Virus synthesis as a tool for assembling large virus vaccines

David Evans, PhD Professor and Vice-Dean Research Dept. of Medical Microbiology & Immunology and Li Ka-Shing Institute of Virology University of Alberta

Disclosures: DE is an author of relevant patents and is entitled to, or receives, financial compensation for his contributions to these intellectual properties. DE is also a paid consultant to Tonix Pharmaceuticals Ltd. These activities have been reported to the University of Alberta as required by COI and COC policies. Poxviruses The world’s first vaccines

Yaba tumour virus Myxoma virus SFV

Sheep pox Monkeypox Vaccinia Variola Cotia virus

Swinepox

Fowlpox

A. moorei EPV C. biennis EPV Smallpox

• Variola virus causes human smallpox • Not as infectious as people think, but mortality is 2-50% • An ancient disease with great historical impact • The earliest medical description is from China (~340 AD) where Ko Hung described “marauder’s pox” and a possible date of ~45 AD Smallpox and the origins of vaccination Variolation predates vaccination by centuries

• Resemblance to camelpox virus suggests variola arose in Asia • By ~1550 Chinese texts described how variolation, with scab material, could immunize against smallpox • Variolation may have much earlier Taoist origins, known secretly for ~500 yrs • Knowledge passed to the middle east and Europe by the 1700s Needham Smallpox eradication • Variolation came to Britain by the early 1700s, but even the most skilled variolator killed 1- 2 in 1,000 • Jenner’s vaccination replaces variolation after the 1790’s • Vaccinia virus was ultimately used to Edward Jenner (1749-1823) eradicate smallpox Vaccinia virus (VACV) The prototypical poxvirus

• Large (~200 kb) double- stranded DNA virus. • The many encoded proteins permit a wide host range and regulate virulence ~250 nm • Origin unclear, not cowpox, possibly a horsepox. • Many different strains were once used worldwide as smallpox vaccines. http://www.twiv.tv/virus-structure/ Application of vaccinia virus research

• Oncolytic viruses (Univ. of Alberta) – EVANS, D.H., and Gammon, D. (2009) “Oncolytic viruses and methods for treating neoplastic disorders”. Issued as US Patent #8,679,509 on 25/04/2014. – Continuation filed in the USA and other countries. Issued as US Patent #9,370,550 on 21/06/2016 and in the EU as #10796626.9 on 19/04/2017. – Currently in GMP manufacturing. • Synthetic poxviruses (Univ. of Alberta & Tonix Pharmaceuticals Ltd.) – Lederman, S, EVANS, D.H., and Noyce, Super-resolution image of a R. (2016) “Synthetic chimeric BSC-40 cell infected with poxviruses”. US provisional application ~4300 fluorescently-tagged vaccinia virus. #62416577, filed on 02/11/2016. RNA

RNA polymerase

GTP, ATP, UTP, CTP

(d)NDP kinases Oncolytic viruses Guanylate kinase dThd NDP GMP Goal: A better treatment (WR182) for bladder cancer Thymidine Ribonucleotide kinase reductase • Mutations in cancer cells (J2) (F4+I4) can render tumours

sensitive to virus infection Thymidylate Thymidylate dGMP • Our approach deletes dTMP dNDP + kinase kinase dUMP vaccinia virus genes to (A48) (A48) Thymidylate make the virus safer and (d)NDP kinases synthase more cancer selective than

existing oncolytics dUTPase dUMP dUTP + dGTP, dATP, dTTP, dCTP • Most oncolytic VACV (F2) encode J2R mutations DNA polymerase • Our virus encode (E9) ribonucleotide reductase Key: (F4L) mutations, why? Red - virus encoded DNA Black – cell Blue - metabolite Gene expression profiles in cancer cells Percent of analyses where expression was altered relative to normal cells

Top 1% of genes upregulated SAMHD1 (313) Top 5% of genes upregulated Top 10% of genes upregulated MYC (376) Top 10% of genes downregulated DGUOK (362) Top 5% of genes downregulated Top 1% of genes downregulated TK2 (352) CMPK1 (273) GUK1 (365) dUTPase (359) TYMP (299) DCTD (370) DCK (361) TMPK1 (359) TYMS (364) TK1 (342) p53R2 (209) R2 (358) R1 (358)

-10 0 10 20 30 Irwin Chad

Percent of Analyses where gene expression in cancer cells was significantly changed relative to normal cells RNA

RNA polymerase

GTP, ATP, UTP, CTP

(d)NDP kinases Oncolytic viruses Guanylate kinase dThd NDP GMP Goal: A better treatment (WR182) for bladder cancer Thymidine Ribonucleotide kinase reductase (J2) (F4+I4) • Product: Our two patents protect UAB-211 encoding Thymidylate Thymidylate dGMP dTMP dNDP + truncated (inactive) forms kinase kinase dUMP of the small R2 (F4) (A48) (A48) Thymidylate (d)NDP kinases subunit of the viral synthase ribonucleotide reductase. dUTPase dUMP dUTP + dGTP, dATP, dTTP, dCTP • DF4L + DJ2R::F4LDR1BD (F2) DNA polymerase (E9) Key: Red - virus encoded DNA Black – cell Blue - metabolite Bladder cancer: An unmet need Common, expensive, and deadly

• The 5th most common cancer in North America; 82,000 cases and 18,000 deaths in 2015 • Immunologically “hot” tumour • Non-muscle invasive bladder cancer is treated by surgery, but 60-70% of cases recur within 2 years and 25% of these progress • Few treatment gains in >30 years. • High system costs. A high recurrence rate necessitates years of subsequent monitoring • Our goal: Develop UAB-211 as a safe and effective early stage treatment for bladder cancer. Pre-clinical example: Immune competent, orthotopic rat model Tumour model: • AY-27 (+/-) luciferase rat cell lines • Cells deposited by catheter into the bladder lumen, 100% tumour take • Tumours confirmed by cystoscopy, palpation, and/or luciferase imaging Virus treatment: • 3 doses of VACV over 3 days • Virus delivered by catheter

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0 0 25 50 75 Days Post Tumor implantation Other observations and current activities See also Potts et al. (2017) EMBO Mol. Medicine

Other features: Patient-derived xenograft

1500 • Cured animals exhibit anti-tumour PBS UV-inactivated Virus immunity (implant resistant and ΔF4L/ΔJ2R

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u • Virus infects BCG-resistant T cancer cells, using a different 0 entry scheme 0 10 20 30 40 50 60 70 80 90 Days Post Treatment Timelines

2017 2018 2019 2020 2021

GMP manufacturing (OHRI) GLP tox? Phase I (NACTRC) Phase 2

Pre-CTA (Health Canada) CTA (Health Canada) Funding: Alberta Innovates + BioCanRx + CIHR UAB-211 construction Current technologies present a technical challenge

• UAB-211 was Parent strain Vaccinia strain WR (wild type) assembled using

standard recom- Knock YFP-gpt Plaque purify Select YFP+ bination methods into F4L locus (∆F4L::YFP-gpt)

• The process is Delete marker Select white Plaque purify with Cre plaques (∆F4L) involved and recombinase required two Knock Plaque purify rounds of marker truncated F4L Select YFP+ (∆F4L ∆J2R::YFP-gpt ∆R1BD removal with Cre gene into J2R F4L ) Delete marker recombinase Select while Plaque purify with Cre plaques (∆F4L ∆J2R::F4L∆R1BD) • Two lox sites are recombinase left behind UAB-211 ∆F4L ∆J2R::F4L∆R1BD Synthetic Orthopoxviruses Using gene synthesis to manufacture a horsepox virus (HPXV) Why horsepox? • The closest molecular relative to vaccinia virus is horsepox • The historical literature suggests Jenner’s vaccine was an equine disease called “Grease” • Might HPXV be a better vaccine or vector, unaffected by 200 years of passage in animals and humans? The problem: The disease is extinct and the one known HPXV strain is unobtainable. The solution: Make it. Vaccinia strains Genome sequencing

TianTan strains

Dryvax

Horsepox

~1% drift Qin et al. J. Virol. (2014) Qin, Liang, and Evans. Virology (2013) Synthetic Orthopoxviruses Using gene synthesis to manufacture a horsepox virus (HPXV) Why horsepox? • The closest molecular relative to vaccinia virus is horsepox • The historical literature suggests Jenner’s vaccine was an equine disease called “Grease” • Might HPXV be a better vaccine or vector, unaffected by 200 years of passage in animals and humans? The problem: The disease is extinct and the one known HPXV strain is unobtainable. The solution: Make it. Reactivating

poxviruses - • Poxvirus DNA is not infectious, but… • A cell infected with one virus 2003;77:7281 .

can recombine and reactivate Virol another • Shope fibroma virus can reactivate vaccinia virus DNA (Yao and Evans, 2003) • The process is recombinogenic and will stitch together

overlapping DNA fragments. H. David J. and Yao, Evans Dan

-

Xiao 7290 Genome assembly (212 kbp) IP: Mostly relates to constructing the telomeres

31.9 kbp YFP-gpt marker (in TK) 8.5 kbp

HPXV YFP-gpt::095

Sequence: GenBank entry DQ792504; DNA: GeneArt VACV Results (WR)

• Multiple clones were recovered Dryvax • The genome sequences were (DPP15) identical to the input DNAs • Our HPXV forms small plaques as reported for the native virus, grows well. CPXV • We deleted the YFP-gpt (B. red) marker and restored the wild-type TK locus. • Product: A synthetic and chimeric horsepox virus HPXV plus the method for making (YFP) other Chordopoxviruses. HPXV: Biological properties Less virulent than Dryvax, produces sterilizing immunity

Intranasal vaccination and challenge Kaplan-Meier survival plot

challenge

challenge Applications Flexible and accurate synthetic virus assembly

• “Designer viruses” bearing multiple genetic modifications • Parallel assembly of candidate vaccines for functional screening purposes • High-throughput assembly of personalized cancer therapeutics • Animal vaccines with superior embedded technologies

Chikungunya particles vectored by a DF4LDJ2R vaccinia virus. Constructed using traditional recombination methods. Inset from Hikke et al. (2016) Biotech. J.. Courtesy Dr. L. Nagata. Next steps What’s still needed?

Ongoing investigations: The most curious fact that these • Assembling other Orthopoxviruses experiments have discovered, is the property which the matter of • R&D relating to telomere structure Grease possesses, of communicating a disease to the human body which and improved reactivation efficiency will prevent the action of the the • Cancer neo-antigens small-pox, whether it be used genuine from its original source, or • Animal vaccines (with the Centre for be made to pass a more circuitous route. Foreign Animal Diseases in Winnipeg) Loy, 1801 • HPXV immunology and manufacturing

What’s still needed? • Regulatory clarity (DURC) • Commercialization strategy (with Dr. S. Lederman/Tonix Pharmaceuticals) Collaborators: Mary Hitt (Dept. of Oncology, University of Alberta) Seth Lederman (Tonix Pharmaceuticals, New York) Ron Moore (Dept. of Surgery, University of Alberta)

Advice and guidance: John Bell (OHRI, Ottawa) Thanks TEC Edmonton Northern Alberta Clinical Trials & Research Centre

Past and present lab members, notably: Don Gammon Chad Irwin Funding: Ryan Noyce Alberta Cancer Foundation Kyle Potts Alberta Innovates (Applied virology fund) David Willer BioCanRx Xiao-Dan Yao Canada Foundation for Innovation Canadian Institutes for Health Research Li Ka-Shing Institute of Virology Natural Sciences & Engineering Research Council Tonix Pharmaceuticals Ltd.