Preclinical characterization of LOXO-783 (LOX-22783), a highly potent, mutant-

selective and brain-penetrant allosteric PI3Kα H1047R inhibitor

Presented at: AACR-NCI-EORTC VIRTUAL INTERNATIONAL CONFERENCE ON MOLECULAR TARGETS AND CANCER

THERAPEUTICS Date: October 7, 2021

Preclinical characterization of LOXO-783 (LOX-22783), a highly potent, mutant-selective and brain-penetrant allosteric PI3Kα H1047R inhibitor

Anke Klippel1, Rui Wang1, Loredana Puca1, Andrew Lee Faber3, Weihua Shen3, Shripad V. Bhagwat3, Kannan Karukurichi1, Feiyu Fred Zhang4, Carmen Perez5, Ramón Rama-Garda5, Ana Ramos5, Yi Zheng3, Zahid Bonday3, James Thomas3, Harold B. Brooks3, Lisa J. Kindler3, Sarah M. Bogner3, Parisa Zolfaghari1, Mark Hicks II1, Sophie Callies6, Brian Mattioni6, Laurie LeBrun2, Jim Durbin6, Erin Anderson2, Christopher G. Mayne2, Edward A. Kesicki1, Gabrielle Kolakowski2, Steven W. Andrews2, Barbara J. Brandhuber2

1Loxo Oncology at Lilly, New York, NY, USA; 2Loxo Oncology at Lilly, Boulder, CO, USA; 3Loxo Oncology at Lilly, Indianapolis, IN, USA; 4Eli Lilly and Company, San Diego, CA, USA; 5Eli Lilly and Company, Alcobendas, Spain; 6Eli Lilly and Company, Indianapolis, IN, USA

Presenting author - Anke Klippel

I have the following financial relationships to disclose:Employee of Loxo Oncology at Lilly and stock-holder of Eli Lilly and Company

I will not discuss off label use and/or investigational use in my presentation

• Phosphoinositide 3-kinase alpha (PI3Kα) H1047R mutations are activating oncogenic events that occur in ~15% of

breast cancers and less commonly in other cancers.

• All approved and investigational PI3Kα inhibitors inhibit both wild-type and mutated PI3Kα with approximate equal

potency. As a result, their efficacy is potentially limited by on-target wild-type PI3Kα mediated toxicity including

dose-limiting hyperglycemia as well as cutaneous and GI toxicity.

• Here we describe the preclinical profile of LOXO-783, a potent, highly mutant-selective and brain-penetrant

allosteric PI3Kα H1047R inhibitor.

Background

LOXO-783 is a potent, highly mutant-selective, & brain penetrant allosteric PI3Kα H1047R inhibitor

LOXO-783 Alpelisib Inavolisib(GDC-0077)

Cell AlphaLISA

pAKT (pS473)

IC50 (nM)

PI3KαH1047R

T47D 3 64 14

SUM185PE 1.3 43 14

MDA-MB-453 5 49 16

PI3Kα WT SK-BR-3 286 128 NT

WT Selectivity ~90x 2.4x NE

Cell proliferation

IC50 (nM)

PI3KαH1047R

T47D 4 396 72

SUM185PE 3.2 356 44

MDA-MB-453 9 727 57

PI3Kα WT SK-BR-3 >300 285 104

WT Selectivity Upper bound of selectivity NE 1x 1.7x

Other propertiesCNS penetrance

Predicted to achieve meaningful brain

exposure

Target occupancy 1-3 h <10 mins <10 mins

5

Breast cancer cell lines used - T47D: PI3KαH1047R/WT (ER+, HER2-); MDA-MB-453: PI3KαH1047R/WT (ER-, HER2-); SUM185PE: PI3KαH1047R/H1047R (ER-, HER2-); SK-BR-3: PI3KαWT/WT (ER-, HER2+) NT: not tested; NE: not estimable

LOXO-783 (Tested at 3 µM)

PI3K isoform Residual enzyme activity(reference normal: 100%)

PI3Kα-H1047R(p110αH1047R/p85α) 5%

PI3Kβ(p110β/p85α) 110%

PI3Kγ(p110γ) 85%

PI3Kδ(p110δ/p85α) 103%

LOXO-783 has excellent selectivity for PI3Kα H1047Rover other PI3K isoforms and non-lipid kinases

• LOXO-783 is exquisitely selective for PI3Kα H1047R – complete enzymatic activity of PI3Kβ, PI3Kγ, PI3Kδ is preserved despite a high concentration of LOXO-783 (3 µM)

• LOXO-783 had no hits in panels of 17 lipid kinases or 368 non-lipid kinases

LOXO-783 selectively inhibits proliferation of PI3KαH1047R cells, but not other PI3Kα mutant or WT breast cancer cells in contrast to alpelisib

TGX-221 = p110β-specific inhibitor EC50 values adjusted for fetal bovine serum (FBS)-protein binding

• LOXO-783 has single digit nanomolar (<5 nM) potency inhibiting proliferation of H1047R breast cancer cell lines, markedly higher cellular potency than alpelisib (150-320 nM)

• LOXO-783 does not inhibit other PI3Kα WT/K111N mutant cell lines, or PTENmut cell lines, in contrast to alpelisib

0 1 2 3 4 5

0

50

100

150

Log Concentration (nM)

Inhi

bitio

n (%

) LOXO-783Alpelisib

BT-474, 10% FBS

0 1 2 3 4

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50

100

150

Log Concentration (nM)

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bitio

n (%

) LOXO-783Alpelisib

T47D, 10% FBS

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100

150

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bitio

n (%

) LOXO-783Alpelisib

SUM185PE, 5% FBS MDA-MB-453, 10% FBS

0 1 2 3 4

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Log Concentration (nM)

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bitio

n (%

)

LOXO-783Alpelisib

ZR-75-1, 10% FBS

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bitio

n (%

) LOXO-783AlpelisibTGX-221

SK-BR-3, 10% FBS

Cell Line (Alteration) LOXO-783 (EC50 nM)

PI3Kα H1047R cell lines

T47D (H1047R/WT) 2-3

SUM185PE (H1047R/H1047R) 2-3

MDA-MB-453 (H1047R/WT) 3-4

PI3Kα K111N, WT, or PTENmut cell lines

SK-BR-3 (WT/WT) 552

BT-474 (K111N/WT) 413

ZR-75-1 (WT/WT, PTENmut) 563

Mutant cell lines

Wild-type cell lines

LOXO-783 selectively inhibits PI3Kα signaling in PI3Kα H1047R mutant cell lines

Cell Line(Alteration)

LOXO-783pAKT inhibition

EC50 (nM)

Selectivity(H1047R : WT)

PI3Kα H1047R cell line

T47D (H1047R/WT) 1.5 - 2.5 NA

PI3Kα K111N or WT cell lines

SK-BR-3 (WT/WT) >1000 (max. tested) >400 - 666x

BT-474 (K111N/WT) >1000 (max. tested) >400 - 666x

• No inhibition of PI3K signaling in WT cells at high LOXO-783 concentrations• LOXO-783 potency in PI3Kα H1047R-mutant cells is not impacted by the presence or absence of WT allele

Treatment time: 4h Treatment time: 4h

pS6 (S235/236)

Vinculin

pAkt (S473)

pAkt (T308)

p110α

DMSO

SK-BR-3 (WT/WT)

Alpe

lisib

500

nM

DMSO

0.6 1.25 2.5 5

T47D (H1047R/WT)Al

pelis

ib20

0 nM LOXO-783 (nM)

125 250 500 1000

LOXO-783 (nM)

FBS-free medium

LOXO-783 demonstrates marked efficacy and a wide therapeutic index in a PI3Kα H1047R breast cancer PDX model

0 3 6 9 12 15 18 21 24 270

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Treatment Days

Tum

or V

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e (m

m3 )

**

ns

***

0 3 6 9 12 15 18 21 24 2720

22

24

26

28

30

Treatment Days

Bod

y w

eigh

t (g)

Vehicle ControlAlpelisib, 40 mpk, QDLOXO-783, 75 mpk, BIDLOXO-783, 150 mpk, BIDBaseline Tumor…..

ns Not Significant

• LOXO-783 shows significant tumor regression in a PDX model derived from a heavily pretreated breast cancer patient with no response to abemaciclib and fulvestrant combination treatment

• LOXO-783 significantly reduced tumor volume compared to the vehicle control at all dose levels

CTG-3401 PDX (H1047R+/-, ER+, HER2-) Body Weight Change

**

*p≤0.001 compared to vehicle control on day 27. Alpelisib is used at the human equivalent dose for this mouse strain (athymic nude). Data are mean ± SEM

Vehicle Control, QD x 14, POAlpelisib, 30 mpk, QD x 14, POAlpelisib, 50 mpk, QD x 14, POLOXO-783, 75 mpk, BID x 14, POLOXO-783, 150 mpk, BID x 14, POLOXO-783, 225 mpk, BID x 14, PO

*p<0.05 compared to alpelisib on day 14. All treatments significantly (p<0.05) reduced tumor volume compared to the vehicle control on day 14. **p<0.05 compared to vehicle. Data are mean ± SEM

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C-p

eptid

e (n

g/m

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cle,

QD

Alpe

lisib

50 m

pk, Q

D

**

75 m

pk, B

ID15

0 m

pk, B

ID22

5 m

pk, B

ID

Vehicle Baseline

LOXO-783, 8h

Alpe

lisib

30 m

pk, Q

D

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6

8

Insu

lin (n

g/m

L)Ve

hicl

e, Q

DAl

pelis

ib30

mpk

, QD

**

75 m

pk, B

ID15

0 m

pk, B

ID22

5 m

pk, B

ID

Vehicle Baseline

LOXO-783, 8h

Alpe

lisib

50 m

pk, Q

D

LOXO-783 achieves significant tumor regression in PI3Kα H1047R breast cancer xenografts without any increases in insulin or C-peptide

SUM185PE (PI3Kα H1047R/H1047R)

*Alpelisib exposure at 50 mpk dose in NSG mice is ~2x higher than human exposure at approved dose

• LOXO-783 shows greater efficacy than alpelisib at doses that do not cause any increase in plasma insulin or C-peptide

Plasma Insulin (ng/mL), NSG Mice(24 Days Repeat Dosing)

Plasma C-peptide (ng/mL), NSG Mice(24 Days Repeat Dosing)

0 2 4 6 8 10 12 140

100

200

300

400

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600

Treatment Days

Tum

or V

olum

e (m

m3 )

**

*

LOXO-783 demonstrates brain penetration in vivo

LOXO-783 CNS free brain penetrance in Rat

LOXO-783 is predicted to achieve meaningful brain target coverage of PI3Kα H1047R

LOXO-783 free brain exposure in NGS Mice

PI3Kα H1047R cell IC90

PI3Kα H1047R cell IC50

Brain and plasma were collected from 3 animals/group/timepoint and test article concentration was determined via LC/MS/MS

0.5h 2h 4h 8h 24h 8h 24h0

20

40

60

CN

S Pe

netra

nce

(%)

LOXO-783(1 mpk, IV)

LOXO-783(100 mpk, PO)

LOXO-783(300 mpk, PO)

LOXO-783, 117 mpk, PO

LOXO-783, 64 mpk, PO

Time (h)

LOXO-783 has intracranial efficacy in preclinical models

0 3 6 9 12108

109

1010

1011

Days Post Treatment

Tot

al F

lux

(p/s

)MDA-MB-453 luc (PI3Kα H1047R/WT) (athymic nude), day 12

Vehicle Control

Alpelisib, 30 mpk, QD

LOXO-783, 75 mpk, BID

LOXO-783, 150 mpk, BID

• LOXO-783 has dose-dependent tumor growth inhibition that exceeds alpelisib administered at maximum tolerated dose

Vehicle ControlAlpelisib, 30 mpk, QDLOXO-783, 75 mpk, BIDLOXO-783, 150 mpk, BID

Presenter: Anke Klippel, aklippel@loxooncology.com

Conclusions

• LOXO-783 is a potent, highly mutant-selective and brain-penetrant allosteric PI3Kα H1047R inhibitor

• LOXO-783 does not inhibit wild-type PI3Kα, other PI3K isoforms, or other kinases

• LOXO-783 causes significant tumor regressions in several ER+, HER2-, PI3Kα H1047R-mutant

breast cancer models without causing significant weight loss or any increase in insulin or C-peptide

• An IND submission is planned for 2022