A L O P E X X Pharmaceuticals, LLC

Transcription

1 A L 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 PA, Washington DC November 2014

2 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 arvard Medical School Promising new alternative for the treatment and prevention of bacterial infections

3 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

4 PNAG: a surface polysaccharide common to multiple and diverse bacterial pathogens Poly N-acetyl glucosamine C C 3 C 2 N C C 3 -linked acetates and succinates C C 2 C 2 C C 2 N C C 3 PNAG-β-1-6-linked polymer of N-acetyl glucosamine residues N-linked acetates C 2 N C C 3 C 2 N 2 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

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

6 Pathogens that make PNAG PNAG Expression Determination Genes for biosynthetic proteins identified and polysaccharide isolated Genes for biosynthetic proteins identified and expression confirmed by immunochemical confirmation Immunolochemical Confirmation nly S. aureus including MRSA S. epidermidis and other coagulase-negative staphylococci E. coli including 0157 and other Shiga-toxin producers Bordetella pertussis, B. parapertussis B. bronchiseptica Y. entercolitica, Y. pseudotuberculosis Burkholderia (including B. mallei) Stenotrophomonas Bacteroides fragilis Bacillus subtilis Borrelia burgdorferi Brucella abortus Clostridium difficle Campylobaccter jejunii Candida albicans Chlamydia trachomatis Francisella tularensis Fungal pathogens elicobacter pylori emophilus ducreyi emophilus influenzae Listeria monocytogenes Mycobacterium tuberculosis Bacterial Species Yersinia pestis Aggregatibacter actinomycetemcomitans Actinobacillus pleuropneumoniae Acinetobacter baumannii Vibrio parahemolyticus Klebsiella Shigella Group B streptococcus S. pneumoniae Vibrio cholerae Enterococcus faecalis Salmonella typhi Plasmodium species Neisseria gonorrhoeae Neisseria meningitides Propionobacterium acnes Streptococcus pyogenes Trichmonas vaginalis/tritrichomonas foetus Rhodococcus equi Streptococcus equi emophilus 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

7 Detection of PNAG expression on bacterial surfaces. Control' (F429)' Control' (F429)' An01PNAG' (F598)' Non1typable'.#influenzae#200' N.#gonorrhoeae#252' E A Non1typable'.#influenzae#140' N.#gonorrhoeae#252' F B N.#gonorrhoeae#FA1090# Non1typable#.#influenzae#75' G C N.#gonorrhoeae#2399# D N.#meningi1dis#serogroup'B# Control'(F429)' An01PNAG'(F598)' Control'+'An01 N.#meningi1dis#serogroup'A# serogroup'a' I J An01PNAG' (F598)' PNAG is intercalated on the surface with the classic capsular polysaccharides Demonstrated by immunochemical confocal PNAG is intercalated on the microscopy, electron microscopy on: surface with the classic. influenzae capsular polysaccharides N. gonorrhoeae Serogroups A & B N. meningitidis Demonstrated by Serogroup 19A S. pneumoniae immunochemical confocal microscopy, electron microscopy PNAG molecules spatially on:area as capsular antigens. influenzae located in the same N. gonorrhoeae Co-staining with anti-serogroup Serogroups A & B N. meningitidis A N. meningitidis Serogroup S. pneumoniae 19A anti-s.pneumoniae serogroup 19A An01PNAG'(F598)'+'an01serogroup'A' An01PNAG'(F598)'+' an01s.#pneumoniae' serogroup'19a'

8 In vivo expression of PNAG by microbial pathogens. uman!mef!samples! S.'pneumoniae'samples'A)D' uman!mef!samples!.'influenzae'(non)typable)'samples'e'&'f' A E Anti S. pneumoniae!chi%nase!!!!!!chi%nase!!!!dispersin!b Anti PNAG Control B Anti S. pneumoniae Chi%nase!!!!!!Chi%nase!!!!!Dispersin!B' Anti PNAG Control G Anti. influenzae F Anti S. pneumoniae 19A Control & DNA stain Anti S. pneumoniae 19A C.#roden1um3GI!infec%on# Anti PNAG & DNA stain Anti S. pneumoniae C.#albicans?kera%%s# DNA stain Control Anti S. pneumoniae verlay DNA stain Anti PNAG verlay M.#tuberculosis#infected!human!lung!%ssue! Phase contrast DNA stain Anti-Mtb Control Phase contrast DNA stain Anti-Mtb Anti-PNAG Anti-PNAG Anti-PNAG Phase contrast N DNA stain Anti-Mtb Anti-PNAG Anti-PNAG Composite Cornea of a mouse with keratitis (J) M. tuberculosis M closely related to pathogenic E. coli C. albicans Composite L Colonic sections from mice ( I) Composite K uman otitis media (E-F) C. rodentium J Infected middle ear fluid (MEF) from humans (A-D) titis media chinchilla model (G-) Non-typable. influenzae I!Chi%nase!!!!!!Chi%nase!!!!Dispersin!B Control Anti PNAG D S. pneumoniae Chi%nase!!!!!!Chi%nase!!!!!Dispersin!B' Control Anti PNAG Anti. influenzae Animal'2' Chi%nase!!!!!Chi%nase!!!!!!Dispersin!B!!!!!Periodate' Anti PNAG Control!Chi%nase!!!!!!Chi%nase!!!!Dispersin!B Control Anti PNAG C Chinchilla!NP!samples! Strain'070' Animal'1' Chi%nase!!!!!Chi%nase!!!!!!Dispersin!B!!!!!Periodate' Control Anti PNAG!Chi%nase!!!!!!Chi%nase!!!!Dispersin!B Anti PNAG Control uman lung tissue (K-N)

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

10 Given the ubiquity of PNAG why don t we all have protective antibodies? Everyone has antibody to PNAG But Natural antibody to PNAG is not opsonic nor protective in animals Among adults examined to date only 2 of 45 had detectable, PNAG-specific opsonic killing antibody titers Titer 1000 psonic 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 Sample of normal human serum 10 umans with severe staphylococcal infections Make opsonic antibodies specific to PNAG, Intensity of exposure to Staphylococci dictates production of these antibodies

11 Modification of PNAG A More Effective Target C 2 N N C 2 C 2 3 C N N C 2 3 C PNAG dpnag C 2 N C 2 C 3 C N 2 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 NT immunogenic antibody response Explains why bacterial infections can occur more than once Percent bacteria killed F598 F628 F mab concentration (µg/ml) uman 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 Antibodies to dpnag bind comparably to both native and dpnag

12 Protection against S. pneumoniae-induced lung infection and Carbapenemase-producing K. pneumoniae 100 F µg F598 Percent survival Time (h) Anti-9GlcN2(TT) F598-50µg F µg F598-25µg Control F µg S. pneumoniae-induced lung infection model F598 administrated intravenously 4 hours before intranasal bacterial challenge (10 8 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 (9GlcN2-TT) All doses resulted in significantly (P<0.05) greater survival compared to control

13 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

14 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 Burkholderia A. baumannii Lethal peritonitis ral infection with Shiga-toxin producing strains Lethal peritonitis Bacteremia Pneumonia 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, NT deacetylated 10+ pathogens studied in > 18 infection models

15 Are we worried about negative effects on normal flora? No effect of antibody to PNAG on normal flora in mouse studies Goats and rabbits vaccinated with dpnag Followed 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

16 Monoclonal Antibody F598 IgG1 mab-binding to PNAG riginated 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 Target strain: S. aureus Mn8

17 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 uman Colon:

18 A Phase 1 pen label Study of F598 Administered in ealthy Adult Volunteers Trial Design bjectives Endpoints Treatment Plan Part 1 Single Dose Determine the safety and tolerance of escalating doses of F598 in healthy adult volunteers Evaluate treatment-related adverse events Evaluate F598 PK and PD Assess the immunogenicity of F598 (AA) pen-label, dose-escalation study Dose Level Dose mg/kg No. Subjects (0.86) (4.30) (8.59) (12.89) (17.18) 4 Sample Size 20 Subjects

19 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? ral pruritis Resolved with observation and anti-histamines (1 subject)

20 pk and anti-drug antibody (AA) Mean F598 serum concentration ( 4 g/ml) mg/kg 5 mg/kg 10 mg/kg 15 mg/kg 20 mg/kg LLQ 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 AA No anti-drug antibodies detected in any subject Includes the 2 subjects with cutaneous reactions Nominal time (days)

21 psonic Activity Titer 1/N Pharmacodynamic assessments performed using opsonphagocytic activity (PA) and opsonophagocytic killing (PK) assays PA flow cytometry using L-60 granulocytes as effector cells and viable xyburst dye-labeled S. aureus as bacterial target PK percentage of bacterial targets killed Excellent correlation between PA and PK assays Antibody retains functional activity to day 50 Possibly considerably longer

22 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 psonic, 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