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  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
• • A—HUMAN NECESSITIES
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  • A61K31/00—Medicinal preparations containing organic active ingredients
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  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
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• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• This disclosure relates to methods for treating an EGFR-mutant cancer in a patient in need thereof by administering to the patient a therapeutically effective amount of at least one RET inhibitor, e.g., at least one selective RET inhibitor, and a therapeutically effective amount of at least one EGFR inhibitor to the patient.
• the disclosure relates to methods for treating an EGFR-mutant cancer in a patient who has been previously treated with at least one EGFR inhibitor, and, in some cases, has acquired resistance to the at least one EGFR inhibitor.
• This disclosure also relates to combination therapies comprising at least one RET inhibitor, e.g., at least one selective RET inhibitor, and at least one EGFR inhibitor.
• the at least one RET inhibitor is a selective RET inhibitor chosen from Compound 1 and pharmaceutically acceptable salts thereof.
• the at least one EGFR inhibitor is chosen from osimertinib and pharmaceutically acceptable salts thereof.
• RET receptor tyrosine kinase
• GDNF glial cell line-derived neurotrophic factors
• GFRa GDNF family receptors-a
• Oncogenic RET rearrangements have been identified in 1-2% of NSCLC (Lipson, D. et al., Nat. Med. 18:382-384 (2012); Takeuchi, K. et ah, Nat. Med. 18:378-381 (2012); Stransky, N. et al., Nat. Commun. 5:4846 (2014)). Oncogenic RET rearrangements generate a constitutively active kinase that promotes tumorigenesis.
• RET-rearranged NSCLC typically has adenocarcinoma histology (though occasionally squamous) and occurs in young, non-smoking patients.
• Compound 1 described herein is a potent and selective inhibitor of RET kinase and oncogenic RET mutants. In cellular systems, Compound 1 inhibits the kinase activity of RET oncogenic mutants with low nanomolar potency.
• malignancies such as thyroid cancer, non-small cell lung cancer (NSCLC), and other advanced solid tumors.
• NSCLC non-small cell lung cancer
• RET fusions may also be implicated in some cases of EGFR-mutant cancer (see Schrock, A.B. et al., J. Thorac. Oncol doi: 10.1016/ j tho.2018.05.027 (published online June 5, 2018)). While certain EGFR inhibitors have been approved in the treatment of cancer, e.g., non-small cell lung cancer (osimertinib), a subset of patients progressing on EGFR inhibitor therapy have acquired gene fusions that cause acquired drug resistance. RET fusions associated with EGFR TKI resistance (see Oxnard, G.R. et al., JAMA Oncology
• EGFR TKI resistance facilitated by RET fusions resembles the bypass track resistance facilitated by MET amplification in EGFR-mutant patients.
• both preclinical and clinical evidence have shown strong responses with by inhibiting both EGFR and MET (Engleman, J.A. et al, Science 316: 1039-43 (2007); Gainor, J.F. et al, J. Thorac. Oncol. 1 l(7):e83-e85 (2016); Ahn, M. et al, J. Thorac. Oncol.
• the following disclosure describes methods for treating an EGFR-mutant cancer in a patient in need thereof by administering to the patient a therapeutically effective amount of at least one RET inhibitor and a therapeutically effective amount of at least one EGFR inhibitor.
• the patient has been previously treated with at least one EGFR inhibitor.
• the patient has acquired resistance to at least one EGFR inhibitor.
• the at least one RET inhibitor is a selective RET inhibitor, e.g., Compound 1 or a pharmaceutically acceptable salt thereof.
• Compound 1 is orally administered to the patient once daily.
• the therapeutically effective amount of the at least one RET inhibitor administered to the patient once daily is 200 mg to 400 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof.
• the therapeutically effective amount of the at least one RET inhibitor administered to the patient once daily is 200 mg to 300 mg of Compound 1 or the weight equivalent of a
• the at least one RET inhibitor is chosen from alectinib, apatinib, BOS172738 (DS-5010), cabozantinib (XL184), dovitinib (TKI258), GSK3179106, GSK3352589, lenvatinib, LOXO-292, SL-1001, TPX-0046, nintedanib, ponatinib, sitravatinib (MGCD516), sorafenib, sunitinib, regorafenib (BAY 73-4506), RXDX-105, vandetanib, XL999, and pharmaceutically acceptable salts of any of the foregoing.
• the at least one RET inhibitor is chosen from alectinib, apatinib, BOS172738 (DS-5010), cabozantinib (XL184), dovitinib (TKI258), GSK3179106, GSK3352589, lenvatinib, LOXO-292, nintedanib, ponatinib, sitravatinib (MGCD516), sorafenib, sunitinib, regorafenib (BAY 73-4506), RXDX-105, vandetanib, XL999, and pharmaceutically acceptable salts of any of the foregoing.
• the at least one EGFR inhibitor is chosen from osimertinib and pharmaceutically acceptable salts thereof. In some embodiments, osimertinib and/or at least one pharmaceutically acceptable salt thereof is orally administered to the patient once daily. In some embodiments, the therapeutically effective amount of the at least one EGFR inhibitor administered to the patient once daily is 80 mg of osimertinib or the weight equivalent of a pharmaceutically acceptable salt thereof. [0014] In some embodiments, the EGFR-mutant cancer is characterized by at least one EGFR mutation chosen from L858R, Dec 19, T790M, C797S, and L792H.
• the EGFR-mutant cancer is characterized by at least one EGFR mutation chosen from T790M, C797S, and L792H. In some embodiments, the EGFR-mutant cancer is characterized by at least two EGFR mutations. In some embodiments, the EGFR-mutant cancer is characterized by three EGFR mutations. In some embodiments, the EGFR-mutant cancer is further characterized by at least one RET-alteration, e.g., a CCDC6-RET fusion. In some embodiments, the EGFR-mutant cancer is lung cancer, e.g., small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC).
• SCLC small cell lung cancer
• NSCLC non-small cell lung cancer
• combination therapies comprising at least one RET inhibitor, e.g., at least one selective RET inhibitor such as Compound 1 and/or a pharmaceutically acceptable salt thereof, and at least one EGFR inhibitor, e.g., osimertinib and/or a pharmaceutically acceptable salt of any of the foregoing.
• at least one RET inhibitor e.g., at least one selective RET inhibitor such as Compound 1 and/or a pharmaceutically acceptable salt thereof
• at least one EGFR inhibitor e.g., osimertinib and/or a pharmaceutically acceptable salt of any of the foregoing.
• Treating a patient, e.g., a human, suffering from an EGFR-mutant cancer with at least one RET inhibitor in combination with at least one EGFR inhibitor may improve therapeutic outcomes in patients with acquired drug resistance to EGFR TKIs.
• Example embodiments of the disclosure further include:
• a method for treating an EGFR-mutant cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of at least one RET inhibitor and a therapeutically effective amount of at least one EGFR inhibitor.
• the at least one RET inhibitor is chosen from alectinib, apatinib, BOS172738 (DS-5010), cabozantinib (XL184), dovitinib (TKI258), GSK3179106, GSK3352589, lenvatinib, LOXO-292, SL-1001, TPX-0046, nintedanib, ponatinib, sitravatinib (MGCD516), sorafenib, sunitinib, regorafenib (BAY 73-4506), RXDX-105, vandetanib, XL999, and pharmaceutically acceptable salts of any of the foregoing.
• lung cancer is chosen from small cell lung cancer and non-small cell lung cancer.
• the at least one RET inhibitor is orally administered to the patient once daily; and the therapeutically effective amount of the at least one RET inhibitor is 200 mg to 400 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof.
• the at least one EGFR inhibitor is chosen from osimertinib and pharmaceutically acceptable salts thereof;
• the at least one EGFR inhibitor is orally administered to the patient once daily; and the therapeutically effective amount of the at least one EGFR inhibitor is 80 mg of osimertinib or the weight equivalent of a pharmaceutically acceptable salt thereof.
• a combination therapy comprising at least one RET inhibitor and at least one EGFR inhibitor.
• RET inhibitor is chosen from alectinib, apatinib, BOS172738 (DS-5010), cabozantinib (XL184), dovitinib (TKI258), GSK3179106, GSK3352589, lenvatinib, LOXO-292, SL-1001, TPX-0046, nintedanib, ponatinib, sitravatinib (MGCD516), sorafenib, sunitinib, regorafenib (BAY 73- 4506), RXDX-105, vandetanib, XL999, and pharmaceutically acceptable salts of any of the foregoing.
• a method for treating a patient suffering from an EGFR-mutant cancer comprising:
• the at least one RET inhibitor is chosen from alectinib, apatinib, BOS172738 (DS-5010), cabozantinib (XL184), dovitinib (TKI258), GSK3179106, GSK3352589, lenvatinib, LOXO-292, SL-1001, TPX-0046, nintedanib, ponatinib, sitravatinib (MGCD516), sorafenib, sunitinib, regorafenib (BAY 73-4506), RXDX-105, vandetanib, XL999, and pharmaceutically acceptable salts of any of the foregoing.
• FIG. 1 is a scan which shows RECIST tumor shrinkage of 78% in a 60-year-old woman with EGFR del 19 and acquired CCDC6-RET fusion after two weeks of treatment with 200 mg once daily of Compound 1 and 80 mg once daily of osimertinib, followed by six weeks of treatment with 300 mg once daily of Compound 1 and 80 mg once daily of osimertinib.
• the serial coronal contrast-enhanced computed-tomography images of the thorax demonstrate a right lower lobe lung mass and pleural nodularity (arrows) seen at baseline (left) with partial response after eight weeks of treatment with Compound 1 and osimertinib (right).
• FIG. 2 demonstrates that CCDC6-RET expressing cell line models were generated by lentiviral infection of PC9 (EGFR del 19) and MGH134 (EGFR L858R/T790M) cells.
• FIG. 3 shows cell proliferation data for PC9 CCDC6 ?£r and MGH 124 ⁇ ( ⁇ )(U, ⁇ ia:i cell s in the presence or absence of osimertinib.
• PC9 and MGH134 cells expressing the CCDC6- RET gene fusion or empty vector (EV) were treated with 1 mM osimertinib or vehicle (VEH) and cell proliferation determined over the course of five days (ratio compared to the beginning of treatment). Data shown are the mean ⁇ s.e.m. of three independent biological replicates.
• FIG. 4A is a western blot for PC9 X and RE9 GG/ c / ⁇ 77/ cells treated with 100 nM afatinib, 1 mM osimertinib, Compound 1, or combinations thereof for 6 hours and harvested for western blotting with the antibodies.
• FIG. 4B is a western blot for MGH 134 /x and MGHl34 CCDC6 ?xr cells treated with 1 pM osimertinib, cabozantinib, and Compound 1 or combinations thereof for 6 hours and harvested for western blotting with the indicated antibodies.
• FIG. 4C shows cell viability after 72 hours for PC9 X and PC9 r7 x / ⁇ 77/ cells treated with Compound 1, or afatinib or osimertinib in the absence or presence of 1 pM Compound 1.
• Compound 1 data is replotted in both panels for comparison purposes. Data are shown as a percentage of vehicle treated control and are the mean ⁇ s.e.m of three independent biological replicates.
• FIG. 5 A is a western blot for PC 9V and PC9 r / V5 / ⁇ / / cells treated with 100 nM afatinib, 1 mM osimertinib, cabozanitinib, or combinations thereof for 6 hours and harvested for western blot analysis.
• FIG. 5B shows cell viability after 72 hours for PC9 X and PC9 r7 3 ⁇ 4 /i/;/ cells treated with cabozantinib, or afatinib or osimertinib in the absence or presence of 1 mM cabozantinib (CAB).
• CAB cabozantinib
• FIG. 5C shows cell viability after 72 hours for MGH 134 /x and MGH 124 ( '( ',)( Y, ⁇ I :I cells treated with a RET inhibitor, cabozantinib, or Compound 1, or osimertinib in the absence or presence of 1 mM RET inhibitor. Data are shown as a percentage of vehicle treated control and are the mean ⁇ s.e.m of three independent biological replicates.
• a“combination therapy” refers to a therapy comprising more than one active agent.
• the two or more active agents can be administered in one dosage form or separate dosage forms. Additionally, the active agents comprising the combination therapy may be administered at the same time (in one or more dosage forms) or at separate times.
• Compound 1 is (lS,4R)-N-((S)-l-(6-(4-fluoro-lH-pyrazol-l-yl)pyridine-3- yl)ethyl)-l-methoxy-4-(4-methyl-6-((5-methyl-lH-pyrazol-3-yl)amino)pyrimidin-2- yl )cy cl ohexanecarb ox ami de :
• Compound 1 (also known as BLU-667 or pralsetinib) entered Phase I clinical trials in the United States for the treatment of patients with thyroid cancer, non-small cell lung cancer, and other advanced solid tumors (NCT03037385).
• NCT03037385 advanced solid tumors
• an“EGFR inhibitor” is a compound which inhibits the activity of EGFR kinase.
• EGFR kinase is wild-type EGFR kinase and/or one or more EGFR-altered kinases (e.g., EGFR fusion, EGFR mutation, or EGFR copy number variation).
• Examples of EGFR inhibitors include, but are not limited to, afatinib, ASP8273, avitinib, brigatinib, cetuximab, dacomitinib, EAI045, erlotinib, gefitinib, HS-10296, icotinib, lapatinib, necitumumab, neratinib, olmutinib, osimertinib, panitumumab, PF- 06747775, rociletinib, and vandetanib.
• a“fusion” is a protein that results from a chromosomal translocation in which two genes are joined with an in-frame coding sequence and results in a chimeric protein.
• a fusion is a chromosomal translocation where the kinase domain of one protein fuses to a dimerization domain of another gene.
• a“RET fusion” is a gene rearrangement. RET rearrangements create a fusion protein juxtaposing the RET kinase domain and a dimerization domain of another protein, creating a constitutively activated dimer which drives tumorigenesis.
• a“RET fusion protein” is the result of a gene rearrangement. RET rearrangements create a fusion protein juxtaposing the RET kinase domain and a dimerization domain of another protein, creating a constitutively activated dimer which drives
• a“RET inhibitor” is a compound which inhibits the activity of RET kinase.
• RET kinase is wild-type RET kinase and/or one or more RET-altered kinases (e.g., RET fusion, RET mutation, or RET copy number variation).
• RET inhibitors include, but are not limited to, alectinib, cabozantinib
• XL184 Compound 1, dovitinib (TKI258), BOS172738 (DS-5010), foretinib, lenvatinib, LOXO-292, ponatinib, RXDX-105, sitravatinib (MGCD516), sorafenib, sunitinib, TAS0286, TPX-0046, SL-1001, and vandetanib.
• RET inhibitors include, but are not limited to, apatinib, AUY-922, DCC-2157, GSK3179106, GSK3352589, motesanib, nintendanib, NVP-AST487, PZ-l, regorafenib (BAY 73-4506), RPI-l, TG101209, SPP86, vatalanib, and XL999.
• RET inhibitors include, but are not limited to, alectinib, cabozantinib (XL184), Compound 1, dovitinib (TKI258), BOS172738 (DS-5010), foretinib, lenvatinib, LOXO-292, ponatinib, RXDX-105, sitravatinib (MGCD516), sorafenib, sunitinib, TAS0286, and vandetanib.
• RET inhibitors include, but are not limited to, apatinib, AUY-922, DCC-2157, GSK3179106, GSK3352589, motesanib, nintendanib, NVP- AST487, PZ-l, regorafenib (BAY 73-4506), RPI-l, TG101209, SPP86, vatalanib, and XL999.
• a“selective RET inhibitor” refers to a compound or a
• a selective RET inhibitor exhibits at least a lO-fold selectivity; at least a l5-fold selectivity; at least a 20-fold selectivity; at least a 30-fold selectivity; at least a 40-fold selectivity; at least a 50-fold selectivity; at least a 60-fold selectivity; at least a 70-fold selectivity; at least a 80-fold selectivity; at least a 90-fold selectivity; at least lOO-fold, at least l25-fold, at least l50-fold, at least l75-fold, or at least 200-fold selectivity for a RET kinase over another kinase.
• a selective RET inhibitor exhibits at least 20-fold selectivity over another kinase, e.g., JAK1. In some embodiments, a selective RET inhibitor exhibits at least 50-fold selectivity over another kinase, e.g. VEGFR-2 or TRKC. In some embodiments, a selective RET inhibitor exhibits at least lOO-fold selectivity over another kinase, e.g., FLT3, JAK2, TRKA, or PDGFR .
• a selective RET inhibitor exhibits at least lOOO-fold selectivity over another kinase, e.g., LIMK1, FGFR1, c- SRC, ML2/MAP3K10, PEAK1, FGFR3, MLK3/MAP3K11, ROS/ROS1, c-KIT, YES/YES 1, FLT4/VEGFR3, JAK3, or TYK2.
• selectivity for a RET kinase over another kinase is measured in a cellular assay.
• selectivity for a RET kinase over another kinase is measured in a biochemical assay.
• Non-limiting examples of selective RET inhibitors include Compound I, SL-1001, and LOXO-292.
• Examples of selective RET inhibitors include Compound 1 and LOXO-292.
• the term“subject” or“patient” refers to organisms to be treated by the methods of the present disclosure. Such organisms include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and in some embodiments, humans.
• mammals e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like
• humans in some embodiments, humans.
• EGFR-mutant cancers include lung cancers (e.g., small cell lung cancer, non-small cell lung cancer, and squamous-cell carcinoma of the lung), anal cancers, colon cancers, thyroid cancers (e.g., papillary thyroid cancer), glioblastoma, epithelial cancers (e.g., epithelial tumors of the head and neck).
• lung cancers e.g., small cell lung cancer, non-small cell lung cancer, and squamous-cell carcinoma of the lung
• anal cancers e.g., colon cancers, thyroid cancers (e.g., papillary thyroid cancer), glioblastoma, epithelial cancers (e.g., epithelial tumors of the head and neck).
• the EGFR-mutant cancer is characterized by at least one EGFR mutation chosen from T790M, C797S, and L792H.
• the EGFR-mutant cancer is characterized by a
• the EGFR-mutant cancer is characterized by a C797S mutation. In some embodiments, the EGFR-mutant cancer is characterized by a L792H mutation. In some embodiments, the EGFR-mutant cancer is characterized by a L858R or Dec 19 mutation. In some embodiments, the EGFR-mutant cancer is characterized by a L858R or Dec 19 mutation and a T790M mutation. In some embodiments, the EGFR- mutant cancer is characterized by a L858R or Dec 19 mutation and a C796S mutation. In some embodiments, the EGFR-mutant cancer is characterized by a L858R or Dec 19 mutation, a T790M mutation, and a C796S mutation.
• the EGFR-mutant cancer is further characterized by at least one RET fusion (e.g., at least one RET fusion listed in Table 1). In some embodiments, the EGFR-mutant cancer is further characterized by CCDC6-RET fusion. In some embodiments, the EGFR-mutant cancer is further characterized by a KIF5B-RET fusion. In some embodiments, the EGFR-mutant cancer if further characterized by a NCOA4-RET fusion.
• a person of ordinary skill in the art may determine if a subject possesses a RET- fusion e.g., using a method selected from hybridization-based methods, amplification-based methods, microarray analysis, flow cytometry analysis, DNA sequencing, next-generation sequencing (NGS), primer extension, PCR, in situ hybridization, fluorescent in situ hybridization, dot blot, and Southern blot.
• primary tumor samples may be collected from a subject.
• the samples are processed, the nucleic acids are isolated using techniques known in the art, then the nucleic acids are sequenced using methods known in the art. Sequences are then mapped to individual exons, and measures of transcriptional expression (such as RPKM, or reads per kilobase per million reads mapped), are quantified.
• Raw sequences and exon array data are available from sources such as TCGA, ICGC, and the NCBI Gene Expression Omnibus (GEO). For a given sample, individual exon coordinates are annotated with gene identifier information, and exons belonging to kinase domains are flagged. The exon levels are then z- score normalized across all tumor samples.
• breakpoint is the boundary at which two different genes are fused. It is sometimes referred to as a“fusion point.” The location where the difference in exon expression is maximal between 5’ and 3’ is the inferred breakpoint of the fusion.
• Thousands of tumor samples can be rapidly profiled in this manner, generating a list of fusion candidates (ranked by t-statistic). High-ranking candidates can then be validated, and fusion partners identified by examining the raw RNA-seq data sets, and identifying chimeric pairs and/or split reads which support the fusion. Candidate fusions can then be experimentally confirmed as described below.
• fusions may be identified by circulating tumor DNA (ctDNA) analysis of plasma (i.e., a liquid biopsy).
• ctDNA circulating tumor DNA
• the EGFR-mutant cancer is a lung cancer.
• the EGFR-mutant cancer is small cell lung cancer.
• the EGFR-mutant cancer is non-small cell lung cancer.
• the EGFR-mutant cancer is squamous-cell carcinoma of the lung.
• the EGFR-mutant cancer is anal cancer.
• the EGFR-mutant cancer is colon cancer.
• the EGFR-mutant cancer is thyroid cancer. In some embodiments, the EGFR-mutant cancer is papillary thyroid cancer.
• the EGFR-mutant cancer is glioblastoma.
• the EGFR-mutant cancer is epithelial cancer. In some embodiments, the EGFR-mutant cancer is an epithelial tumor of the head or neck.
• the patient suffering from an EGFR-mutant cancer has previously been treated with at least one EGFR inhibitor (e.g., osimertinib and/or
• the patient suffering from an EGFR-mutant cancer has acquired resistance to at least one EGFR inhibitor (e.g., osimertinib and/or pharmaceutically acceptable salts thereof).
• at least one EGFR inhibitor e.g., osimertinib and/or pharmaceutically acceptable salts thereof.
• the phrase“therapeutically effective amount” refers to the amount of an active agent (e.g., Compound 1 or a pharmaceutically acceptable salt thereof) sufficient to effect beneficial or desired results.
• a therapeutically effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a specific formulation or administration route.
• the phrase“weight equivalent of a pharmaceutically acceptable salt thereof’ in reference to a specific compound includes the weight of both the compound and the associated salt.
• TAGRISSO® tablets contain 47.7 or 95.4 mg of osimertinib mesylate, which is the weight equivalent of 40 or 80 mg of osimertinib, respectively.
• pharmaceutically acceptable salts of osimertinib mesylate include osimertinib besylate, osimertinib hydrochloride, etc.
• the term“treating” includes any effect, e.g., lessening, reducing, modulating, ameliorating, or eliminating, that results in the improvement of the condition, disease, disorder, and the like, or ameliorating a symptom thereof.
• RET inhibitors that may be utilized in some embodiments include those that are well-known in the art, e.g., alectinib, apatinib, AUY-922, cabozantinib (XL184), Compound 1, DCC-2157, dovitinib (TKI258), BOS172738 (DS-5010), foretinib, GSK3179106, GSK3352589, lenvatinib, LOXO-292, TPX-0046, SL-1001, motesanib, nintendanib, NVP- AST487, ponatinib, PZ-l, regorafenib (BAY 73-4506), RPI-l, RXDX-105, TG101209, sitravatinib (MGCD516), sorafenib, sunitinib, RPI-l, TAS0286, TG101209, SPP86, vatalani
• the RET inhibitor is a multikinase inhibitor that was originally designed to target other kinases, e.g., vascular endothelial growth factor receptor 2 (VEGFR-2), tyrosine-protein kinase MET, and/or EGFR, that inhibits the other kinases more potently than RET, e.g., cabozantinib, vandetanib, sunitinib, levatinib, regorafenib, and RXDX-105.
• multikinase inhibitors with RET activity are poor inhibitors of RET with gatekeeper mutations at the V804 residue, e.g., V804L and V804M.
• the at least one RET inhibitor is a selective RET inhibitor.
• the selective inhibitor was designed for highly potent and selective targeting of wild-type (WT) RET and oncogenic mutant forms of RET, e.g., prevalent RET alterations, including RET fusions (e.g., KIF5B-RET, CCDC6-RET), and RET activating mutations (e.g., C634W, M918T, V804L/M), while maintaining selectivity against other human kinases.
• a selective RET inhibitor has activity against multiple oncogenic mutant forms of RET, regardless of tumor type.
• the equipotent activity of a selective RET inhibitor across multiple oncogenic mutant forms of RET differentiates the selective RET inhibitor from a multikinase inhibitor with RET inhibitory activity.
• the at least one RET inhibitor is a selective RET inhibitor.
• Compound l is a RET-selective inhibitor that only inhibits wild-type RET and one or more mutant forms of RET and has little inhibitory activity against other kinases.
• Selective RET inhibitors that may be utilized in some embodiments include those that are well-known in the art, e.g., compounds disclosed in WO 2016/127074, WO 2017/011776, WO 2017/079140, WO 2017/161269,
• the at least one RET inhibitor is a selective RET inhibitor chosen from:
• the at least one RET inhibitor is a selective RET inhibitor chosen from: 4-(6-(4-benzylpiperazin-l-yl)pyri din-3 -yl)-6-(2- morpholinoethoxy)pyrazolo[l,5-a]pyridine-3-carbonitrile; 6-(2-hydroxyethoxy)-4-(6-(6-((6- methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3. l .
• the at least one RET inhibitor is a selective RET inhibitor chosen from: N-(l-(5-(3-cyano-6-(2-hydroxy-2-methylpropoxy)pyrazolo[l,5-a]pyridin-4- yl)pyri din-2 -yl)-4-methylpiperidin-4-yl)benzamide; 6-ethoxy-4-(6-(4-hydroxy-4-(pyri din-2 - ylmethyl)piperidin-l-yl)pyridin-3-yl)pyrazolo[l,5-a]pyridine-3-carbonitrile; 6-(2-hydroxy-2- methylpropoxy)-4-(6-(3-(pyridin-2-yloxy)azetidin-l-yl)pyridin-3-yl)pyrazolo[l,5-a]pyridine- 3-carbonitrile; 6-(2-hydroxy-2-methylpropoxy)-4-(6-(4-((6-methoxypyridazin-3-
• the at least one RET inhibitor is administered once daily. In some embodiments, the at least one RET inhibitor is administered orally. In some embodiments, the at least one RET inhibitor is orally administered once daily.
• the at least one RET inhibitor is chosen from Compound 1 and pharmaceutically acceptable salts thereof.
• the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 200 mg to 400 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof.
• the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 200 mg, 205 mg, 210 mg, 215 mg, 220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265 mg, 270 mg, 275 mg, 280 mg, 285 mg, 290 mg, 295 mg, 300 mg, 305 mg, 310 mg, 315 mg, 320 mg, 325 mg, 330 mg, 335 mg,
• the at least one RET inhibitor is chosen from Compound 1 and pharmaceutically acceptable salts thereof.
• the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 200 mg to 400 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof.
• the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 200 mg, 205 mg, 210 mg, 215 mg, 220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265 mg, 270 mg, 275 mg,
• the at least one RET inhibitor is chosen from Compound 1 and pharmaceutically acceptable salts thereof.
• the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 200 mg to 300 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof.
• the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 200 mg, 205 mg, 210 mg, 215 mg, 220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265 mg, 270 mg, 275 mg, 280 mg, 285 mg, 290 mg, 295 mg, or 300 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof.
• the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 200 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 205 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 210 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 215 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof.
• the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 220 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 225 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 230 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 235 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof.
• the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 240 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 245 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 250 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 255 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof.
• the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 260 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 265 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 270 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 275 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof.
• the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 280 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 285 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 290 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 295 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof.
• the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 300 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 305 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 310 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 315 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof.
• the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 320 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 325 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 330 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 335 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof.
• the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 340 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 345 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 350 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 355 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof.
• the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 360 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 365 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 370 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 375 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof.
• the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 380 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 385 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 390 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 395 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor orally administered once daily is 400 mg of Compound 1 or the weight equivalent of a pharmaceutically acceptable salt thereof.
• the at least one EGFR inhibitor is chosen from afatinib, ASP8273, avitinib, brigatinib, cetuximab, dacomitinib, EAI045, erlotinib, gefitinib, HS- 10296, icotinib, lapatinib, necitumumab, soloartinib, neratinib, olmutinib, osimertinib, panitumumab, PF-06747775, EGF816, YH5448, avitinib, rociletinib, vandetanib, and pharmaceutically acceptable salts of any of the foregoing.
• the at least one EGFR inhibitor is a selective inhibitor.
• a selective EGFR inhibitor was designed for highly potent and selective targeting of oncogenic mutant forms of EGFR, e.g., exon 19 deletion, L858R, T790M.
• a selective EGFR inhibitor has activity against multiple oncogenic mutant forms of EGFR, regardless of tumor type.
• the equipotent activity of a selective EGFR inhibitor across multiple oncogenic mutant forms of EGFR differentiates a selective EGFR inhibitor from a multikinase inhibitor with EGFR inhibitory activity.
• a selective EGFR inhibitor is a third generation EGFR inhibitor.
• a selective EGFR inhibitor has activity against oncogenic mutant forms of EGFR, including exon 19 deletion, L858R, and T790M.
• a selective EGFR inhibitor includes osimertinib, rociletinib, olmutinib, EGF816, PF-06747775, YH5448, and avitinib.
• the selective EGFR inhibitor does not have activity against WT EGFR.
• a selective EGFR inhibitor is not a covalent inhibitor.
• the at least one EGFR inhibitor is administered once daily. In some embodiments, the at least one EGFR inhibitor is administered orally. In some embodiments, the at least one EGFR inhibitor is orally administered once daily.
• the at least one EGFR inhibitor is chosen from osimertinib and pharmaceutically acceptable salts thereof.
• the therapeutically effective amount of the at least one EGFR inhibitor orally administered once daily is 80 mg of osimertinib or the weight equivalent of a pharmaceutically acceptable salt thereof.
• the active agent can be administered as a pharmaceutical formulation, wherein the active agent is combined with one or more pharmaceutically acceptable excipients or carriers.
• the active agent may be formulated for administration in any convenient way for use in human or veterinary medicine.
• the compound included in the pharmaceutical preparation may be active itself, or may be a prodrug, e.g., capable of being converted to an active compound in a physiological setting.
• phrases“pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• Examples of pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol;
• polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol
• esters such as ethyl oleate and ethyl laurate
• agar such as agar
• buffering agents such as magnesium hydroxide and aluminum hydroxide
• antioxidants examples include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
• water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite, and the like
• oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluen
• Solid dosage forms can include one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia;
• pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia;
• humectants such as glycerol
• disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate;
• solution retarding agents such as paraffin
• absorption accelerators such as quaternary ammonium compounds
• wetting agents such as, for example, cetyl alcohol and glycerol monostearate
• absorbents such as kaolin and bentonite clay
• lubricants such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof
• coloring agents such as paraffin
• absorption accelerators such as quaternary ammonium compounds
• wetting agents such as, for example, cetyl alcohol and glycerol monostearate
• absorbents such as kaolin and bentonite clay
• lubricants such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof
• coloring agents such as paraffin
• absorption accelerators such as quaternary ammonium compounds
• wetting agents such as,
• Liquid dosage forms can include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
• the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan, and mixtures thereof.
• inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers,
• Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
• suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
• Ointments, pastes, creams, and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof.
• excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof.
• Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances.
• Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
• Dosage forms for the topical or transdermal administration of Compound 1 include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants.
• the active compound may be mixed under sterile conditions with a
• pharmaceutically acceptable carrier and with any preservatives, buffers, or propellants that may be required.
• Compound 1 When Compound 1 is administered as a pharmaceutical, to humans and animals, it can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (such as 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
• the formulations can be administered topically, orally, transdermally, rectally, vaginally, parentally, intranasally, intrapulmonary, intraocularly, intravenously,
• intramuscularly intraarterially, intrathecally, intracapsularly, intradermally, intraperitoneally, subcutaneously, subcuticularly, or by inhalation.
• Afatinib, osimertinib, and cabozantinib were purchased from Selleck Chemicals and resuspended in DMSO.
• Phospho-EGFR (Y1068), EGFR, pBRAF (Ser445), RET, pAKT (Ser473), ART, pMEKl/2 (Ser2l7/22l), MEK1/2, pERKl/2 (Thr202/204), ERK1/2, and Actin antibodies were purchased from Cell Signaling Technology.
• pRET (Y1062) antibody was obtained from Abeam.
• RNA from cell lines was extracted using an RNeasy Mini Kit (Qiagen). RNA (1 pg) was reverse transcribed using SuperscriptTM II Reverse Transcriptase
• CCDC6-RET, PCBP2-BRAF, and TBP were PCR amplified using the following primers: CCDC6 exon 1 F- CGGACAGCGCCAGCG, RET exon 19 R- GCATTATTACAGTCCACCAGCG; (PCBP2- BRAF Primer 1) PCBP2 exon 6 F- AGGT GGAT GC AAGATC AAGG, BRAF exon 13 R- TAGCCAGTTGTGGCTTTGTG; (PCBP2-BRAF Primer 2) PCBP2 exon 2 F- C GGT GT GATT G A AGGT GGAT , BRAF exon 18 R- ACAGGAAACGCACCATATCC; TBP F- CCCATGACTCCCATGACC, TBP R- TTTACAACCAAGATTCACTGTGG.
• the PCR products were confirmed by agarose gel electrophoresis. Following amplification, Sanger sequencing was performed.
• PC9 and MGH134 cell lines are known in the art (Hata, A.N. et ak, Nat. Med. 22(3):262-69 (2016)). Cells were cultured in RPMI1640 (Life Technologies) supplemented with 10% FBS. MGH845-1 cells were additionally cultured in 150 nM osimertinib. All cells were routinely tested and verified to be free of mycoplasma contamination.
• a CCDC6-RET fusion construct was synthesized by GenScript and ligated into the pLENTI6/V5-D-TOPO vector using the ViraPower Lentiviral Directional TOPO
• Lentivirus was generated by transfecting the pLENTI6 constructs and packaging plasmids into 293FT cells (Life Technologies). Virus production, collection, and infection were completed following the manufacturer's protocol. Transduced cells were selected in blasticidin (10-20 mg/mL) for one week.
• tissue and plasma samples exhibiting CCDC6-RET rearrangement came from separate donors, indicating that RET rearrangements are a low frequency but recurrent finding in EGFR-mutant patients with AR to osimertinib.
• SFA solid fusion assay
• FISH RET fluorescence in situ hybridization
• Example 3 CCDC6-RET expression in EGFR-mutant NSCLC cell lines confers resistance to EGFR inhibitors.
• CCDC6-RET fusion expressing cell line models were generated by lentiviral infection of PC9 (EGFR del 19) and MGH134 (EGFR L858R/T790M) cells (FIG. 2).
• CCDC6-RET expression alone did not lead to increased activation of downstream MAPK (phospho-ERKl/2) or PI3K (phospho-AKT) signaling at baseline; however, RET, ERK1/2, and AKT phosphorylation was retained in the presence of afatinib or osimertinib in both PC9 CCDC6 i?£r and MGH 1 4 ( r!X 6 RI:i cell s (FIGs. 4A-4B).
• expression of the CCDC6-RET fusion can confer resistance to EGFR- inhibitors in EGFR-mutant NSCLCs.
• Example 4 Acquired resistance resulting from CCDC6-RET expression can be overcome by EGFR plus RET.
• PC9CCDC6-RET cells generated as above, were treated with Compound 1 in the absence or presence of EGFR inhibitors.
• Treatment with Compound 1 alone suppressed RET phosphorylation, but did not decrease downstream ERK or ART phosphorylation (FIG. 4A).
• Combined treatment with Compound 1 and either osimertinib or afatinib completely suppressed both phospho-ERK and phospho-AKT and decreased cell viability to a similar level as parental cells treated with EGFR TKI (FIG. 4C). Similar results were observed in MGH 1 4 CCDC6 RET cell S (FIGS. 4B, 5C).
• PC9 Y 7 3 ⁇ 4 - /i/:/ and MORP 34 GG/ c - /c:/ cells were sensitive to EGFR TKI + cabozantinib, a multi-kinase inhibitor with RET activity (FIGs. 4B, 5A-5C). Taken together, these data demonstrate that acquired resistance resulting from the CCDC6-RET fusions can be overcome by dual EGFR plus RET blockade.
• Example 5 Study of Compound 1 and Osimertinib for Metastatic Non-Small Cell Lung Cancer with RET Fusion-Mediated Resistance to EGFR Inhibition (Combination study for metastatic NSCLC with RET-mediated resistance to EGFR inhibition)
• This study is an open-label, Phase 1/2 study designed to evaluate the safety, tolerability, antitumor activity, PK, and pharmacodynamics of the potent and selective RET inhibitor, Compound 1, in combination with the third-generation EGFR inhibitor,
• a dose-escalation study is conducted to assess the safety and tolerability of the combination of Compound 1 and osimertinib, and to identify the maximum tolerated dose (MTD) and/or recommended Phase 2 dose (RP2D).
• MTD maximum tolerated dose
• R2D recommended Phase 2 dose
• the overall safety profile of combination treatment with Compound 1 and osimertinib is assessed by the type, frequency, severity, timing, and relationship to study drug of any adverse events, serious adverse events, changes in vital signs, ECGs, and safety laboratory tests.
• ORR overall response rate
• the study also assesses additional measures of anti cancer activity, including duration of response (DOR), disease control rate (DCR), clinical benefit rate (CBR), progression-free survival (PFS), and overall survival (OS).
• DOR duration of response
• DCR disease control rate
• CBR clinical benefit rate
• PFS progression-free survival
• OS overall survival
• DOR is defined as the number of months from the time criteria are first met for either CR or PR, until the first date that progressive disease (PD) is objectively documented for patients with confirmed CR or PR;
• DCR is defined as the proportion of patients who experience a best response of stable disease (SD), partial response (PR), or complete response (CR) according to RECIST 1.1; partial response (PR), or complete response (CR) according to RECIST 1.1; and
• PFS is defined as the number of months from first dose of study treatment to the earlier of PD or death due to any cause.
• PK parameters of Compound 1 include: population-derived estimates including maximum plasma drug concentration (Cmax), area under the plasma concentration versus time curve from time 0 to 24 hours postdose (AUC0-24), plasma drug concentration at 24 hours post-dose (C24) at steady-state; and type, frequency, severity, timing, and relationship to study drug of any AEs, serious adverse events (SAEs), changes in vital signs, ECGs, and safety laboratory tests.
• Cmax maximum plasma drug concentration
• AUC0-24 area under the plasma concentration versus time curve from time 0 to 24 hours postdose
• C24 plasma drug concentration at 24 hours post-dose
• SAEs serious adverse events
• the study includes a standard 3+3 dose-escalation portion to identify the MTD and/or RP2D of Compound 1 when given in combination with osimertinib, followed by an expansion phase to assess ORR and other measures of clinical activity. All patients enrolled in the Phase 1 study portion must begin osimertinib treatment at the approved starting dose of 80 mg/day. In the Phase 2 study portion, patients who experienced toxicity with prior osimertinib treatment may initiate osimertinib at a lower starting dose if necessary. The Compound 1 dose levels are evaluated in dose escalation of 200 mg, 300 mg, and 400 mg. Patients in the expansion phase receive the RP2D as determined from dose-escalation.
• RET fusion status is determined by local or central assessment using a tumor or blood sample taken at the time of (or following) progression of disease on an EGFR inhibitor.
• Patients may continue to receive study treatment until precluded by toxicity, noncompliance, withdrawal of consent, death, or closure of the study. Patients who experience RECIST l. l-defmed progression of disease but continue to experience clinical benefit in the opinion of the treating investigator may continue study therapy with approval.
• Compound 1 is permanently discontinued, the patient is considered to have completed the study treatment period; and other anticancer therapy (including, if appropriate, osimertinib) is received as subsequent therapy during survival follow-up. Patients who require permanent discontinuation of osimertinib may continue Compound 1 monotherapy after approval.
• the patient population includes participants that: are > 18 years of age at the time of signing the informed consent; have pathologically confirmed, definitively diagnosed, metastatic EGFR-mutant NSCLC; have at least one target lesion evaluable by RECIST 1.1; for Phase 1 only: have radiologically documented disease progression during or after previous treatment with any 2nd or 3rd generation EGFR inhibitor TKI; for Phase 2 only: have radiologically documented disease progression during or after previous treatment with osimertinib; have oncogenic RET fusion, as detected by local or central testing of tumor tissue or circulating tumor nucleic acid in blood, using a sample as described above (for patients with RET status determined locally for eligibility, the patient must also consent to submission of blood and tissue samples for retrospective confirmation of RET status by central testing); are willing to provide archived tumor tissue (if a sample obtained following progression of disease during or after previous treatment with osimertinib is available) or, if appropriate archived tumor tissue is not available, is willing to undergo a pre
• the patient population excludes participants that: have any additional known primary driver alteration (other than the original EGFR mutation and RET fusion), including but not limited to targetable mutations of ALK, ROS1, MET, and BRAF; have any past medical history of interstitial lung disease (ILD) or interstitial pneumonitis, radiation pneumonitis that required steroid treatment, or any evidence of clinically active ILD within 28 days prior to enrollment; have central nervous system (CNS) metastases or a primary CNS tumor that is associated with progressive neurological symptoms or requires increasing doses of corticosteroids to control the CNS disease (if a patient requires corticosteroids for management of CNS disease, the dose must have been stable for the 2 weeks preceding C1D1); have had any anti-PD-l/PDL-l/CTLA therapy within 6 months, and any other anticancer therapy (including both systemic therapy and radiotherapy) within 14 days or 5 half-lives prior to the first dose of study drug, whichever is shorter (excluding prior osimer
• Hemoglobin ⁇ 9.0 g/dL red blood cell transfusion and erythropoietin may be used to reach at least 9.0 g/dL, but must have been administered at least 2 weeks prior to the first dose of study drug;
• Phase 1 dose-escalation phase A sample size of up to 18 patients is included in the Phase 1 dose-escalation phase.
• the total number of participants enrolled in the dose-escalation is dependent upon the observed safety profile, which determines the number of participants per dose cohort as well as the number of cohorts required to confirm the recommended Phase 2 dose (RP2D).
• R2D Phase 2 dose
• the sample size at stage 1 is 10 patients (30% of the total sample size) by assuming the null hypothesis response rate 5% and the alternative response rate 25% with one sided alpha 0.025 and power 90%.
• the cumulative sample size from stage 1 and stage 2 is 33.
• the trial is stopped due to failure to reject the null hypothesis if the response rate is no more than 1/10 (10%). Otherwise, the trial continues to stage 2.
• the null hypothesis is rejected if there are at least 5 responders among all 33 patients.
• the Response Evaluable Population includes all patients who have measurable disease at baseline, receive at least one dose of each study treatment (Compound 1 and osimertinib), and have an evaluable post-baseline tumor response assessment.
• the REP is utilized for the primary analyses of ORR, DCR and CBR.
• the Safety Population including all patients who receive at least one dose of Compound 1, is utilized for the remaining efficacy endpoints and safety.
• Progression-Free Survival is analyzed using the Kaplan-Meier method. If a patient does not experience progressive disease or death, then the patient is censored at the time of the last response assessment.
• Safety analyses consists of data summaries for clinical and laboratory parameters and AEs. The number and percentage of patients experiencing one or more AEs are summarized by the relationship to study drug and severity based on NCI CTCAE v 5.0. Adverse events are coded using the Medical Dictionary for Regulatory Activities (MedDRA). Laboratory parameters are summarized using descriptive statistics, by post-treatment shifts relative to baseline, and data listings of clinically significant abnormalities. Vital signs and ECG data are summarized using descriptive statistics. Compound 1 plasma concentration data is tabulated with descriptive statistics. Compound 1 exposure parameters are correlated with safety endpoints and antitumor activity.

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