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• Joe Gozgit• Ribon Therapeutics

RBN-2397: A potent and selective small molecule inhibitor of PARP7 that induces tumor-derived antitumor immunity dependent on CD8 T cells

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Disclosure Statement• I am an employee and shareholder of Ribon Therapeutics

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Targeting PARP7 to Restore Tumor-Derived Type I Signaling is a Novel Therapeutic Strategy in Cancer

• Engaging cytosolic nucleic acid sensing and the Type I interferon (IFN) response is an emerging therapeutic strategy

• Stimulate production of cytokines to promote an adaptive immune response

• Currently, most approaches involve agonistic modulation of the tumor microenvironment

• PARP7 is a monoPARP regulated by cancer relevant stresses

• Amplified in cancers with strong smoking association

• Acts as a “brake” on cytosolic nucleic acid sensing and suppresses Type I IFN signaling

Targeting a negative regulator of tumor-produced Type I IFN is a novel therapeutic strategy

PARP7 acts as a fundamental regulator of intrinsic stress support pathways and is a novel tumor

vulnerability in cancer cells

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Aberrant nucleic acids can be detected by various sensing mechanisms including cGAS/STING and RIG-I

PARP7 Acts as a Brake on Cytosolic Nucleic Acid Sensing and the Type I IFN Response

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Suppression of IFN-β by PARP7

Overexpression of PARP7 suppresses IFN-βresponse to dsDNA

Synthetic dsDNA

PARP7 has been reported to negatively regulate the Type I response by interacting with TBK1 during viral

infection (Yamada et al., 2016)

HEK293T cells transfected with PARP7 treated with synthetic double stranded (ds)-DNA for 24 hours

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RBN-2397 is a Potent and Selective Inhibitor of PARP7

• RBN-2397 is a potent inhibitor of PARP7• Binds to PARP7 in the NAD+ binding pocket

with key interactions in adenosine sub-pocket driving potency and selectivity

• Sub-nanomolar biochemical activity

• RBN-2397 displays selectivity to PARP7• >50-fold selective vs. PARP family• No inhibition in kinase panel (1 µM)

• Drug-like properties support oral dosing in humans

• First in human Phase I multi-center clinical trial is underway (NCT04053673)

NNH

F3CO

HNO N

O

N N

NCF3

Structure of RBN-2397

Co-crystal structure of RBN-2397 bound to PARP12/7

PARP12 was used as a surrogate for PARP7. Four labeled residues were mutated from PARP12 to match the PARP7 sequence

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PARP7 (MAR)IC50 = 0.002 µM

PARP1 (PAR)IC50 = 0.6 µM

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PARP1 (PAR)IC50 = 0.003 µM

PARP7 (MAR)IC50 = 2.6 µM

RBN-2397 Potently and Selectively Inhibits PARP7-Dependent Activity Compared to PARP1

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Vyas et al., 2013

• PARPs regulate their cellular function by modifying target proteins with ADP-ribose

• PolyPARPs (e.g., PARP1) attach polymers of ADP-ribose units (PARylation)

• MonoPARPs (e.g., PARP7) modify proteins with a single unit of ADP-ribose (MARylation)

PARP family consists of 17 members:2 subclasses based on catalytic activity

RBN-2397 potently inhibits MARylation with a 300-fold window over PARylation

PARP1-H2O2 activated Hela cells (PAR)PARP7-overexpressing SK-MES-1 cells (MAR)

PARP1 inhibitor: Niraparib

PARP7 inhibitor: RBN-2397

Lu et al., 2019

PARMAR

24-hour treatment

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RBN-2397 Restores Cytosolic Nucleic Acid Sensing in the Mouse CT26 Cancer Cell Line

Selective induction of CXCL10

RBN-2397 restores Type I IFN response in CT26 cells

Induction of pSTAT1

Restoration of Type I IFN response is measured by an increase in STAT1 phosphorylation and interferon

stimulated genes (ISGs)

PARP7 inhibition “releases the brake” on cytosolic nucleic acid sensing and induces Type I IFNs

e.g. CXCL10

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RBN-2397Inactive analogOlaparib

Compound (nM) 24-hour treatment

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RBN-2397 Restores Cytosolic Nucleic Acid Sensing Dependent on Pattern Recognition Receptor Signaling

RBN-2397 restores Type I IFN signaling through pattern recognition receptor pathway

Pharmacological inhibitors used to investigate the role of PARP7 in suppressing Type I IFNs

e.g. CXCL10

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XX

X

JAKTBK1 STING

BX795: TBK1 inhibitorRuxolitinib: JAK inhibitorC-178: STING inhibitor

Blockade of:

24-hour treatment

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RBN-2397 Induces Tumor-Specific Adaptive Immune Memory in CT26 Syngeneic Model with Durable Complete Responses

CT26 re-challenge 4T1 re-challenge

• Once daily oral dosing of RBN-2397 in CT26 tumor-bearing BALB/c mice

• Tumor-free mice were monitored for 60 days

• Tumor-free mice re-challenged with CT26 and subsequently 4T1 cells

• All tumor-free mice rejected CT26 cells but not 4T1, demonstrating induction of tumor-specific adaptive immune memory

Primary Efficacy: RBN-2397 induces durable regressions Re-challenge of tumor-free mice: Rejection of CT26 cells

TF: Tumor free miceAll groups co-dosed with ABT

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CT26 mouse model Innate immune cells

Adaptive immune cells

Mouse Strain

RBN-2397Efficacy NK Mac B T DC

BALB/cTumor

regression + + + + +

NOG

~50% TGI - -a - - -a

Adaptive Immune Response is Indispensable for RBN-2397 Antitumor Activity

RBN-2397 shows substantially reduced activity in CT26 tumor-bearing immunodeficient mice

Characterization of immune cell populations present in BALB/c and NOG mice

+: Present; -: Absenta: Reduced macrophage and dendritic cell function

NOG: Immunodeficient

All groups co-dosed with ABT

9 tumor-free mice

BALB/c: Immunocompetent

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Robust Depletion of Immune Cell Populations in CT26 Tumor-Bearing Mice

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Tumor implantation

Administration of blocking antibodies to deplete CD4, CD8

and NK cells

RBN-2397 dosing

Efficacy readout

Monitor immune cell populations

BALB/c

Scheme for immune cell depletion in CT26-tumor-bearing BALB/c mice

Specific depletion of immune cell populations

Blood absolute counts

Tumor absolute counts

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CD8 T Cells Are Essential for the Antitumor Immunity Induced by RBN-2397 in the CT26 Syngeneic Model

CD4 T cell depletion had no effect on antitumor activity

NK cell depletion had no effect on antitumor activity

CD8 T cell depletion attenuated antitumor activity

Triple depletion was not different than CD8 T cell single depletion

All groups co-dosed with ABT

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RBN-2397 Induces Type I IFN Signaling and Enhances Immune Markers in CT26 Tumors

Increase in GrzB on CD8 T cells

RBN-2397 shows dose-dependent effects on PD markers in CT26 tumors

RBN-2397 enhances antigen presentation and T cell activation in tumor-infiltrating immune cells

Increase in MHCII on DC1 cells Increase in CD86 on DC1 cells

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CT26 tumor-bearing mice administered as single oral dose.All groups co-dosed with ABT

CT26 tumor-bearing mice dosed with RBN-2397 500 mg/kg. Tumors collected on days 3, 6 & 12.

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IFN-β CXCL10 IFIT1 MX10

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• Ablation of tumor TBK1 nearly eliminates the antitumor activity of RBN-2397

• Tumor-produced IFN-β is the source of the innate immune activation and crucial for antitumor activity

• Blockade of tumor and host IFNAR1 signaling prevents the antitumor activity of RBN-2397

• Suggests contribution of immune system through activation of IFN signaling in immune cells by tumor-produced IFN-β

All groups co-dosed with ABT

Type I IFN Produced by Tumors in Response to RBN-2397 Plays a Major Role in the Development of Durable Antitumor Immunity in CT26 Model

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Tumor TBK1

Tumor + Host IFNAR1

Tumor IFNAR1

CT26 wild type CT26 TBK1 KO CT26 IFNAR1 KO + anti-IFNAR1

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RBN-2397 is the First Potent and Selective PARP7 Inhibitor to Enter Clinical Development

• Targeting PARP7 to restore tumor-derived Type I signaling is a novel therapeutic strategy in cancer

• Inhibition of PARP7 induces antitumor immunity dependent on tumor-produced Type I IFN and CD8 T cells

• RBN-2397 is the first agent targeting this cancer vulnerability to enter clinical development

Complete regressions and antitumor immunity as a single agent

IFNUncloaks the tumor cells and recruits immune cells

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PARP7 acts as a “brake” on cytosolic nucleic acid sensing and suppresses the Type I IFN response

Ribon PARP7 abstracts at AACR 2021#381: PARP7 expression in cancer#1021: PARP7 inhibitor mechanism of action studies

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Acknowledgements

Victoria RichonAndy SantospagoLaurie SchenkelRichard SchroederPrashant ShambharkarJeff SongTad StewartKerren SwingerLuke UtleyZacharenia VarsamisMelissa VasbinderTim WigleJodie Wong

Ryan AboEllen BambergDanielle BlackwellRichard BushellAnne CheungW. David ChurchLisa ClearyDavid CordoBryan DorseyJennifer DowningJoseph GozgitLinette GreyBin GuiHeike KeilhackPeter KimDanielle KnightKaiko Kunii

Team Ribon:Kevin KuntzKristy Kuplast-BarrJenkins LemeraChang LiuAlvin LuAhmed MadyChristina MajerKristen McEachernMaegan MikulaElena MinissaleJason MoJennifer MolinaSunaina NayakMario NiepelSudha ParasuramanNicholas PerlYue Ren

Paul ChangLee KrausTimothy Mitchison

James AudiaLarry LaskyPatricia Rao

Founders and Advisors:

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