Small-molecule-regulated gene circuit for controlling ...

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Small-molecule-regulated gene circuit for controlling cytokine expression in cell therapiesMichelle Hung1, Divya Israni2, Huishan Li2, Yin Yin Chong1, Poornima Ramkumar1, Allison Drain2, Allison Quach1, Mengxi Tian1, Rishi Savur1, Niran Almudhfar1, Chen-Ting Lee1, Abla Bakir1, Edwin Cruz1, Carmina Blanco1, Travis Wood1, Mario Lorente1, Brandon Lee1, Brett Kiedaisch1, Russell M. Gordley1, Marcela Guzman Ayala1, Ahmad Khalil1, Gary Lee1, Tim Lu1

1 Senti Biosciences, Inc.

2 Boston University

1

Presented by: Michelle Hung

May 14, 2021

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SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES

Disclosures

o Michelle Hung is a paid employee of Senti Biosciences, Inc

o This presentation included verbal remarks by the presenter that are not included here

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Challenge


Executive Summary

Our Solution

Design and Build Regulator Dial – a gene circuit to regulate production of potent immune effectors using FDA-approved small molecule drugs

Test Regulator Dial in primary immune cells and Learn how different properties of the gene circuit can be optimized to achieve desired results

Optimized Regulator Dial gene circuit enabled Grazoprevir dose-dependent control of IL-12 production in vivo

Safely improve efficacy of cell-based immunotherapies for solid tumors

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PersistenceImmune Cell

Recruitment and Tumor Killing

Direct Killing

Engineered Cell Engineered Cells Cancer Cell

Engineered CellCancer Cell

Attack

Why arm cell-based immunotherapies with cytokines?

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Controlling IL-12 arming in cell-based immunotherapies


The benefit of controlling the arming of cell-based immunotherapies

Dose Controlled Armed CAR-immune cells (IV)OFF

Dose Small Molecule Drug (Oral)

Little/no immune effectors secreted in absence of small molecule drug

Presence of small molecule triggers controlled secretion of immune effectors in the tumor

ON

o IL-12 is a highly potent immune activator with the potential to stimulate the tumor immunity cycle

o Overexpressing IL-12 from adoptive T cell therapies using a poorly regulated promoter has resulted in significant clinical toxicities (Zhang et al., Clin. Can. Res. 2015)

o Narrow therapeutic window associated with IL-12 has limited success to date

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Regulator Dial Transcription Factor Parts Controlled Payload

Regulator Dial is intended to enable control of gene expression via FDA approved small molecules

o Regulator Dial Transcription Factor uses a highly specific ZF DNA binding domain linked to an activator that drives target gene expression without impacting the rest of the transcriptome

o Controlled payload is a cytokine driven by a promoter that contains a binding site for the Regulator Dial DNA binding domain

cytokine payload

Clinically-driven design of synthetic gene regulatory programs in human cells. Divya V. Israni, Hui-Shan Li, Keith A. Gagnon, Jeffry D. Sander, Kole T. Roybal, J. Keith Joung, Wilson W. Wong, Ahmad S. Khalil. bioRxiv 2021.02.22.432371

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o Regulator DNA binding domain is linked to the transcriptional activation domain by a protease cleavable linker

o When the protease is active, the DNA binding domain is not linked to the transcriptional activation domain, and is unable to activate transcription

o Cytokine payload is OFF

cytokine payload


Absence of Small Molecule Drug

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o NS3i small molecule suppresses protease activity, resulting in an intact transcription factor

o Regulator Dial transcription factor can activate transcription

o Cytokine payload is ON


cytokine payload

+ NS3 inhibitor


Presence of Small Molecule Drug

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Ideal properties of Regulator Dial



o Versatile: transcriptional regulation has the potential to control any payload of interest; can regulate multiple payloads

o Safe: small molecule is FDA approved (hepatitis drug); NS3 protease incorporates mutations to avoid immunogenicity

o Dose Dependent: payload level depends on small molecule dose

o Convenient: orally-dosed favorable PK profile


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Can Regulator Dial control gene expression in primary immune cells?

Fluorescent reporter

Cells transduced with lentivirus encoding Regulator Dial

- ASV + ASV

o NS3 inhibitors are a family of well tolerated, orally available, FDA-approved small molecules

o Here, Asunaprevir (ASV) was the NS3 inhibitor used

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Regulator Dial induced a 3.3-fold increase in fluorescent reporter upon small molecule treatment

- ASV + ASV

initial Regulator Dial

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+ small molecule


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- ASV + ASV

High basal activity is potentially toxic for potent immune effectors such as IL-12

initial Regulator Dial

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o Initial regulator dial promoter had high basal activity, which could be potentially toxic if a potent immune effector such as IL-12 is being controlled

o We identified alternative minimal promoters that could have lower basal activity

Can promoter optimization reduce basal activity of the Regulator Dial?

optimize

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Can promoter optimization reduce basal activity of the Regulator Dial?

optimized

Basal expression was reduced by 20-fold upon promoter optimization

- ASV

leak

y Reg

ulat

or D

ial

optim

ized

Reg

ulat

or D

ial

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optimized

Optimized Regulator Dial induced an 11-fold increase in fluorescent reporter upon small molecule treatment

- ASV + ASV

How well does the optimized Regulator Dial induce gene expression?

tuned Regulator Dial

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Regulator Dial Small molecule sensitivity

Regulator Dial small molecule sensitivity

0.01 0.1 10

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Asunaprevir NS3i Concentration (uM)

Flu

ore

scent re

port

er

(AU

)

Regulator Dial

small molecule sensitivity


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Regulator Dial small molecule sensitivity

0.01 0.1 10

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Flu

ore

scent re

port

er

(AU

)

Regulator Dial

small molecule sensitivityASV Cmax


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Senti’s Design, Build, Test, Learn cycle was applied to improve Regulator Dial small molecule sensitivity


0.01 0.1 10

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(AU

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Regulator Dial

small molecule sensitivityASV Cmax

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ASV Cmax

0.01 0.1 10

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scent re

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(AU

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Regulator Dial


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ASV Cmax

0.01 0.1 10

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scent re

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(AU

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Regulator Dial


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0.01 0.1 10

0

20000

40000

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80000

uM Asunaprevir

Flu

ore

scent re

port

er

(AU

) Asunaprevir sensitivity

Regulator Dial sensitivity to alternative NS3 inhibitors

Multiple NS3i small molecules were chosen based on IC50s against NS3 and human PK data

ASV Cmax

0.01 0.1 10

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uM Danoprevir

Flu

ore

scent re

port

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(AU

) Danoprevir sensitivity

DPV Cmax

0.01 0.1 10

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uM Grazoprevir

Flu

ore

scent re

port

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(AU

) Grazoprevir sensitivity

GRZ Cmax

Regulator Dial showed high sensitivity to Grazoprevir at clinically relevant levels

Asunaprevir

sensitivity

Danoprevir

sensitivity

Grazoprevir

sensitivity

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Regulator Dial enables control of IL-12 production in primary immune cells

Regulator Dial dose-dependently controlled IL-12 production in primary immune cells at clinically relevant concentrations of Grazoprevir

IL-12IL-12

IL-12

0.01 0.1 10

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25000

uM Grazoprevir

pg/m

L IL-1

2

Cmin Cmax

Regulator Dial control of IL-12

Cmin to Cmax are the average minimal and maximal serum concentrations of GRZ in patients treated daily with the clinical dose (ZEPATIER ®)

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In Vivo Regulator Dial control of hIL-12 production

0 1 2 3 4 8

collect plasma

cardiac puncture

Day

Regulator Dial cells

dosing

Grazoprevir dosing

Vehicle 50mg/kg 75mg/kg10

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IL12 p

g/m

L

Day 4

Vehicle 50mg/kg 75mg/kg10

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IL12 p

g/m

L

Day 8

IL12

Exp

ress

ion

Lev

el (p

g/m

L)

IL12

Exp

ress

ion

Lev

el (p

g/m

L)

Regulator Dial enabled dose dependent and reversible IL-12 production in vivo

In vivo

Study Design hIL-12 in mouse plasma

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Armed, Controlled Therapy enabled by Regulator Dial


Regulator Dial could enable us to control expression of potent immune effectors for immune cell therapies

Regulator Dial has the potential to enable the following benefits:

o Optimized for safe, low expression of potent cytokines in the absence of small molecule drug and strong, dose dependent induction of cytokine production in the presence of small molecule drug

o Versatile to regulate potentially any payload of interest

o Convenient regulation by orally dosed, FDA-approved small molecule

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Acknowledgements

Thank you to the fantastic team at Senti Biosciences

and our collaborators in the Khalil labat Boston University!

See our other Senti Abstracts:Title: Precise Targeting of AML with First-in-Class OR / NOT Logic-Gated Gene Circuits in CAR-NK CellsGarrison et al. (abstract 77)Title: Precise Tumor Targeting with NOT Logic-Gated Chimeric Antigen Receptor Gene CircuitsFrankel et al. (abstract 960)

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