Cogent Biosciences : Bezuclastinib Selectivity to KIT A Loop Mutations, Minimal Brain Penetration, and Favorable PK Properties In Preclinical Models

Bezuclastinib Selectivity to KIT A-Loop Mutations, Minimal Brain Penetration, and Favorable PK Properties In Preclinical Models

American Initiative in Mast Cell Diseases (AIM)

2022 AIM Physician and Investigator Conference

Salt Lake City, Utah, May 22, 2022

KIT Activation Loop Mutants are Key Targets for Systemic Mastocytosis and Refractory GIST

• KIT mutations serve as driver mutations in up to 80% of gastrointestinal stromal tumors (GIST) and in over 90% of systemic mastocytosis (SM)1,2
• In GIST, patients often relapse after front-line imatinib treatment due to secondary mutations in ATP-binding domain (ABD) or Activation Loop (AL)3

• 1. Second-linesunitinib is active against ABD mutations, but not AL mutations
• Inhibitors targeting AL mutations, notably D816V (a common AL mutation in SM), have shown activity in the treatment of advanced SM, but off-target toxicities of available compounds may limit optimal clinical dosing4, 5
• Bezuclastinib is a novel type I TKI that was developed as a highly potent and selective inhibitor of KIT D816V

ABD= ATP-Binding Domain; AL= Activation Loop

1. Klug LR et al., Nature Reviews Clinical Oncology, 2022:1-14; 2. Shomali W, Gotlib J. Hematology. 2018;2018(1):127-136; 3. Napolitano A, Vincenzi B, British Journal of Cancer. 2019;120(6):577-578; 4. RyDAPT [package insert]. East Hanover, New Jersey: Novartis Pharmaceuticals; 2021; 5. AYVAKIT [package insert]. Cambridge, MA: BluePrint Medicines; 2021.

Guarnieri et al, AACR Annual Meeting 2022.	2

Bezuclastinib is a Potent Inhibitor of KIT D816V, an Activation Loop Mutation

	Cell IC50 (nM)
Compound
KIT V560G/D816V
	(HMC 1.2)

			Bezuclastinib	14
			Avapritinib		13
			BLU-263		6
			Ripretinib		54
			Imatinib		>1000
			Sunitinib		>1000
			Regorafenib		>1000
HMC1.2 human mast cells (V560G/D816V) were treated with inhibitors for 1	IC50 values from ELISA in (A) in nM are represented for
hour followed by analysis for phosphorylated c-KIT ELISA (R&D Systems)	bezuclastinib and other KIT inhibitors

Guarnieri et al, AACR Annual Meeting 2022.	3

Bezuclastinib Demonstrates Superior Selectivity Against Closely Related

Kinases

• Bezuclastinib demonstrates no activity on closely related kinases, unlike other KIT inhibitors
• Inhibition of these closely related kinases have been linked to off-target toxicities, such as edema and pleural effusions1,2

Compound			Cell IC50 (nM)
PDGFRα	PDGFRß		CSF1R	FLT3	KDR
Bezuclastinib	>10,000	>10,000		>10,000	>1000	>1000
Avapritinib	53	10		249	305	>1000
BLU-263	21	6		161	345	>1000
Ripretinib	20	34		312	534	110
Imatinib	75	247		1027	>1000	>1000
Sunitinib	23	14		313	1	4
Regorafenib	138	1180		473	237	101

The table displays IC50 values (nM) for the closely related kinase assays. Color key displays where the fold change of these values vs. on-target KIT activity falls. On- target KIT activity was calculated with the following information for each KIT inhibitor: Bezuclastinib (KIT D816V = 14nM), Avapritinib (KIT D816V = 13nM), BLU-263 (KIT D816V = 6nM, Ripretinib (KIT D816V = 54nM), Imatinib (KIT V560G, HMC.1.1 cells = 10.7nM3), Sunitinib (KIT JMD/T670I GIST T1 5R cells = 8.8nM), and Regorafenib (KIT K642E = 20nM4)

Key: Fold change from on-target KIT activity

≤ 10x

10x-30x30x-50x50x-100x > 100x

Color key displays where the fold change of these values vs. on-target KIT activity falls. On-target KIT activity was based on potency presented in prior slide.

1. Giles FJ et al, Leukemia. 2009;23(10):1698-1707; 2. Liu S, Kurzrock R. Seminars in Oncology. 2015;42(6):863-875 3. Smith BD et al., Cancer Cell. 2019;35(5):738-751;

1. Wilhelm S et al,Molecular Cancer Therapeutics. 2007;6(11_Supplement): B260-B260;

Guarnieri et al, AACR Annual Meeting 2022.	4

Bezuclastinib Demonstrates Minimal Brain Penetration

17 May 2022 Final Draft

25 mg/kg Bezuclastinib	2000
10000
	1500

30 mg/kg BLU-263

2000

1 0 0 0 0

1500

ng/mL

1000
100
10
0	10	20	30
		Time (hr)

1000

500

0

Plasma Brain

Day 3 (4hr)

ng/mL

ng/mL	1 0 0 0
			1000
	1 0 0			500
	1 0			0
	0	1 0	2 0	3 0	Plasma Brain
			Time (hr)

t1/2 (h)	Cmax (ng/mL)	AUClast (ng∙hr/mL)	Brain:Plasma ratio
7	2592 ± 364	21509 ± 2558	0.07

t1/2 (h)	Cmax (ng/mL)	AUClast (ng∙hr/mL)	Brain:Plasma ratio
13	672 ± 102	6980 ± 1156	2.0

t1/2 (h)	Cmax (ng/mL)	AUClast (ng∙hr/mL)	Brain:Plasma ratio
4	1470 ± 222	16200 ± 414	0.18

• Bezuclastinib shows minimal brain penetration with brain to plasma ratio of 0.07 compared to 2.0 for avapritinib
• The absence of brain penetration is a preferred feature for a KIT A-Loop inhibitor given the CNS-related adverse events that have been observed in this targeted class1,2
• In a separate neurobehavioral (CNS) safety pharmacology study, rats were treated with oral doses of 0, 5, 25, or 100 mg/kg of bezuclastinib. No effect on behavioral endpoints were observed in this study, or in repeat dose toxicology studies (Data on File)

To assess brain distribution, male Sprague Dawley rats were administered 25 mg/kg bezuclastinib SDD, 5 mg/kg avapritinib, or 30 mg/kg BLU-263 by oral gavage. Dose levels were selected to correlate with clinical exposures reported in human clinical studies. Plasma samples were collected after a single dose and assessed for drug concentration by LC-MS/MS. Animals were dose administered for 2 additional days and plasma/brain harvested 4 hr post final dose. This repeat-dose administration - rather than single dose- allowed for a proper survey of steady state brain levels.

1. RyDAPT [package insert]. East Hanover, New Jersey: Novartis Pharmaceuticals; 2021; 2. AYVAKIT [package insert]. Cambridge, MA: BluePrint Medicines; 2021

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