Introduction

The development of LOXO-195, a second generation TRK kinase inhibitor that overcomes acquired resistance to first generation inhibitors in patients with TRK-fusion cancers

J. Blake1, G.R. Kolakowski1, B. Tuch2, K. Ebata2, B. Brandhuber1, S. Winski1, K. Bouhana1, N. Nanda2, W.I. Wu1, A. Parker1, R. Hamor1, P.D. Larsen1, F. Sullivan1, W. DeWolf1, N. Neitzel1, L. Wollenberg1, S. Smith2, S. Andrews1, S.M. Rothenberg2. 1Array BioPharma, Drug Discovery, Boulder, CO, USA, 2Loxo Oncology, Research and Development, Stamford, CT, USA.

ENA-0491/Poster No. 442

TRK in cancer

Oncogenic NTRK gene fusions occur at low frequency, but in a wide spectrum oftumor types in patients. Early clinical trials of LOXO-101, a specific TRK tyrosinekinase inhibitor (TKI), and entrectinib, a multi-kinase inhibitor (MKI) with anti-TRKactivity, have demonstrated tumor responses in TRK-fusion patients. Unfortunately,acquired resistance has been reported in entrectinib-treated patients, mediated bysecondary mutations in the TRK fusion that may directly interfere with drug binding.Although resistance mutations have not yet been reported in LOXO 101-treatedpatients, entrectinib-resistant mutations may be cross-resistant to LOXO-101.

Two classes of acquired resistance mutations have been reported in patients todate: substitutions in the kinase “solvent front” (TRKA G595R, TRKC G623R) or the“xDFG” motif (TRKA G667C). No clinically available TRK inhibitors have been shownto overcome resistance mutations that have been reported in patients. Notably,homologous mutations in other fusion kinases (e.g. ALK, ROS1) confer acquiredresistance to the relevant first generation TKIs (e.g. crizotinib); solvent-frontmutations in particular are poorly addressed by currently available second generationinhibitors (e.g. ceritinib, alectinib).

We have identified LOXO-195 as a second generation TRK inhibitor with high oralbioavailability and favorable PK in animals. LOXO-195 demonstrated potent inhibitionof TRK fusions, including critical acquired resistance mutations, in enzyme andcellular assays, with minimal activity against other kinases. In diverse TRK fusionmouse models, LOXO-195 inhibited phospho-ERK and caused dramatic tumor growthinhibition, superior to first generation TRK inhibitors, without significant toxicity.Remarkably, LOXO-195 could overcome challenging solvent-front resistance mutationsidentified in both TRKA and TRKC fusions that have arisen in patients treated withentrectinib. Therefore, LOXO-195 has the potential to address a critical unmet clinicalneed for patients whose tumors have progressed after treatment with first generationTRK inhibitors. LOXO-195 is anticipated to enter the clinic in 2017.

SummaryMethods

• LOXO-195 was identified as a potent and selective second generation TRK inhibitorwith activity against wild-type TRK kinases and key resistance mutations, with highselectivity and oral bioavailability, and favorable pharmacokinetics (PK) in animals.

• In vitro and in vivo evaluations, including enzyme and cell-based assays, PK/PD(pharmacodynamics) correlations, drug metabolism characterization, and non-clinical safety evaluation were performed using standard methods.

• Efficacy and PK/PD studies were performed with TRK-dependent tumor models,including allografts of NIH 3T3 cells expressing a delta-Ig2-TRKA (TRKA)activating mutation, without/with the G595R and G667C resistance mutations, inaccordance with IACUC and Guide for Laboratory Animal Care and Use guidelines.

Dose-dependent inhibition of TRK and tumor growth in diverse activated TRK mouse models

Results

We set out to identify a second generation TRK inhibitor that is unaffected byacquired resistance mutations identified in patients, while maintaining LOXO-101-likeselectivity and excellent drug-like properties. Here, we present LOXO-195, a secondgeneration, potent and specific TRK TKI with unique activity against acquiredresistance mutations, including difficult-to-treat substitutions in the solvent front.

Figure 2. Structural modeling of entrectinib and LOXO-101 interactionswith TRKA-G595R and TRKC-G623R (solvent front), and TRKA-G667C andTRKC-G696A (xDFG). Red circles indicate clashes.

LOXO-195 possesses high potency against TRK resistance mutations

Figure 3. Wild type or mutant TRKs were expressed in the context ofTRKA or ETV6-NTRK3. For TRKA constructs, inhibition of signaling wasdetermined by examining pTRK levels in cell lysates by ELISA assay aftertreatment of cells with the indicated agents for one hour (upper). Sincereagents are not available to evaluate pTRK in the context of the ETV6-NTRK3 fusion, pERK levels by flow cytometry were used as a downstreammarker of TRK activation. (lower). n≥3.

Figure 6. Twice daily treatment with LOXO-195, but not LOXO-101,caused dose-dependent inhibition of tumor growth for all TRKAallografts, including TRKA (wild type), solvent-front (TRKA-G595R) andxDFG (TRKA-G667C) (upper), without decreasing body weight (middle).pERK levels in tumor lysates after dosing NIH 3T3-TRKA-G595Rallografts with LOXO-101 or LOXO-195 were determined by immunoblotand compared to drug plasma levels (lower).

Figure 1. Left Normal TRK biology. Middle Chromosomal rearrangementsproduce oncogenic TRK fusions that activate the TRK kinase. RightAcquired resistance to first generation TRK TKIs.

ERK

AKT

Neurotrophin receptors

• TrkA (NTRK1)

Pain, thermoregulation

• TrkB (NTRK2)

Movement, memory, mood, appetite, body weight

• TrkC (NTRK3)

Proprioception

Normal TRK Proteins TRK Fusions Acquired resistance

• Ligand binding domain replaced by 5’ fusion partner; highly expressed by promoter of 5’ fusion gene

• Ligand-independent activation

first generation TRK TKI

first generation TRK TKI

TrkA kinase domainLMNA

Initial tumor response

Tumor progression

LMNA TrkA kinase domain

G595R G667C

Biopsy, sequence

V e h i c l e

3 0 m g / k g B I D L O X O - 1 9 5

1 0 0 m g / k g B I D L O X O - 1 9 5

3 0 0 m g / k g B I D L O X O - 1 9 5

6 0 m g / k g Q D L O X O - 1 0 1

LOXO-195 displays high selectivity across the kinome

Figure 4. Upper Greater than 100-fold selectivity was observed for > 95%of 228 purified kinase domains in radiometric kinase assays for bothLOXO-101 and LOXO-195 when tested at 1 mM. Entrectinib is shown forcomparison. Lower Percent of control values for select kinases commonlyinhibited by MKIs. Note: TRKC was not included in this analysis.

KinaseLOXO-195

% control

TRKA 0.5

TRKB 0

EGFR 100

KIT 100

Met 100

PDGFRalpha 100

PDGFRbeta 100

KDR (VEGFR2) 100

V e h i c l e

3 0 m g / k g B I D L O X O - 1 9 5

1 0 0 m g / k g B I D L O X O - 1 9 5

3 0 0 m g / k g B I D L O X O - 1 9 5

6 0 m g / k g Q D L O X O - 1 0 1

G595R G623RG667C G696AEntr

ect

inib

Entre

ctinib

TRKA TRKC

LOXO-195 LOXO-101 Entrectinib

TRKB TRKBTRKA

TRKBTRKATRKA

LOXO-195 is selectively cytotoxic to TRK-fusion cells

Figure 5. Each cell line was treated with 10 concentrations of LOXO-195 intriplicate for 72 hours, followed by DAPI staining and cell counting.

Cell

Count

EC50 (mM

)

TRK-fusion (n=3)No TRK-fusion (n=84))

10

1

0.1

0.01

0.001

0.0001

ERK AKT

RTK5’ PartnerPromoter

5’ Partner TrkA A/B/C

Tyr

Tyr

T R K A

0 . 0 1 0 . 1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0

0

2 5

5 0

7 5

1 0 0

D r u g C o n c e n t r a t i o n ( n M )

pT

RK

(

% o

f C

on

tr

ol)

E T V 6 - N T R K 3

0 . 0 1 0 . 1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0

0

2 5

5 0

7 5

1 0 0

D r u g C o n c e n t r a t i o n ( n M )

pE

RK

(

% o

f C

on

tr

ol)

T R K A G 5 9 5 R

0 . 0 1 0 . 1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0

0

2 5

5 0

7 5

1 0 0

D r u g C o n c e n t r a t i o n ( n M )

pT

RK

(

% o

f C

on

tr

ol)

E T V 6 - N T R K 3 G 6 2 3 R

0 . 0 1 0 . 1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0

0

2 5

5 0

7 5

1 0 0

D r u g C o n c e n t r a t i o n ( n M )

pE

RK

(

% o

f C

on

tr

ol)

T R K A G 6 6 7 C

0 . 0 1 0 . 1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0

0

2 5

5 0

7 5

1 0 0

D r u g C o n c e n t r a t i o n ( n M )

pT

RK

(

% o

f C

on

tr

ol)

E T V 6 - N T R K 3 G 6 9 6 A

0 . 0 1 0 . 1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0

0

2 5

5 0

7 5

1 0 0

D r u g C o n c e n t r a t i o n ( n M )

pE

RK

(

% o

f C

on

tr

ol)

L O X O - 1 9 5

L O X O - 1 0 1

E n t r e c t i n i b

L O X O - 1 9 5

L O X O - 1 0 1

E n t r e c t i n i b

G595R G667C G696AG623R

LO

XO

-101

TRKA TRKC

LO

XO

-101

X-ray crystal structures and models of TRK proteins and acquired resistance mutations

0

2 0

4 0

1 1 0

6 0

1 0 0

1 0 m g / k g

3 0 m g / k g

1 0 0 m g / k g

6 0 m g / k g

N I H 3 T 3

T R K A G 5 9 5 R

L O X O - 1 9 5 / - 1 0 1 P l a s m a C o n c . ( m g / m L )

pE

RK

in

T

um

or

(P

er

ce

nt

o

f

Co

nt

ro

l)

V e h

3

L O X O - 1 0 1

L O X O - 1 9 5

0 2 4 6 8 1 0 1 2

0

1 0 0 0

2 0 0 0

3 0 0 0

N I H 3 T 3

T R K A

D a y

Tu

mo

r

Vo

lu

me

(m

ea

n

S

EM

,

mm

3

)

N I H 3 T 3

T R K A G 5 9 5 R

0 2 4 6 8 1 0 1 2

0

1 0 0 0

2 0 0 0

3 0 0 0

D a y

Tu

mo

r

Vo

lu

me

(m

ea

n

S

EM

,

mm

3

)

0 2 4 6 8 1 0 1 2

0

1 0 0 0

2 0 0 0

3 0 0 0

N I H 3 T 3

T R K A G 6 6 7 C

D a y

Tu

mo

r

Vo

lu

me

(m

ea

n

S

EM

,

mm

3

)

0 2 4 6 8 1 0 1 2

0

1 0

2 0

3 0

N I H 3 T 3

T R K A

D a y

Bo

dy

We

ig

ht

(m

ea

n

S

EM

,

g)

0 2 4 6 8 1 0 1 2

0

1 0

2 0

3 0

N I H 3 T 3

T R K A G 5 9 5 R

D a y

Bo

dy

We

ig

ht

(m

ea

n

S

EM

,

g)

0 2 4 6 8 1 0 1 2

0

1 0

2 0

3 0

N I H 3 T 3

T R K A G 6 6 7 C

D a y

Bo

dy

We

ig

ht

(m

ea

n

S

EM

,

g)