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 O H C H 2 produced by major bacterial, fungal H O PNAG- β -1-6-linked polymer O O of N-acetyl glucosamine H O and protozoal parasites C H H N H H C O residues 3 C H 2 C H H H O H C O 3 H O – Present as a capsular antigen O O H N H H surrounding the outside of the cell C H C 2 O H H C H H C 3 O O CH 2 • Capsules well known targets of effective vaccines and H O CH 2 O O-linked C O H O H passive therapies H N H acetates and OH C H 2 H H C H O succinates N-linked H C O 3 H O acetates • Employed by bacteria to H O O H N H 2 H H – Facilitate adherence to biomaterials – tissue, prostheses – Protect the bacterial cell from host defenses • Critical virulence factor

Pathogens that make PNAG PNAG Expression Bacterial Species Determination § S. aureus including MRSA § Yersinia pestis Genes for biosynthetic proteins § S. epidermidis and other coagulase-negative § Aggregatibacter actinomycetemcomitans identified and polysaccharide staphylococci isolated § Actinobacillus pleuropneumoniae § E. coli including 0157 and other Shiga-toxin § Acinetobacter baumannii producers § Vibrio parahemolyticus § Bordetella pertussis, § Klebsiella Genes for biosynthetic proteins § B. parapertussis § Shigella identified and expression confirmed by immunochemical confirmation § B. bronchiseptica § Group B streptococcus § Y. entercolitica, § S. pneumoniae § Y. pseudotuberculosis § Vibrio cholerae § Burkholderia (including B. mallei) § Enterococcus faecalis • Initially identified on a limited number of bacterial species § Stenotrophomonas § Salmonella typhi • 4-gene biosynthetic locus identified § Bacteroides fragilis § Plasmodium species Immunolochemical Confirmation • Polysaccharide isolated § Bacillus subtilis § Neisseria gonorrhoeae Only § Borrelia burgdorferi § Neisseria meningitides § Brucella abortus § Propionobacterium acnes • PNAG found to be chemically identical across species § Clostridium difficle • Streptococcus pyogenes § Campylobaccter jejunii • Trichmonas vaginalis/Tritrichomonas foetus • Small variations in acetylation levels of the amino groups § Candida albicans § Rhodococcus equi • Variations in the amount of O-linked acetates and succinates § Chlamydia trachomatis § Streptococcus equi § Francisella tularensis § Hemophilus parasuis § Fungal pathogens § Salmonella cholerasuis • Recent large expansion of organisms known to express PNAG § Helicobacter pylori § Streptococcus suis § Hemophilus ducreyi § Streptococcus uberis § Hemophilus influenzae § Streptococcus dysgalactiae § Listeria monocytogenes § Staphylococcus pseudintermedius § Mycobacterium tuberculosis 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 PNAG Expression Bacterial Species Determination § S. aureus including MRSA § Yersinia pestis Genes for biosynthetic proteins § S. epidermidis and other coagulase-negative § Aggregatibacter actinomycetemcomitans identified and polysaccharide staphylococci isolated § Actinobacillus pleuropneumoniae § E. coli including 0157 and other Shiga-toxin § Acinetobacter baumannii producers § Vibrio parahemolyticus § Bordetella pertussis, § Klebsiella Genes for biosynthetic proteins § B. parapertussis § Shigella identified and expression confirmed by immunochemical confirmation § B. bronchiseptica § Group B streptococcus § Y. entercolitica, § S. pneumoniae § Y. pseudotuberculosis § Vibrio cholerae § Burkholderia (including B. mallei) § Enterococcus faecalis § Stenotrophomonas § Salmonella typhi § Bacteroides fragilis § Plasmodium species Immunolochemical Confirmation § Bacillus subtilis Only § Neisseria gonorrhoeae § Borrelia burgdorferi § Neisseria meningitides § Brucella abortus § Propionobacterium acnes § Clostridium difficle • Streptococcus pyogenes § Campylobaccter jejunii • Trichmonas vaginalis/Tritrichomonas foetus § Candida albicans § Rhodococcus equi § Chlamydia trachomatis § Streptococcus equi § Francisella tularensis § Hemophilus parasuis § Fungal pathogens § Salmonella cholerasuis § Helicobacter pylori § Streptococcus suis § Hemophilus ducreyi § Streptococcus uberis § Hemophilus influenzae § Streptococcus dysgalactiae § Listeria monocytogenes § Staphylococcus pseudintermedius § Mycobacterium tuberculosis Wide range of bacteria, fungi and protozoa shown to produce PNAG but lack an identifiable genetic loci • Proc. Natl Acad of Sci, June 2013

Detection of PNAG expression on bacterial surfaces . • PNAG is intercalated on the surface with the classic capsular polysaccharides • Demonstrated by immunochemical confocal PNAG is intercalated on the Control' An01PNAG' Control' An01PNAG' microscopy, electron microscopy on: (F429)' (F598)' (F429)' (F598)' surface with the classic N.#gonorrhoeae# 252' Non1typable'H .#influenzae# 200' • H. influenzae capsular polysaccharides E A • N. gonorrhoeae • Serogroups A & B N. meningitidis N.#gonorrhoeae# 252' Non1typable' H.#influenzae# 140' • Demonstrated by • Serogroup 19A S. pneumoniae F B immunochemical confocal • PNAG molecules spatially located in the same microscopy, electron microscopy N.#gonorrhoeae# FA1090 # Non1typable #H.#influenzae# 75' on: area as capsular antigens • H. influenzae G C • Co-staining with • N. gonorrhoeae • anti-serogroup A N. meningitidis • Serogroups A & B N. meningitidis N.#gonorrhoeae# 2399 # N.#meningi1dis# serogroup'B # • • Serogroup 19A S. pneumoniae anti- S.pneumoniae serogroup 19A H D Control'(F429)' An01PNAG'(F598)' Control'+'An01 An01PNAG'(F598)'+'an01serogroup'A' N.#meningi1dis# serogroup'A # serogroup'A' An01PNAG'(F598)'+' an01 S.#pneumoniae ' serogroup'19A' I J

I n vivo expression of PNAG by microbial pathogens. Human!MEF!samples! Human!MEF!samples! Chinchilla!NP!samples! Strain'070' !Chi%nase!!!!!!Chi%nase!!!!Dispersin!B S.'pneumoniae' samples'A)D' Animal'1' H.'influenzae' (non)typable)'samples'E'&'F' Chi%nase!!!!!!Chi%nase!!!!!Dispersin!B ' Chi%nase!!!!!Chi%nase!!!!!!Dispersin!B!!!!!Periodate ' Control Anti PNAG Control Anti PNAG Anti PNAG Control • S. pneumoniae G E A Anti H. influenzae Anti S. pneumoniae 19A Anti S. pneumoniae • Infected middle ear fluid (MEF) from !Chi%nase!!!!!!Chi%nase!!!!Dispersin!B Animal'2' Chi%nase!!!!!Chi%nase!!!!!!Dispersin!B!!!!!Periodate ' Anti PNAG Chi%nase!!!!!!Chi%nase!!!!!Dispersin!B ' Control humans (A-D) Control Anti PNAG Control Anti PNAG B Anti S. pneumoniae F H Anti H. influenzae • Otitis media chinchilla model (G-H) Anti S. pneumoniae 19A !Chi%nase!!!!!!Chi%nase!!!!Dispersin!B Control Anti PNAG C.#roden1um3 GI!infec%on # • Non-typable H. influenzae Control & DNA stain Anti PNAG & DNA stain C Anti S. pneumoniae • Human otitis media (E-F) I !Chi%nase!!!!!!Chi%nase!!!!Dispersin!B Control Anti PNAG • C. rodentium C.#albicans ?kera%%s # D DNA stain Control Overlay DNA stain Anti PNAG Overlay Anti S. pneumoniae • Colonic sections from mice ( I) J M.#tuberculosis# infected!human!lung!%ssue! • closely related to pathogenic E. coli Phase contrast DNA stain Anti-Mtb Control Composite • K C. albicans • Cornea of a mouse with keratitis (J) Phase contrast DNA stain Anti-Mtb Anti-PNAG Anti-PNAG Composite L • M. tuberculosis Anti-PNAG • Human lung tissue (K-N) M Phase contrast DNA stain Anti-Mtb Anti-PNAG Composite Anti-PNAG N

Clinical Relevance of PNAG Loss of Target Reduces Virulence of Bacteria 10 3 S. aureus Mn8 P<.001 P<.05 cfu/ml blood 10 2 10 1 0 Strain: WT Comp Δ ica WT Comp Δ ica Time: 2 hours 4 hours the blood • Loss of PNAG production decreases survival of S. aureus in • Loss or mutation cripples the bacterium’s ability to cause infection Andrea Kropec A , Infection and Immunity 73:6868–6876, 2005