TPX-0005 – 4-hydroxytamoxifen

Repotrectinib (TPX-0005) is a next generation ROS1/TRK/ALK inhibitor that potently inhibits ROS1/TRK/ALK solvent front mutations

Alexander Drilon,1* Sai-Hong Ignatius Ou,2* Byoung Chul Cho,3 Dong-Wan Kim,4 Jeeyun Lee,5 Jessica J. Lin,6 Viola W. Zhu,2 Myung-Ju Ahn,5 D. Ross Camidge,7 Judy Nguyen,1 Dayong Zhai,8 Wei Deng,8 Zhongdong Huang,8 Evan Rogers,8 Juliet Liu,8 Jeff Whitten,8 John K. Lim,8 Shanna Stopatschinskaja,8 David M. Hyman1, Robert C. Doebele,7 J. Jean Cui,8& Alice T. Shaw6&

1Memorial Sloan Kettering Cancer Center, Weill Cornell Medical College, New York, NY, USA; 2Chao Family Comprehensive Cancer Center, University of California Irvine School of Medicine, Orange, CA, USA; 3Yonsei Cancer Center, Severance Hospital, Yonsei University College of Medicine, Seoul, Republic of Korea; 4Seoul National University Hospital, Seoul, Republic of Korea; 5Samsung Medical Center,
Sungkyunkwan University School of Medicine, Seoul, Republic of Korea;

6Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA;

7University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA 8TP

Therapeutics Inc, San Diego, CA, USA

*contributed equally, &corresponding authors

RUNNING TITLE: Repotrectinib inhibits ROS1/TRK/ALK solvent front mutations

KEYWORDS: solvent front mutation, ALK, ROS1, TRK, acquired resistance

CORRESPONDING AUTHORS:

Alice T. Shaw, MD PhD, Massachusetts General Hospital, 32 Fruit Street, Boston, MA 02114, 617-643-0563 (phone), 617-726-0453 (fax), email: [email protected]
J. Jean Cui, PhD, TP Therapeutics, Inc.,10628 Science Center Drive, Ste. 225, San Diego, CA 92121, (858) 926-5251 (phone), email: [email protected]

CONFLICT OF INTEREST STATEMENT: A Drilon has served as a compensated consultant or received honoraria for TP Therapeutics, Loxo Oncology, Helsinn, AstraZeneca, Roche/Genentech, Pfizer, Takeda/Ariad, Blueprint Medicines, and Beigene. D. Zhai, W. Deng, Z. Huang, E. Rogers, J Liu, J Whitten, J Lim, and S Stopatschinskaja are employees of and have ownership interest in TP Therapeutics. J Cui is co-founder and chief scientific officer of TP Therapeutics, and has ownership in TP Therapeutics. SHI Ou serves on the Scientific Advisory Board of TP Therapeutics and has stock ownership in TP Therapeutics. VWZ has received honoraria from AstraZeneca, Roche-Foundation Medicine, Roche/Genentech, Takeda, and Biocept, and consulting fees from TP Therapeutics. A Shaw has served as a compensated consultant or received honoraria from Pfizer, Novartis, Genentech/Roche, Ignyta, Blueprint Medicine, LOXO, Daiichi-Sankyo, Ariad/Takeda, Chugai, Taiho Pharmaceuticals, EMD Serono, KSQ Therapeutics, Foundation Medicine, Natera, and TP Therapeutics. The remaining authors have no financial interests to declare.

ABSTRACT

The use of tyrosine kinase inhibitors (TKIs) with activity against ALK, ROS1, or TRKA-C can result in significant clinical benefit in patients with diverse tumors harboring ALK, ROS1 or NTRK1-3 rearrangements; however, resistance invariably develops. The emergence of on-target kinase domain mutations represents a major mechanism of acquired resistance. Solvent front substitutions such as ALK G1202R, ROS1 G2032R or D2033N, TRKA G595R, and TRKC G623R are among the most recalcitrant of these mechanisms. Repotrectinib (TPX-0005) is a rationally-designed, low molecular weight, macrocyclic TKI that is selective and highly potent against ROS1, TRKA-C, and ALK. Importantly, repotrectinib exhibits activity against a variety of solvent front substitutions in vitro and in vivo. As clinical proof of concept, in an on-going first-in-human phase 1/2 trial, repotrectinib achieved confirmed responses in patients with ROS1 or NTRK3 fusion-positive cancers who had relapsed on earlier generation TKIs due to ROS1 or TRKC solvent front substitution-mediated resistance.

STATEMENT OF SIGNIFICANCE

Repotrectinib (TPX-0005), a next generation ROS1, pan-TRK, and ALK tyrosine kinase inhibitor, overcomes resistance due to acquired solvent front mutations involving ROS1, NTRK1-3, and ALK. Repotrectinib may represent an effective therapeutic option for patients with ROS1-, NTRK1-3-, or ALK-rearranged malignancies who have progressed on earlier generation TKIs.

INTRODUCTION

The development and approval of earlier-generation tyrosine kinase inhibitors (TKIs) for the treatment of ALK- and ROS1-rearranged non-small cell lung cancers (NSCLCs) led to the appreciation of receptor tyrosine kinase fusions as targetable oncogenic driver alterations in a diverse group of solid malignancies (1-4). More recently, rearrangements involving NTRK1, NTRK2, and NTRK3, encoding TRKA, TRKB, and TRKC, respectively, have been identified as oncogenic drivers in a variety of different tumor types. NTRK-rearranged cancers are exquisitely sensitive to targeted therapies that inhibit TRK regardless of age and tumor origin (5,6).

Despite the clinical activity of TKIs, resistance invariably develops. Moreover, particularly recalcitrant mechanisms of on-target resistance can emerge in fusion- positive tumors, especially after exposure to potent TKIs. The acquisition of mutations resulting in amino acid substitutions at the kinase solvent front represents an example of such a mechanism. These substitutions that occur secondary to solvent front mutations (SFMs) include ALK G1202R in ALK-rearranged tumors, ROS1 G2032R and ROS1 D2033N in ROS1-rearranged tumors, and TRKA G595R and TRKC G623R in NTRK1- and NTRK3-rearranged tumors, respectively (7-17). In ALK-rearranged cancers, ALK G1202R is commonly observed after treatment with more potent earlier- generation agents, and can occur in approximately a third of patients who relapse on second-generation ALK TKIs such as alectinib and ceritinib (10). Similarly, ROS1 G2032R can also emerge in about one-third of patients after crizotinib failure, with an additional 5% of patients developing another solvent front substitution, ROS1 D2033N (14,15). Solvent front substitutions were reported in NTRK-rearranged cancers after

entrectinib treatment, and mediated resistance in the majority (7 out of 10 tested) of

NTRK-rearranged cancers after larotrectinib treatment (5,6).

In this report we describe the pre-clinical activity of repotrectinib (TPX-0005), a rationally designed next-generation TKI developed to potently inhibit clinically recalcitrant solvent front substitutions involving ROS1, TRKA-C and ALK, in addition to wildtype ROS1, TRKA-C and ALK and other clinically relevant non-solvent-front mutations. We also demonstrate proof-of-principle clinical activity in an ongoing phase 1/2 clinical trial investigating the safety and efficacy of repotrectinib (www.clinicaltrials.gov:
NCT03093116).

RESULTS

Design and structure

Many currently available ALK, ROS1, and TRKA-C inhibitors are ATP-competitive type I TKIs that have an ATP adenine-equivalent kinase hinge binder (Figure S1). These agents have an extra motif extending to the solvent area, which represents a shared liability in the setting of on-target resistance mediated by the acquisition of SFMs (Figures S2 & S3). Repotrectinib was designed to overcome clinical resistance mutations, especially SFMs (Figures S4-S6 and Table S1). Structurally, the drug is a rigid, three-dimensional macrocycle that precisely anchors the molecule in the adenine binding site with a bioactive binding conformation predefined to avoid the entropy penalty after binding. Furthermore, repotrectinib is smaller in size compared to currently available ROS1, TRKA-C and ALK inhibitors in clinic, with favourable central nervous system (CNS) drug-like properties for human brain penetration (Table S2A, B). This

novel design was aimed to target both wildtype and SFM kinases (Table S3) as well as other resistance mutations identified in the clinic as demonstrated by its preclinical activity against various ALK-mutant kinases (Table S4). Structural modeling predicts that repotrectinib can accommodate the bulky, positively-charged arginine side chain in the solvent front without any steric clashes. These solvent front substitutions include ROS1 G2032R and ROS1 D2033N , TRKA G595R, TRKB G639R, TRKC G623R, and ALK G1202R (Figure 1).

Enzyme and cell-based activity

Repotrectinib inhibited the kinase activity of wildtype ROS1, TRKA-C, and ALK, and their SFMs with IC50 values in the range of 0.071-4.46 nM (Table S3). Based on the high potency against ROS1/TRKA-C/ALK, the kinase selectivity of repotrectinib was screened at 100 nM against 395 distinct kinases. Screening hits were further evaluated for kinase inhibition IC50 values (Table S5). Repotrectinib showed high potency against ROS1 and TRKA-C with approximately 15-fold selectivity over ALK. In addition, repotrectinib inhibited JAK2 and multiple SRC family members in biochemical assays (Figure 2A).

Because each kinase has a distinct binding affinity (Km) with ATP and a unique conformation in cells, kinase selectivity was further evaluated at the cellular level. Repotrectinib inhibited the phosphorylation of ROS1 and TRKA with IC50s of <1 nM in NIH3T3 CD74-ROS1 and KM12 cells, and ALK with IC50 of <3 nM in Karpas-299 cells (Figure S7). Repotrectinib inhibited JAK2 substrate STAT5 phosphorylation (IC50 158 nM) in SET2 cells, and SRC phosphorylation (IC50 89 nM) in H2228 cells (Figure S7). Overall, repotrectinib demonstrated high selectivity for ROS1, TRKA-C, and ALK in cell- based phosphorylation assays. Ba/F3 cells were engineered to express wild type fusion proteins (CD74-ROS1, LMNA- TRKA, ETV6-TRKB, ETV6-TRKC, and EML4-ALK variant 1) or corresponding solvent front substitutions (ROS1 G2032R, ROS1 D2033N, TRKA G595R, TRKB G639R, TRKC G623R, TRKC G623E and ALK G1202R). In models harboring wildtype ROS1 fusion proteins, repotrectinib and lorlatinib were more potent (IC50s <0.2 nM and 0.2 nM) compared to crizotinib, entrectinib, ceritinib, brigatinib, cabozantinib, and ensartinib (Figure 2B). As predicted, ROS1 G2032R and ROS1 D2033N rendered resistance to crizotinib, entrectinib, ceritinib, brigatinib, and ensartinib. While lorlatinib retained activity against these mutations in preclinical studies (7), repotrectinib and cabozantinib were substantially more potent than lorlatinib against ROS1 G2032R with IC50s of 3.3 nM and 11 nM, respectively, compared to an IC50 of 160.7 nM for lorlatinib. For ROS1 D2033N, repotrectinib was slightly less potent than cabozantinib (1.3 nM versus 0.2 nM), but more potent than lorlatinib (3.3 nM) (Figure S8A and Table S6A). In Ba/F3 models harboring wild type TRKA-C fusion proteins, repotrectinib was more potent (IC50 <0.2 nM) compared to larotrectinib and entrectinib (Figures 2C). The drug retained potent activity against TRKA G595R (IC50 0.4 nM), TRKB G639R (IC50 0.6 nM), TRKC G623R (IC50 0.2 nM) and TRKC G623E (IC50 1.4 nM), whereas these substitutions rendered resistance to larotrectinib and entrectinib. In addition, repotrectinib was 46- and 62-fold more potent than entrectinib and larotrectinib, respectively, at inhibiting KM12 cell proliferation (IC50 0.2 nM) (Figure S8B, Table S6B). Similarly, in Ba/F3 models harboring wild type EML4-ALK variant 1, repotrectinib (IC50 27 nM) was more potent than crizotinib, had comparable activity with alectinib, ceritinib and brigatinib, and was less potent than lorlatinib (Figure 2D); however, the drug retained potent activity against ALK G1202R (IC50 63.6 nM), comparable to that seen with lorlatinib (IC50 41.5 nM). In addition, repotrectinib inhibited Karpas-299 cell proliferation (IC50 23.7 nM) with a similar potency as alectinib (Figure S8C, Table S6C). Overall, repotrectinib was a potent ROS1/TRK/ALK inhibitor in both enzymatic and cellular assays, and selectively cytotoxic to engineered cell lines with ROS1, NTRK or ALK rearrangements, while having IC50 >1000 nM for parental Ba/F3 cells (Table S6C) and cell lines with other oncogene drivers (Table S6D)

In vivo activity

The in vivo activity of repotrectinib was tested against representative SFMs, ROS1 G2032R, TRKA G595R, and ALK G1202R, using mouse xenograft models. In SCID/Beige mice bearing Ba/F3 CD74-ROS1 wildtype or CD74-ROS1 G2032R xenograft tumors, the twice daily (BID) dosing with 15 or 75 mg/kg/dose of crystalline repotrectinib resulted in a free Ctrough of 2.2 and 13.3 nM, and tumor growth inhibitons (TGIs) of 197% and 200% against CD74-ROS1 wildtype tumors (Figure 3A) and 99% and 200% against CD74-ROS1 G2032R tumors (Figure 3B), respectively, without body weight loss (Figure S9A). A free Ctrough of 13.3 nM led to complete tumor regression in ROS1 WT and G2032R tumor models. The mean trough plasma concentration is summarized in Table S7.

In athymic nude mice bearing NIH3T3 LMNA-TRKA wildtype xenograft tumors, a micronized crystalline formulation of repotrectinib was used for BID dosing at 3 or 15 mg/kg/dose leading to a free Ctrough of 3.5 and 22.7 nM, and TGIs of 100% and 128%, respectively (Figure 3C), while entrectinib achieved TGI of 98% at 15 mg/kg BID dosing. The treatment with micronized crystalline repotrectinib at 3, 15, or 60 mg/kg/dose BID resulted in a free Ctrough of 3.5, 22.7 and 173.5 nM, leading to TGIs of 56%, 97%, and 123%, respectively in TRKA G595R tumor model (Figure 3D) without body weight loss (Figure S9B), while entrectinib had 78% TGI at 60 mg/kg BID. A free Ctrough of 22.7 nM was required to achieve 97% TGI in NIH3T3 LMNA-TRKA G595R tumor model. The mean trough plasma concentration is summarized in Table S7.

The activity of repotrectinib was investigated in SCID/Beige mice bearing Ba/F3 EML4- ALK v1 wildtype or EML4-ALK v1 G1202R xenograft tumors. BID dosing with 15 or 75 mg/kg of the crystalline repotrectinib resulted in TGIs of 75% and 154% against EML4- ALK v1 wildtype tumors (Figure 3E), and 56% and 99% against EML4-ALK v1 G1202R tumors (Figure 3F), respectively without body weight loss (Figure S9C). Therefore, a free Ctrough of 13.3 nM was required to achieve 99% TGI in Ba/F3 EML4-ALK v1 G1202R tumor model. The mean trough plasma concentration is summarized in Table S7.

Proof-of-concept clinical activity

Repotrectinib is being investigated in an on-going first-in-human dose escalation phase 1/2 clinical trial (NCT03093116) in patients with advanced ALK-, ROS1-, or NTRK1-3- rearranged cancers.

ETV6-NTRK3 G623E+ mammary analogue secretory carcinoma (MASC)

A 44 year-old man presented in October of 2012 with a right parotid mass. He underwent a right parotidectomy and a MASC was identified. His history was thereafter marked by recurrent local disease requiring two subsequent resections and chemoradiation. In 2015, he developed recurrent local disease in addition to metastatic disease involving the lungs. Genomic profiling later revealed an ETV6-NTRK3 rearrangement. Crizotinib was initiated with a best response of progressive disease.
The patient was then enrolled onto a clinical trial of entrectinib which he received for six months with partial response, followed by doxorubicin on progression of disease, and later entrectinib and trametinib which the patient received for two months with progressive disease. He was then enrolled onto the phase I trial of repotrectinib (40 mg once daily). A pre-treatment biopsy revealed persistence of an in-frame ETV6-NTRK3 rearrangement containing the kinase domain of NTRK3 and an NTRK3 G623E mutation that was not present on a pre-entrectinib biopsy. A rapid and dramatic response to repotrectinib was achieved within the first few days of administration, noted as substantial shrinkage of externally visible disease involving the right mandibular area (Figures 4A&4B). Plasma exposure indicated the Ctrough (144 nM) was above the IC90 (80 nM) needed to inhibit TRKC G623E Ba/F3 cell growth (Figure S10A). Radiologic imaging after 8 weeks of therapy revealed a partial response (PR) with shrinkage of multiple areas of disease involving the right cheek, cervical lymph nodes, chest wall nodules, and pulmonary and pleural nodules, followed by a confirmed PR with 82% tumor shrinkage by RECIST v1.1. The patient started showing slow progression involving certain lesions after 6 months of treatment and repotrectinib dose was gradually escalated to 160 mg BID as permitted by the protocol. He remains on therapy
at 13+ months at the time of manuscript submission with no dose-limiting toxicities and

only mild grade 1 peripheral sensory neuropathy. The clinical outcome is consistent with the cell-based inhibitory assay (Figure 2C) and the mice xenograft study ( Figure S10B).

CD74-ROS1 G2032R+ non-small cell lung cancer (NSCLC)

A 41-year-old Asian female never-smoker presented with stage IV NSCLC with a large pleural effusion, pleural-based nodules and multiple enlarged mediastinal and supraclavicular lymph nodes. Pleural effusion cytology and supraclavicular lymph node fine needle aspirate cytology both revealed adenocarcinoma. Molecular profiling identified a CD74-ROS1 rearrangement. MRI of the brain was negative for intracranial metastasis at the time of diagnosis. She received one cycle of carboplatin/pemetrexed/bevacizumab while awaiting insurance authorization for crizotinib. She started on crizotinib in June 2016 and had a durable response for 12 months, at which time imaging showed increasing mediastinal lymphadenopathy (Figure 4C). Repeat biopsy and comprehensive genomic profiling at Foundation Medicine revealed CD74-ROS1 and a ROS1 G2032R SFM. The patient subsequently enrolled on the phase 1 repotrectinib trial and received repotrectinib 160 mg once daily. Pre-repotrectinib brain imaging revealed multiple, clinically asymtomatic supratentorial and infratentorial brain metastases (Figure 4D). She tolerated repotrectinib treatment well with mild grade 1 ataxia, paresthesias, and nausea early in her course that resolved with supportive care, and ongoing mild grade 1 perioral numbness and dysgeusia. A confirmed PR (-36.6%) by RECIST v1.1 was achieved which is ongoing at 8 months at the time of manuscript submission. Of note, this patient also responded to repotrectinib in the central nervous system (CNS) (Figure 4D&4E). The PK revealed

the Ctrough (425 nM) was above the IC90 (100 nM) needed to inhibit ROS1 G2032R Ba/F3 cell proliferation (Figure S10C).

DISCUSSION

In cancers driven by gene fusions, sequential TKI therapy has emerged as a paradigm for maintaining disease control in the face of continued dependence on the fusion oncoprotein. The emergence of solvent front mutations (SFMs) during the course of treatment, especially following more potent TKIs, represents a significant therapeutic challenge (9-19). Repotrectinib, by targeting the center of the ATP binding site with a small, three-dimensional macrocyclic structure, effectively circumvents steric hindrance from solvent front substitutions. Consistent with this, in multiple preclinical models, repotrectinib demonstrated potent anti-proliferative activity against wildtype fusion proteins involving ROS1, TRKA, TRKB, TRKC, and ALK, and their corresponding SFMs in cellular inhibitory assays and xenograft models.

For ROS1, both ceritinib (19) and entrectinib (20) have demonstrated clinical activity in crizotinib-naïve ROS1+ NSCLC patients. Additionally, cabozantinib has demonstrated activity against ROS1 SFMs (14,21). Lorlatinib is a next generation ALK/ROS1 inhibitor with potent inhibitory activity against wildtype ROS1. In an ongoing phase 1/2 study, lorlatinib demonstrated variable clinical activity in crizotinib-refractory ROS1+ NSCLC patients (22,23). Lorlatinib can inhibit the ROS1 gatekeeper substitution L2026M in vitro but is not as potent against the solvent front substitution ROS1 G2032R (7). Indeed, repotrectinib exhibits more potent inhibitory activity against wild type ROS1 and SFMs than all other ROS1 TKIs with the exception of cabozantinib against D2033N (0.2 nM

[cabozantinib] versus 1.3 nM [repotrectinib]). Overall, repotrectinib exhibited potent anti- proliferative activity in Ba/F3 cells transduced with the oncogenic driver CD74-ROS1, and effectively inhibited ROS1 G2032R (3.3 nM) and D2033N (1.3 nM) (Table S6A).
Consistent with these results, repotrectinib induced a durable and ongoing response in a crizotinib-refractory patient with a ROS1-rearranged tumor harboring ROS1 G2032R.

Currently, no targeted therapy is approved for NTRK-rearranged cancers. The first- generation agent larotrectinib demonstrated marked antitumor activity in patients with NTRK-rearranged cancers (6). Similarly, entrectinib is active in NTRK-rearranged cancer patients (5). On-target resistance frequently emerges with the use of either agent, including the acquisition of SFMs in 7 out of 10 patients treated with larotrectinib (6), although a larger data set is required to determine the true frequency of on-target resistance in this context. Repotrectinib is highly potent against TRKA-C solvent front substitutions preclinically, and a dramatic response to therapy was observed in a patient with an NTRK3 fusion-positive tumor harboring a SFM. It remains to be determined whether there are any differences in the clinical activity between repotrectinib and LOXO-195, another next generation TRK inhibitor currently in clinical development (24).

While repotrectinib is 15-fold less potent against ALK than ROS1 in enzymatic assays, repotrectinib has superior ALK inhibitory activity compared with the first generation ALK inhibitor crizotinib and compares favorably in its ALK G1202R inhibitory activity against currently approved second-generation ALK inhibitors (ceritinib, alectinib, brigatinib). The current phase 1 trial will investigate the clinical activity of repotrectinib in ALK- rearranged malignancies.

Finally, CNS progression is a significant clinical challenge in oncogene-driven lung cancers, including ALK- and ROS1-rearranged NSCLC. The available next-generation ALK inhibitors have significantly improved CNS activity over crizotinib (25). While the incidence of CNS metastasis may be lower in ROS1 fusion-positive compared to ALK fusion-positive lung cancer patients, the incidence of CNS metastases increases with time on treatment (15). As shown in the preliminary clinical data presented in this report, repotrectinib has demonstrated proof-of-concept CNS activity against untreated brain metastases in a patient with a crizotinib-refractory, ROS1 fusion-positive lung cancer.

In summary, this is the first report on the design and preclinical evaluation of the novel next generation ROS1/TRK/ALK TKI, repotrectinib. Repotrectinib was specifically designed to overcome refractory SFMs which commonly emerge in patients with ROS1/NTRK/ALK-rearranged malignancies who have relapsed on currently available TKIs. In an ongoing first-in-human dose-escalation trial, repotrectinib demonstrated antitumor activity in patients with ROS1- or NTRK3-rearranged tumors that harbor resistant SFMs. While safety, dosing and clinical efficacy are still being established, these findings suggest that repotrectinib could represent an effective treatment option for ROS1/NTRK-rearranged malignancies, including those harboring resistant SFMs. The clinical application of repotrectinib for TKI-refractory, ALK fusion positive patients needs to be further defined in the on-going trial.

METHODS

Preclinical studies

Structural modeling

Structural modeling of ALK, ROS1 and TRK solvent front mutants in complex with repotrectinib (TPX-0005) were carried out with X-ray co-crystal structures of ALK (PDB 4CLJ), ROS1 (PDB 3ZBF and 4UXL), TRKA (PDB 4AOJ), TRKB (PDB 4AT3) and
TRKC (PDB 4YMJ) using Schrodinger Maestro software. (Supplemental information)

Enzyme assays

The enzymatic kinase inhibitory activities of repotrectinib was evaluated at Reaction Biology Corporation using the radiolabeled HotSpot kinase assay platform, and kinase selectivity was first evaluated using the KINOMEscan® site-directed competition binding assay against 456 human kinases at DiscoeRx. See Supplemental information for detailed information.

Cell lines and assays

Human lung cancer cell line NCI-H2228 was obtained from ATCC (2014, Manassas, VA). Cell lines NIH3T3 and Ba/F3 were purchased from DSMZ (2015, German Collection of Microorganisms and Cell Culture, Braunschweig, Germany). Karpas-299 cell line was purchased and licenced from Sigma (2015, Cambridge). KM12 cell line was obtained from NCI. (2015, Frederick Cancer DCTD Tumor Cell Repository). NCI- H2228, Karpas-299, and KM12 cells were authenticated by confirmation of the presence of each fusion (EML4-ALK, NPM-ALK or TPM3-TRKA). NIH3T3 and Ba/F3 cells were not authenticated. Cell lines were confirmed to be mycoplasma-free
(Biomiga) and were used between 3-10 passages. The genes of EML4-ALK (variant 1),

CD74-ROS1, LMNA-TRKA , ETV6-TRKB, ETV6-TRKC, and the solvent front mutant genes of EML4-ALK G1202R, CD74-ROS1 G2032R, CD74-ROS1 G2033N, LMNA- TRKA G595R, ETV6-TRKB G639R, ETV6-TRKC G623R, and ETV6-TRKC G623E
were synthesized at GenScript and cloned into pCDH-CMV-MCS-EF1-Puro plasmid (System Biosciences, Inc). The corresponding Ba/F3 and NIH3T3 stable cells were generated by transducing Ba/F3 cells and NIH3T3 cells with lentivirus containing the desired fusion gene or mutant. Please refer to Supplemental information for detailed procedures.
For cellular phosphorylation assays, half a million cells per well were seeded in 24 well plate for 24 hrs, and then treated with compounds for 4 hours. For cell proliferation assays, two thousand cells per well were seeded in 384 well white plate for 24 hrs, and then treated with compounds for 72 hours. Cell proliferation was measured using CellTiter-Glo luciferase-based ATP detection assay (Promega) following the manufactures’s protocol. Please refer to Supplemental information for detailed procedures.

In vivo xenograft studies

All animal studies were conducted in accordance with the guidelines as published in the Guide for the Care and Use of Laboratory Animals. Mice were maintained and used in accordance with the animal protocol EB15-013 (approved by Explora BioLabs’ Institutional Animal Care and Use Committee). Five million cells in 100 L serum-free medium supplemented with 50% Matrigel (Corning, Inc.) were implanted subcutaneously in the right flank region of the mouse. Mice were randomized by tumor size into treatment groups when tumor volume reached about 100-200 mm3 and

repotrectinib were administered orally (BID) at determined dosage. Tumor size and body weight were measured on designated days. Please refer to Supplemental information for the detailed information.

Treatment plan design and conduct

Repotrectinib is being investigated in a first-in-human dose escalation study (NCT- 03093116). The study has been conducted in accordance with recognized ethical guidelines such as the Declaration of Helsinki. The protocol has been approved by the institutional review boards at each participating site. Written informed consent was obtained from all the patients before screening. Details of eligibility criteria are listed at the www.clinicaltrials.gov.

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ACKNOWLEDGMENTS: Funding was provided by TP Therapeutics, Inc.

FIGURE LEGENDS

Figure 1. Binding of repotrectinib to ROS1, TRKA-C and ALK kinases with solvent front substitutions. Crizotinib (pink) has a basic piperidine group that extends to the solvent front area, thus clashing with ROS1, TRKA-C and ALK solvent front substitutions (located at the C-terminus of the ATP adenine binding hinge) that mediate resistance to therapy. Similar to crizotinib, other currently available ROS1, TRKA-C and ALK inhibitors carry an extra motif that likewise extends to the solvent area that sterically clashes with solvent front substitutions. In order to avoid this steric inteference, repotrectinib was designed to be much smaller in size (MW 355.37) than the currently available ROS1, TRKA-C and ALK inhibitors. As shown here, repotrectinib (green) has a rigid three dimensional macrocyclic structure that precisely anchors the molecule completely inside the ATP adenosine binding site of the cocrystal structure of ROS1, TRKA-C and ALK with a predefined bioactive binding conformation. Structural modeling studies indicate steric clashes do not occur between repotrectinib and solvent front mutations involving ROS1, TRKA-C and ALK.

Figure 2. Selectivity and in vitro antiproliferative activity of repotrectinib. A. The selectivity of repotrectinib for 395 distinct kinases is shown in this kinome tree. The selectivity index (SI) is defined as the kinase IC50 value devided by the ROS1 IC50 value (0.071 nM) and is depicted by the size of the circles: the largest circle is ROS1 with an SI value of 1, followed by TRKA, TRKB and TRKC with SI values between 1 and 10, followed by ALK, JAK2, and FYN with SI values between 10 and 20. Kinases with SI values greater than 20 but less than 250 are LYN, YES1, FGR, TXK, ARK5, SRC, DDR1 and FAK. B. Anti-proliferation activity of repotrectinib, crizotinib, lorlatinib,
entrectinib, ceritinib, brigatinib, cabozantinib and ensartinib against Ba/F3 cells

engineered with CD74-ROS1 WT, CD74-ROS1 G2032R, or CD74-ROS1 D2033N

fusion proteins, respectively. C. Anti-proliferation activity of repotrectinib, entrectinib and larotrectinib against Ba/F3 cells engineered with LMNA-TRKA WT, LMNA-TRKA G595R, ETV6-TRKB WT, ETV6-TRKB G639R, ETV6-TRKC WT, ETV6-TRKC G623R,
or ETV6-TRKC G623E fusion proteins, respectively. D. Anti-proliferation activity of repotrectinib, crizotinib, ceritinib, alectinib, brigatinib and lorlatinib against Ba/F3 cells engineered with EML4-ALK WT or EML4-ALK G1202R fusion proteins.

Figure 3. In vivo antitumor activity of repotrectinib. The antitumor activity of repotrectinib in the following fusion-positive xenograft models, several of which harbor solvent front mutations, is shown: A. Ba/F3 CD74-ROS1 wild type (WT) in SCID/Beige mice, B. Ba/F3 CD74-ROS1 G2032R in SCID/Beige mice, C. NIH3T3 LMNA-TRKA WT
in athymic nude mice, D. NIH3T3 LMNA-TRKA G595R in athymic nude mice, E. Ba/F3 EML4-ALK v1 WT in SCID/Beige mice, F. Ba/F3 EML4-ALK v1 G1202R in SCID/Beige mice. The comparative activity of entrectinib is included in Figures 3C and 3D.
Graphs of tumor volume changes in response to treatment with vehicle and various doses of ropotrectinib or entrectinib are shown on the left. Each corresponding waterfall plot on the right represents the degree of xenograft response in one mouse. Tumor regression or stabilization was observed with repotrectinib in all models, including resistant models harboring solvent front mutations.

Figure 4. Response to repotrectinib in solvent front substitution-containing cancers. A. A brisk and dramatic response to repotrectinib was observed in externally visible tumor from a patient with an ETV6-NTRK3-rearranged mammary analogue

secretory carcinoma (MASC) harboring an NTRK3 G623E solvent front mutation acquired after prior treatment with entrectinib. B. Combined computed and positron emission tomography images show a concurrent metabolic and radiologic response (regression of dermal and subcutaneous metastases) to therapy that was subsequently confirmed with follow up imaging. The patient remains on repotrectinib at more than 13 months. C. Similarly, a confirmed radiologic response to therapy was achieved in a patient with a CD74-ROS1-rearranged non-small cell lung cancer (NSCLC) that acquired a ROS1 G2032R solvent front mutation after prior treatment with crizotinib.
Regression of a subcarinal node is shown. D and E. The intracranial activity of repotrectinib is shown, with magnetic resonance imaging demonstrating regression or disappearance of multiple previously untreated supratentorial and infratentorial brain metastases. The patient remains on repotrectinib at more than 8 months since treatment initiation.

Author Manuscript Published OnlineFirst on August 9, 2018; DOI: 10.1158/2159-8290.CD-18-0484 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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Author Manuscript Published OnlineFirst on August 9, 2018; DOI: 10.1158/2159-8290.CD-18-0484 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Repotrectinib (TPX-0005) is a next generation ROS1/TRK/ALK inhibitor that potently inhibits ROS1/TRK/ALK solvent front mutations
Alexander Drilon, Sai-Hong I. Ou, Byoung Chul Cho, et al.
Cancer Discov Published OnlineFirst August 9, 2018.

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