Synbodies as Anti- Infective Agents

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1 Synbodies as Anti- Infective Agents MIPT Stephen Albert Johnston Center for Innovations in Medicine

2 Current Center for Innovations in Medicine Projects OBJECTIVE INVENTION COMPANY Health Monitoring/ Immunosignatures HealthTell, Inc Early Diagnosis Universal Preventative Frameshift Antigens Calviri, LLC Cancer Vaccine NextGen Antibiotics, Anti-Virals Synbodies 2

3 Kd1 xkd2 = Kd12 3

Synbody Affinity Ligands Peptide 1 Peptide 2 AKT1 K D = 1.

Synthesis In vitro discovery Diehnelt, et.

(2011) 1473 Intensity 4.0 3.5 3.0 2.5 2.0 1.

4 Synbody Affinity Ligands Peptide 1 Peptide 2 AKT1 K D = 1.5 nm TNF-a K D = 23 nm Linker Orthogonal Group Antibody Alternative Chemical Synthesis In vitro discovery Diehnelt, et. al PLoS One 5 (2010) e10728 Staphylococcus aureus Gupta, et al Bioconj Chem 22 (2011) 1473 Intensity Influenza anti-na mab Influenza Synbodies K D = 0.9 nm Concentration (M) Gupta, et al (in preparation) Domenyuk, et. al PLoS One 8 (2013) e

5 Virus Synbody Development Process Protein Bacteria Bind to peptide library Low affinity peptides Affinity improved peptide High affinity Synbody 5

Antibiotic Resistance Threats in the United States,

6 Global Antibiotic Resistance Crisis US Centers for Disease Control World Health Organization CDC Antibiotic Resistance Threats in the United States, 2013 WHO, Antimicrobial Resistance Global Report on Surveillance,

Few Antibiotic Classes Nature, May 1 2014 Recent approvals have been modifications of old scaffolds Tedizolid Linezolid

7 Few Antibiotic Classes Nature, May Recent approvals have been modifications of old scaffolds Tedizolid Linezolid derivative Oritavancin vancomycin derivative Ceftolozane / tazobactam cephalosporin / b-lactamase inhibitor combination 7

8 Bacteria Always Develop Resistance Resistant S. aureus Nature, May

9 Antibacterial Development Bacteria Binding Peptides / Killing Peptides Lead peptides Activity Improved peptides Antibacterial Synbody Platform for rapid development of new antibiotics Screened 13 different Gram-positive and Gram-negatives 9

10 Problem: How to Display Ab Diversity Antibody Diversity 10 9 Different Ab/person Peptide Sequence Space In 3 x 10 5 peptides

identifies binding peptides and lytic peptides Synbody constructed from

11 Staphylococcus aureus Synbody Neg ctrl Neg ctrl Binding Peptide Binding peptide SA Growth Inhibition Lytic peptide Peptide array screening identifies binding peptides and lytic peptides Synbody constructed from binding and lytic peptide has higher activity Domenyuk, et. al PLoS One 8 (2013) e

12 Antibacterial Peptide Problems Low Activity / High Toxicity MIC = um high dose needed for in vivo activity Poor Protease Stability Short t 1/2 rapid degradation high dose needed Too expensive Long history of failure Nature, My

Amino Acid Substitution Approach Increase S.

13 Amino Acid Substitution Approach Increase S. aureus activity Increase selectivity vs P. aeruginosa Reduce Hemolysis Identify amino acid substitutions that improve performance of peptide arm Improved peptide Diehnelt, et al In preparation Improved Synthesis by Conjugation Improved Synbody (1) 13

14 Activity / Toxicity Improvement (1) Hemolysis (H 50 ) MIC (1) MSSA MRSA (USA300) 12.5 um 12.5 um P. aeruginosa PAO1 > 50 um P. aeruginosa PA14 > 50 um > 500 um Therapeutic Index (TI) 40 CFU / ml Synbody (1) is bactericidal at 2x MIC x MIC 10^7 CFU / ml (t=0) 10^8 CFU / ml (t=0) Synbody active against MSSA and MRSA Improved synbody is bactericidal (>3log10 reduction) at 2x MIC Increased S. aureus activity, selectivity and lower hemolysis Diehnelt, et al In preparation 14

15 Antibacterial Peptide Hurdles Low Activity / High Toxicity Poor Protease Stability Selective D-amino Acid Substitution Too expensive L-Lys = $2.48 / g D-Lys = $19.64 / g (7.9x L-Lys) b-lys = $610 / g (245x L-Lys) 15

16 Low Cost Protease Stability Improvement Selective D-AA substitution (2) D-Arg, D-Lys Inhibitory Peptide (3) Initial S. aureus synbody rapidly degraded in serum (t 1/2 < 10 min) Prepared new synbody with 2 copies of D-Arg, D-Lys peptide D-Arg,D-Lys substitution improved t 1/2 to 140 min Diehnelt, et al In preparation 16

17 In vitro Stable Synbody MIC (3) MSSA 12.5 um MRSA (USA300) 6.25 um t 1/2 H min > 500 um TI 80 Bactericidal with low toxicity Active after 6 hours serum incubation CFU / ml Active after 6 hours in serum Time (min) Ly-Ly-H2 Cells + Sera Incubated in fresh mouse serum at 2x MIC Aliquots removed and added to protease inhibitor Each time point is added to an equal volume of MSSA, incubated overnight, plated, and colonies counted Diehnelt, et al In preparation 17

18 CFU / ml Growth Phase Independent Ly-Ly-Sc0 Killing Possible Membrane Targeting MOA ~10^6 CFU / ml (t=12) ~10^8 CFU / ml (t=12) Open Circle Open Triangle Square Black Triangle x MIC Starting Density 10 6 CFU/mL 10 7 CFU/mL 10 8 CFU/mL 10 9 CFU/mL Antibiotics that target actively dividing cells show growth phase dependent activity ~10^7 CFU / ml (t=0) ~10^9 CFU / ml (t=0) Rapid bactericidal activity indicative of membrane disruption CFU / ml Rapidly Bactericidal MSSA x MIC x MIC x MIC MSSA 12.5 Ly-Ly-Sc0 Time (hr) 6.25 um Ly-Ly-Sc0 25 um Ly-Ly-Sc0 Difficult to develop resistance against membrane targeted antibiotics 18

19 In vivo Testing MRSA Air 2 4 H o ur Pouch Po uch, Fem a e B A L B /c Model x 10^7 C F U s 2L 42 A 4 HC Hour,N = 8 m uch, ice/g Fem Fem ro a e up a e B BA AL B L B /c /c V C,#42 o r #14 D x 10^7 x C CF UF Us s L AL AC C,N,N = 8 = 8 m m ice/g ro ro up up V ieh VC C,#42 e t p o ep r o #14 r tide,100mm #14 D Dieh e t e t p at ep p ep t0 tid tid e,100mm at at t0 t0 Prepared a new synbody (14) with a larger scaffold to improve in vivo performance MRSA MIC = 25 mm Tested (14) at 4x MIC and negative control synbody (42) in a MRSA air pouch infection model(pamela Hall - U. New Mexico College of Pharmacy) (14) halted weight loss and reduced bacterial burden in a mouse model Po uch avage L o g C F U s /m L % W eight L o s s * VC Po Po uch uch avage avage L o g C F U s /m /m L #42 VC # VC *** 6 6 % W eight L o s s eight # VC VC 6 6 ** VC K id ey L o g C F U s /m L #42 #14 # # VC #14 *** *** #42 #14 K id ey L o g C F U s /m /m L K id ey #42 VC = 1x PBS *p<0.05 **p<0.01 ***p<0.001 #42 #14 44 ** 3 3 # VC VC #42 #42 ** ** #14 #14 Diehnelt, et al In preparation 19

20 Synbody Antibiotics Summary System to produce bactericidal and bacteriostatic antibiotics D-AA substitution for low cost protease stability Conjugation approach to synbody production Amino acid substitution to alter activity, selectivity and toxicity Rapid discovery and early stage development of new antibiotic candidates 20

21 Antiviral Synbody Development Virus Peptide Array Screen Candidate Peptides Synbody Library Synbody Screen viruses to identify binding peptides Screened 14 different enveloped and non-enveloped One-pot reaction conjugation strategy to produce synbody library Screen synbodies for protection in a cellular inhibition assay 21

22 Influenza Virus 3,000 to 52,000 deaths annually (US) 1918 Spanish Flu ~50,000,000 deaths worldwide H5N1 avian flu H7N9 avian flu Tao and Zheng Science (22 Nov 2012) science Enveloped, -ssrna nm diameter Current Treatments Seasonal Vaccine Tamiflu / Relenza 22

array Screened live virus, formalin inactivated, and UV

23 Peptide Discovery UV inactivated H1N1 Peptides in Common vs Sample type Live H1N1* Screened A/PR/8/34 H1N1 against 10,000 peptide array Screened live virus, formalin inactivated, and UV inactivated virus Good correlation between peptides selected with each sample 23

24 Peptides Scaffold Synbody Production HPLC Analysis of Reaction Scaffold Peptide Synbody (t=2 hr) Synbody (pure) Conjugation strategy with bi-functional scaffold (maleimide conjugation) Rapid, high yield reaction Modular multiple scaffolds can be prepared Generation of multiple synbodies from single reaction 24

25 In vitro Inhibition Assays Cytopathic Reduction Assay Transform of CPE - 04/6/11 Plaque Reduction Assay % Cytopathic Effect log [Synbody] % Virus Syn 5 IC 50 = 17 mm IC 50 = 17 um R2 = 0.78 ** log [Synbody] * Untreated Syn 5 Syn 75 Synbody IC 50 (um) 4.4 um 18 um Synbody candidates were screened for reduction of A/PR/8/34 killing of MDCK cells Syn (5) selected for development Synbody 5 tested in plaque reduction assay and reduced number of plaques with similar IC 50 25

26 Ladder Naïve Sera 5min 15min 30min 1hr 2hr 4hr 6hr Empty 10ng WT 100ng WT Protease Stability of Syn (5) Synbody Control Syn (5) Syn (5) incubated in fresh mouse sera At each time point, aliquot removed and added to protease inhibitor Synbody pulled down using streptavidin coated magnetic beads Recovered synbody detected by Western Blot Syn (5) showed little degradation by serum proteases after 6 hours 26 26

27 Toxicity Testing of Syn (5) In Vitro Toxicity Screening In Vivo Toxicity Test CC 50 RBCs (red blood cells) HEK293 (kidney) C3A (liver) THP-1 (monocyte) Hs68 (foreskin fibroblast) > 500 um > 100 um > 500 um > 500 um > 500 um Low in vitro toxicity BALB/c mice were administered either PBS (black) or 50 mg/kg (red) twice per day (i.p.) for 5 days No weight loss or other signs of acute toxicity observed 27

28 A) In vivo Challenge Syn (5) Weight Loss Survival Percent survival PBS + Flu PBS Alone Synbody + Flu Time BALB/c mice (n=10) were challenged with 1x LD95 of A/PR/8/34 Treated with either PBS (blue) or 50 mg/kg synbody (red) twice per day (i.p.) for 5 days Synbody increased survival but not significant (p = 0.06). Why? 28

29 Molecular Target of Synbody? 29

30 25 um Syn (5) 2.5 um Syn (5) 250 nm Syn (5) 25 nm Syn (5) 2.5 nm Syn (5) H1N1 Only 25 um Neg Syn Immune Sera No Virus Syn (5) Does Not Act on HA Does not Block Hemagglutination Does not Bind HA SPR of HA to Syn (5) 30

31 Syn (5) Binds Nucleoprotein (NP) SPR of NP Pull-down of NP Pull-down of NP from 2 different viruses A/PR/8/1934 (H1N1) 60 Response (RU) Time (sec) Signal K D = 63 nm R 2 = NP (nm) A/Sydney/5/1997 (H3N2) Virus Beads Synbody NP is highly abundant and conserved across Influenza A strains Syn (5) binds recombinant NP and NP from 2 different Influenza A strains 31

32 Syn (5) Inhibits Multiple Influenza Strains % Virus H1N1 Reduces NP Positive Plaques A/PR/8/ Reduces NP Positive Plaques from 2 strains % Virus * 60 ** ** 40 ** log Syn [M] :1,000 Polyclonal Sera * 20 0 ** ** A/PR/8/34 A/CA/7/09 Synbody Nucleozin Immune Sera *p < 0.05 **p < 0.01 NP is >93% conserved between A/PR/8/34 and A/CA/7/09 Syn (5) reduces NP positive plaques from both strains while Nucleozin (known NP) inhibitor does not due to an inactivating NP mutation Synbody possibly inhibits virus through NP 32

33 Influenza Summary Developed synbody that inhibited A/PR/8/34 in vitro and provided partial protection in vivo Synbody binds NP which is difficult to inhibit due to abundance and intracellular location 33

34 Conclusions Synbody platform can generate antibacterials and antivirals for therapeutic development Currently improving performance of synbody and integrating 330,000 peptide library in the discovery step 34

35 Rapid Anti-Infective Development DARPA funded project to develop a platform that could make a therapeutic to an unknown pathogen in 7 days Potential use in an Ebola outbreak scenario 35

36 10K, 124K and 330K Peptide Arrays Available Full Immunosignature Analysis on Samples Provided - Diagnostics - Vaccine Development - Epitope Binning of Monoclonals - Therapeutic Response Evaluation Synbodies Created to Provided Targets Custom Arrays Synthesized

37 Acknowledgements Chris Diehnelt John Lainson Andrey Loskutov Valeriy Domenyuk Nidhi Gupta Bart Legutki Phillip Stafford Zhan-Gong Zhao CIDV Aurelie Crabbe Cheryl Nickerson University of New Mexico Pamela Hall Seth Daly Funding DARPA New Antibiotics for Superbugs Crowdfunding Funds to SAJ 37

ynthetic and Systems iology orum on Microbial Threats arch Stephen Albert Johnston

ynthetic and Systems iology orum on Microbial Threats arch 14-15 2011 Stephen Albert Johnston Stephen.johnston@asu.edu HTP Screening Systems Based on Synthetic Genes and Proteins Sequence Genes Proteins