg/kg of a standard antibiotic, cefotaxime

• Retro-orbital challenge with Carbapenemase+ K. pneumoniae
• Mice were treated 24h before infection with
• MAb F598
• Polyclonal antisera to PNAG vaccine (9GlcNH2-TT)
• All doses resulted in significantly (P<0.05) greater survival

compared to control

F598

Targeting dPNAG can prevent infections from a broad range of pathogens

Lethal peritonitis from S. aureus Corneal keratitis from C. albicans Reduction in levels of N. meningitidis in the brain Lethal systemic infection from malaria P. berghei ANKA

Summary of Protection Data for Antibody and/or Vaccine Targeting dPNAG Published Results

Pathogen Animal Infection Model

• S. aureus

Mouse

• lethal peritonitis
• Bacteremia
• skin infection
• corneal keratitis

E coli

• Lethal peritonitis
• Oral infection with Shiga-toxin producing strains

Burkholderia

• Lethal peritonitis
• Bacteremia
• Pneumonia
• A. baumannii
• N. meningitidis serogroup B
• Neonatal infection
• S. pyogenes
• Lethal infections
• L. monocytogenes
• Lethal infections
• S. pneumoniae
• Pneumonia
• Corneal infections
• C. albicans
• Keratitis
• P. berghei
• Malaria
• S. aureus

Sheep

• Mastitis
• Lethal infection (Amorena)
• Protection associated with achieving titer, vaccine used

purified PNAG, NOT deacetylated

10+ pathogens studied in > 18 infection models

Are we worried about negative effects

• n normal flora?
• No effect of antibody to PNAG on normal flora in mouse

studies

• Goats and rabbits vaccinated with dPNAG

– Followed 12-36 months with no untoward effects noted

• Natural antibodies to normal microbial antigens are very

important in protection against infection

– About 5% of healthy humans do develop natural opsonic/protective antibody to PNAG

• Phase I evaluation of MAb F598 in humans revealed no

signs or symptoms associated with disruption of normal flora

• Originated from B cells of a patient 3

years after S. aureus infection

• B cells were transformed with EBV and

cloned

• Screened for antibody to PNAG and

dPNAG

• Engineered to be IgG1 antibodies
• Better bacterial killing
• Three candidate antibodies identified
• F598, F628 and F630
• F598 had best binding to native and

deacetylated PNAG

• Clinical candidate

Monoclonal Antibody F598

IgG1 mAb-binding to PNAG

Target strain: S. aureus Mn8

Tissue Cross Reactivity Results

• Target bacterial antigen not known to exist in

mammalian species

• Related to chitin
• principal component of arthropod

exoskeletons

• Tissue cross reactivity studies
• Panel of 32 primate and 33 human normal

tissues

• All tissues negative

PNAG-positive S. epidermidis bacterial cells

Normal Kidney Human Colon:

A Phase 1 Open label Study of F598 Administered in Healthy Adult Volunteers Trial Design

Objectives Determine the safety and tolerance of escalating doses of F598 in healthy adult volunteers Endpoints

• Evaluate treatment-related adverse events
• Evaluate F598 PK and PD
• Assess the immunogenicity of F598 (HAHA)

Treatment Plan Open-label, dose-escalation study Part 1 Single Dose Sample Size 20 Subjects

Dose Level Dose mg/kg

• No. Subjects

1 1.0 (0.86) 4 2 5.0 (4.30) 4 3 10.0(8.59) 4 4 15.0 (12.89) 4 5 20.0 (17.18) 4

Phase I Trial Results

• 20 subjects treated
• Generally safe and well tolerated
• 2 possible cutaneous reactions at 20 mg

cohort observed

– Grade 1 rash 8 hrs after infusion – ? Oral pruritis – Resolved with observation and anti-histamines (1 subject)

pK and anti-drug antibody (HAHA)

Nominal time (days)

5 10 15 20 25 30 35 40 45 50

Mean F598 serum concentration (4 g/mL)

0.1 1 10 100 1000

1 mg/kg 5 mg/kg 10 mg/kg 15 mg/kg 20 mg/kg LLOQ

• pK
• Long elimination half-life
• 468h to 714 h (~20- 30 days)
• Antibody detected up to day 50
• F598 exposure increased in a

close to dose proportional manner

• HAHA
• No anti-drug antibodies detected

in any subject

• Includes the 2 subjects with

cutaneous reactions

Opsonic Activity

Titer ¡1/N ¡

• Pharmacodynamic assessments performed using opsonphagocytic activity (OPA) and
• psonophagocytic killing (OPK) assays
• OPA
• flow cytometry using HL-60 granulocytes as effector cells and viable Oxyburst dye-labeled S. aureus as

bacterial target

• OPK
• percentage of bacterial targets killed
• Excellent correlation between OPA and OPK assays
• Antibody retains functional activity to day 50
• Possibly considerably longer

Targeting PNAG

• PNAG is expressed on a broad range of bacterial, fungal and

protozoal pathogens

– lack of identifiable genetic locus for the antigen indicative of a possible evolutionary convergent acquisition of PNAG synthesis

• Antibodies against native PNAG do not induce protective

immunity

• Vaccines or antibodies that target dPNAG are able to provide

protective immunity

– Opsonic, protective epitope is associated with non-acetylated or backbone portion of the molecule

• Phase I trial of monoclonal antibody F598

– Safe and well tolerated

• 2 minor cutaneous reactions

– ½-life ~ 28 days – Functional (opsonic) activity noted to at least day 50

• Phase II pilot and proof-of-concept trials with F598 to begin in 2015