ALK mutations and uses thereof

Abstract

Provided herein are mutant anaplastic lymphoma kinase (ALK) nucleic acid molecules and polypeptides, methods related to detecting mutant ALK nucleic acid molecules and polypeptides in cancer, as well as methods of treatment, uses, systems, and non-transitory computer-readable storage media related thereto. A mutant ALK nucleic acid molecule or polypeptide of the present disclosure can be used to identify cancers that are more likely to be resistant to ALK-targeted therapies, or individuals that may benefit from a change in ALK- targeted therapy-based treatment.

Description

Docket No: 197102019040 ALK MUTATIONS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the priority benefit of U.S. Provisional Application No. 63 / 678,367, filed on August 1, 2024, the contents of which are incorporated herein by reference in their entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (197102019040seqlist.xml; Size: 9,205 bytes; and Date of Creation: July 29, 2025) is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] Provided herein are mutant anaplastic lymphoma kinase (ALK) nucleic acid molecules and polypeptides, methods related to detecting such mutant ALK nucleic acid molecules and polypeptides, as well as methods of diagnosis / treatment and uses related thereto. BACKGROUND

[0004] The anaplastic lymphoma kinase (ALK) gene (also known as CD246) encodes a receptor tyrosine kinase. ALK is a member of the insulin receptor superfamily. ALK activation induces downstream pathways associated with cell survival, angiogenesis, and cell proliferation (Grande et al., Molecular cancer therapeutics, vol. 10,4 (2011): 569-79). ALK is a known oncogene that has been associated with cancerous phenotypes, including inflammatory myofibroblastic tumors, neuroblastoma, lung cancer, non-Hodgkin’s lymphoma, and anaplastic large cell lymphoma, among others. Chromosomal rearrangements involving the ALK gene have been found in certain cancers, and have been characterized as oncogenic (see, e.g., Hallberg et al., Annals of oncology, vol. 27 Suppl 3 (2016): iii4-iii15; Ignatius et al., JTO clinical and research reports, vol. 1,1100015, 2020; and Holla, V.R. et al. (2017) Cold Spring Harb Mol Case Stud. 3(1):a001115). ALK gene fusions have also been associated with sensitivity of cancer to ALK inhibitors, such as crizotinib, ceritinib, alectinib, lorlatinib, or entrectinib (see, e.g., Ali et al., USCAP Abstract 18682015; Mansfield et al., Annals of oncology, vol. 27,11 (2016): 2111-2117; Yakirevich et al., Clinical cancer research, vol. 22,15 (2016): 3831-40; Subbiah et al., Journal of hematology & oncology, vol. 866, 2015; Amatu et al., British journal of cancer vol. 113,12 (2015): 1730-4; Lee et al., Oncotarget vol. 6,27 (2015): 24320-32; Shan et al., Journal of thoracic oncology, vol. 10,6 1MOFO-358156824Docket No: 197102019040 (2015): e37-9; Ali et al., The oncologist, vol. 21,6 (2016): 762-70; and Ou et al., Journal of thoracic oncology, vol. 9,12 (2014): 1821-5).

[0005] While treatment with an ALK-targeted therapy (e.g., a tyrosine kinase inhibitor (TKI) such as crizotinib, alectinib, ceritinib, ensartinib, brigatinib, or lorlatinib) has been shown to provide clinical responses and improved survival in cancers with activating ALK rearrangements (e.g., ALK+ cancers), such treatment can lead to the emergence of acquired resistance (Peng, L. et al. (2022) Front Oncol. 12:863461). Acquired resistance can include on-target (e.g., mutations in an ALK gene) as well as off-target alterations.

[0006] Thus, there is a need in the art for characterizing the cancer landscape of ALK mutations, particularly those arising in cancers treated with an ALK-targeted therapy. Detection of such mutations may help guide treatment decisions in patients with ALK+ cancers, potentially predicting and / or detecting resistance to ALK-targeted therapies and informing when to change therapies.

[0007] All references cited herein, including patents, patent applications, and publications, are hereby incorporated by reference in their entirety. To the extent that any reference incorporated by reference conflicts with the instant disclosure, the instant disclosure shall control. SUMMARY OF THE INVENTION

[0008] In one aspect, provided herein is a method of predicting resistance of a cancer in an individual to a treatment comprising an anaplastic lymphoma kinase (ALK)-targeted therapy, the method comprising acquiring knowledge of a mutant ALK nucleic acid molecule or mutant ALK polypeptide in a sample obtained from an individual, wherein the mutant ALK nucleic acid molecule encodes an ALK polypeptide that comprises, or the mutant ALK polypeptide comprises, a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; wherein responsive to the acquisition of said knowledge, the cancer is predicted as more likely to be resistant to a treatment comprising an ALK- targeted therapy, as compared to a cancer that does not comprise the mutant ALK nucleic acid molecule or polypeptide.

[0009] In another aspect, provided herein is a method of predicting resistance of a cancer in an individual to a treatment comprising an ALK-targeted therapy, the method comprising detecting a mutant ALK nucleic acid molecule or mutant ALK polypeptide in a sample 2MOFO-358156824Docket No: 197102019040 obtained from an individual, wherein the mutant ALK nucleic acid molecule encodes an ALK polypeptide that comprises, or the mutant ALK polypeptide comprises, a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; wherein responsive to the detection, the cancer is predicted as more likely to be resistant to a treatment comprising an ALK-targeted therapy, as compared to a cancer that does not comprise the mutant ALK nucleic acid molecule or polypeptide, based at least in part on the detection.

[0010] In another aspect, provided herein is a method of monitoring for resistance of a cancer in an individual to a treatment comprising an ALK-targeted therapy, the method comprising acquiring knowledge of a mutant ALK nucleic acid molecule or mutant ALK polypeptide in a sample obtained from an individual, wherein the mutant ALK nucleic acid molecule encodes an ALK polypeptide that comprises, or the mutant ALK polypeptide comprises, a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; wherein presence of the mutant ALK nucleic acid molecule or polypeptide indicates resistance to a treatment comprising an ALK-targeted therapy. In another aspect, provided herein is a method of screening for resistance of a cancer in an individual to a treatment comprising an ALK-targeted therapy, the method comprising acquiring knowledge of a mutant ALK nucleic acid molecule or mutant ALK polypeptide in a sample obtained from an individual, wherein the mutant ALK nucleic acid molecule encodes an ALK polypeptide that comprises, or the mutant ALK polypeptide comprises, a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; wherein presence of the mutant ALK nucleic acid molecule or polypeptide indicates resistance to a treatment comprising an ALK-targeted therapy.

[0011] In another aspect, provided herein is a method of monitoring for resistance of a cancer in an individual to a treatment comprising an ALK-targeted therapy, the method comprising detecting a mutant ALK nucleic acid molecule or mutant ALK polypeptide in a sample obtained from an individual, wherein the mutant ALK nucleic acid molecule encodes an ALK polypeptide that comprises, or the mutant ALK polypeptide comprises, a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; wherein presence of the mutant ALK nucleic acid molecule or polypeptide indicates resistance to a treatment comprising an ALK-targeted therapy. In another aspect, provided herein is a method of 3MOFO-358156824Docket No: 197102019040 screening for resistance of a cancer in an individual to a treatment comprising an ALK- targeted therapy, the method comprising detecting a mutant ALK nucleic acid molecule or mutant ALK polypeptide in a sample obtained from an individual, wherein the mutant ALK nucleic acid molecule encodes an ALK polypeptide that comprises, or the mutant ALK polypeptide comprises, a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; wherein presence of the mutant ALK nucleic acid molecule or polypeptide indicates resistance to a treatment comprising an ALK-targeted therapy.

[0012] In some embodiments according to any one of the embodiments disclosed herein, the individual has received a prior treatment comprising an ALK-targeted therapy.

[0013] In another aspect, provided herein is a method of treating or delaying progression of cancer, comprising: administering to the individual a treatment comprising a first ALK- targeted therapy; acquiring knowledge of a mutant ALK nucleic acid molecule or mutant ALK polypeptide in a sample obtained from an individual (e.g., obtained after administration of the first ALK-targeted therapy), wherein the mutant ALK nucleic acid molecule encodes an ALK polypeptide that comprises, or the mutant ALK polypeptide comprises, a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; and administering (e.g., after the acquisition of knowledge of the mutant ALK nucleic acid molecule or mutant ALK polypeptide in the sample) to the individual a treatment comprising a second ALK-targeted therapy different from the first ALK-targeted therapy.

[0014] In another aspect, provided herein is a method of treating or delaying progression of cancer, comprising: administering to the individual a treatment comprising a first ALK- targeted therapy; detecting a mutant ALK nucleic acid molecule or mutant ALK polypeptide in a sample obtained from an individual (e.g., obtained after administration of the first ALK- targeted therapy), wherein the mutant ALK nucleic acid molecule encodes an ALK polypeptide that comprises, or the mutant ALK polypeptide comprises, a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; and administering (e.g., after detecting the mutant ALK nucleic acid molecule or mutant ALK polypeptide in the sample) to the individual a treatment comprising a second ALK-targeted therapy different from the first ALK-targeted therapy. 4MOFO-358156824Docket No: 197102019040

[0015] In some embodiments according to any one of the embodiments disclosed herein, the methods further comprise ceasing administration of the first ALK-targeted therapy (e.g., after acquiring knowledge of or detecting the mutant ALK nucleic acid molecule or mutant ALK polypeptide in the sample).

[0016] In another aspect, provided herein is a method of treating or delaying progression of cancer, comprising: acquiring knowledge of a mutant ALK nucleic acid molecule or mutant ALK polypeptide in a sample obtained from an individual, wherein the mutant ALK nucleic acid molecule encodes an ALK polypeptide that comprises, or the mutant ALK polypeptide comprises, a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; and administering to the individual a treatment comprising an ALK-targeted therapy.

[0017] In another aspect, provided herein is a method of treating or delaying progression of cancer, comprising: detecting a mutant ALK nucleic acid molecule or mutant ALK polypeptide in a sample obtained from an individual, wherein the mutant ALK nucleic acid molecule encodes an ALK polypeptide that comprises, or the mutant ALK polypeptide comprises, a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; and administering to the individual a treatment comprising an ALK-targeted therapy.

[0018] In some embodiments according to any one of the embodiments disclosed herein, the individual has received a prior treatment comprising a different ALK-targeted therapy. In some embodiments, the methods further comprise ceasing the prior treatment comprising the different ALK-targeted therapy (e.g., prior to the administration of the treatment). In some embodiments, the ALK-targeted therapy is administered to the individual after acquiring knowledge of or detecting the mutant ALK nucleic acid molecule or mutant ALK polypeptide in the sample. In some embodiments, the ALK-targeted therapy is administered to the individual prior to acquiring knowledge of or detecting the mutant ALK nucleic acid molecule or mutant ALK polypeptide in the sample. In some embodiments, acquiring knowledge of or detecting the mutant ALK nucleic acid molecule or mutant ALK polypeptide in the sample occurs before and after administration of an ALK-targeted therapy to the individual.

[0019] In some embodiments according to any one of the embodiments disclosed herein, the ALK-targeted therapy administered to the individual is selected based at least in part on acquisition of knowledge, or detection, of the mutant ALK nucleic acid molecule or mutant ALK polypeptide. In some embodiments, the prior treatment comprises crizotinib, and the ALK-targeted therapy comprises alectinib, brigatinib, ceritinib, ensartinib, or lorlatinib. In 5MOFO-358156824Docket No: 197102019040 some embodiments, the prior treatment comprises crizotinib, alectinib, brigatinib, ceritinib, or ensartinib, and the ALK-targeted therapy comprises lorlatinib.

[0020] In some embodiments according to any one of the embodiments disclosed herein, the cancer further comprises an ALK nucleic acid molecule that encodes an ALK polypeptide comprising, or comprises an ALK polypeptide comprising, a second ALK mutation associated with resistance (e.g., acquired resistance) to an ALK-targeted therapy. In some embodiments, the methods further comprise acquiring knowledge of an ALK nucleic acid molecule that encodes an ALK polypeptide comprising, or comprises an ALK polypeptide comprising, a second ALK mutation associated with resistance (e.g., acquired resistance) to an ALK-targeted therapy in a sample obtained from the individual. In some embodiments, the methods further comprise detecting an ALK nucleic acid molecule that encodes an ALK polypeptide comprising, or comprises an ALK polypeptide comprising, a second ALK mutation associated with resistance (e.g., acquired resistance) to an ALK-targeted therapy in a sample obtained from the individual. In some embodiments, the second ALK mutation associated with acquired resistance to an ALK-targeted therapy is selected from the group consisting of T1151M, T1151R, C1156Y, I1171N, I1171T, I1171S, I1171H, I1171V, F1174X, V1180L, R1192P, L1196M, L1198F, G1202X, G1202del, D1203N, S1206Y, E1210K, and G1269A, amino acid numbering based on SEQ ID NO:2. In some embodiments, the second ALK mutation associated with acquired resistance to an ALK- targeted therapy is selected from the group consisting of T1151M, L1152R, C1156Y, I1171T, F1174 (any missense), R1192P, L1196M, G1202R, G1269A, S1206Y, and E1210K, amino acid numbering based on SEQ ID NO:2, and optionally the second ALK mutation is associated with resistance to a first-generation ALK TKI, e.g., crizotinib. In some embodiments, the second ALK mutation associated with acquired resistance to an ALK- targeted therapy is selected from the group consisting of T1151R, I1171N, I1171T, I1171S, I1171H, I1171V, F1174C, V1180L, G1202 (any missense), G1202R, G1202del, and D1203N, amino acid numbering based on SEQ ID NO:2, and optionally the second ALK mutation is associated with resistance to a second-generation ALK TKI, e.g., ceritinib, brigatinib, or alectinib. In some embodiments, the second ALK mutation associated with acquired resistance to an ALK-targeted therapy comprises a set of multiple ALK mutations selected from the group consisting of C1156Y + L1198F, L1196M + D1203N, L1196M + G1202R, G1202R + G1269A, G1202R + F1174L, L1196M + F1174L, amino acid numbering based on SEQ ID NO:2, and optionally the second ALK mutation is associated with resistance to a third-generation ALK TKI, e.g., lorlatinib. In some embodiments, the 6MOFO-358156824Docket No: 197102019040 methods further comprise acquiring knowledge of a nucleic acid molecule that encodes, or a polypeptide comprising, a mutation in one or more genes selected from the group consisting of AXL, BRAF, MAP2K1, MET, NF2, PIK3CA, and NRAS in a sample obtained from the individual. In some embodiments, the methods further comprise detecting a nucleic acid molecule that encodes, or a polypeptide comprising, a mutation in one or more genes selected from the group consisting of AXL, BRAF, MAP2K1, MET, NF2, PIK3CA, and NRAS in a sample obtained from the individual. In some embodiments, the methods further comprise acquiring knowledge of a nucleic acid molecule that encodes, or a polypeptide comprising, a mutation in one or more genes associated with off-target resistance (e.g., acquired resistance) to an ALK-targeted therapy. In some embodiments, the methods further comprise detecting a nucleic acid molecule that encodes, or a polypeptide comprising, a mutation in one or more genes associated with off-target resistance (e.g., acquired resistance) to an ALK-targeted therapy. In some embodiments, the methods further comprise generating a report indicating presence of the mutant ALK nucleic acid molecule or ALK polypeptide in the sample. In some embodiments, the methods further comprise generating a molecular profile for the individual based at least in part on presence of the mutant ALK nucleic acid molecule or ALK polypeptide in the sample. In some embodiments, the cancer further comprises a gene fusion, gene rearrangement, gene amplification, or activating mutation in an ALK gene. In some embodiments, the methods further comprise acquiring knowledge of a gene fusion, gene rearrangement, gene amplification, or activating mutation in an ALK gene in a sample obtained from the individual. In some embodiments, the methods further comprise detecting a gene fusion, gene rearrangement, gene amplification, or activating mutation in an ALK gene in a sample obtained from the individual.

[0021] In some embodiments according to any one of the embodiments disclosed herein, the ALK-targeted therapy is crizotinib, alectinib, brigatinib, ceritinib, ensartinib, lorlatinib, TPX- 0131, NVL-655, entrectinib, repotrectinib, belizatinib, TQ-B3139, WX-0593, PLB-1003, SAF-189s, CT-707, gilteritinib, XMU-MP-5, AZD3463, CEP-37440, ASP3026, KRCA- 0008, or TAE684. In some embodiments, the ALK-targeted therapy comprises a tyrosine kinase inhibitor (TKI). In some embodiments, the ALK-targeted therapy comprises an ALK proteolysis-targeting chimeric (PROTAC) degrader.

[0022] In some embodiments according to any one of the embodiments disclosed herein, the cancer is lung cancer, anaplastic large cell lymphoma (ALCL), neuroblastoma, an inflammatory myofibroblastic tumor (IMT), spitzoid tumor, colorectal cancer, B-cell lymphoma, ovarian cancer, non-Hodgkin’s lymphoma (NHL), or thyroid cancer. In some 7MOFO-358156824Docket No: 197102019040 embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is an ALK+ cancer, e.g., an ALK-rearrangement positive (ALK+) cancer.

[0023] In some embodiments according to any one of the embodiments disclosed herein, the mutant ALK nucleic acid molecule is detected in the sample by one or more of: a nucleic acid hybridization assay, an amplification-based assay, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay, real-time PCR, a screening analysis, fluorescence in situ hybridization (FISH), spectral karyotyping, multicolor FISH (mFISH), comparative genomic hybridization, in situ hybridization, sequence-specific priming (SSP) PCR, high-performance liquid chromatography (HPLC), mass-spectrometric genotyping, or sequencing. In some embodiments, the sequencing comprises a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique; and optionally wherein the massively parallel sequencing (MPS) technique comprises next-generation sequencing (NGS). In some embodiments, the methods further comprise selectively enriching for one or more nucleic acids in the sample comprising nucleotide sequences corresponding to the mutant ALK nucleic acid molecule; wherein the selectively enriching produces an enriched sample. In some embodiments, the methods further comprise sequencing the enriched sample. In some embodiments, detecting the mutant ALK nucleic acid molecule in the sample comprises: providing a plurality of nucleic acid molecules obtained from the sample, wherein the plurality of nucleic acid molecules comprises nucleic acid molecules corresponding to the mutant ALK nucleic acid molecule; optionally, ligating one or more adapters onto one or more nucleic acid molecules from the plurality of nucleic acid molecules; optionally, amplifying the one or more ligated nucleic acid molecules from the plurality of nucleic acid molecules; optionally, capturing amplified nucleic acid molecules from the amplified nucleic acid molecules; sequencing, by a sequencer, the captured nucleic acid molecules to obtain a plurality of sequence reads that represent the captured nucleic acid molecules, wherein one or more of the plurality of sequence reads correspond to the mutant ALK nucleic acid molecule; analyzing the plurality of sequence reads; and based on the analysis, detecting the mutant ALK nucleic acid molecule in the sample. In some embodiments, the methods further comprise receiving, at one or more processors, sequence read data for the plurality of sequence reads. In some embodiments, analyzing the plurality of sequence reads comprises identifying, using the one or more processors, the presence or absence of sequence reads corresponding to the mutant ALK nucleic acid molecule. In some embodiments, the amplified nucleic acid molecules are captured by hybridization with one or 8MOFO-358156824Docket No: 197102019040 more bait molecules. In some embodiments, the one or more adapters comprise amplification primers, flow cell adapter sequences, substrate adapter sequences, sample index sequences, or unique molecular identifier (UMI) sequences. In some embodiments, detecting the mutant ALK nucleic acid molecule in the sample comprises: providing a plurality of nucleic acid molecules obtained from the sample, wherein the plurality of nucleic acid molecules comprises nucleic acid molecules corresponding to the mutant ALK nucleic acid molecule; preparing a nucleic acid sequencing library from the plurality of nucleic acid molecules in the sample; amplifying said library; selectively enriching for one or more nucleic acid molecules comprising nucleotide sequences corresponding to the mutant ALK nucleic acid molecule in said library to produce an enriched sample; sequencing the enriched sample, thereby producing a plurality of sequence reads; analyzing the plurality of sequence reads for the presence of the mutant ALK nucleic acid molecule; and detecting, based on the analyzing step, the mutant ALK nucleic acid molecule in the sample from the individual. In some embodiments, the plurality of nucleic acid molecules comprises a mixture of cancer nucleic acid molecules and non-cancer nucleic acid molecules. In some embodiments, the cancer nucleic acid molecules are derived from a tumor portion of a heterogeneous tissue biopsy sample, and the non-cancer nucleic acid molecules are derived from a normal portion of the heterogeneous tissue biopsy sample. In some embodiments, the sample comprises a liquid biopsy sample, and wherein the cancer nucleic acid molecules are derived from a circulating tumor DNA (ctDNA) fraction of the liquid biopsy sample, and the non-cancer nucleic acid molecules are derived from a non-tumor fraction of the liquid biopsy sample. In some embodiments, the selectively enriching comprises: combining one or more bait molecules with the sample or library, thereby hybridizing the one or more bait molecules to one or more nucleic acids in the sample or library comprising nucleotide sequences corresponding to the mutant ALK nucleic acid molecule and producing nucleic acid hybrids; and isolating the nucleic acid hybrids to produce the enriched sample. In some embodiments, the one or more bait molecules comprise a capture nucleic acid molecule configured to hybridize to a nucleotide sequence corresponding to the mutant ALK nucleic acid molecule. In some embodiments, the capture nucleic acid molecule comprises between about 10 and about 30 nucleotides, between about 50 and about 1000 nucleotides, between about 100 and about 500 nucleotides, between about 100 and about 300 nucleotides, or between about 100 and about 200 nucleotides. In some embodiments, the one or more bait molecules are conjugated to an affinity reagent or to a detection reagent. In some embodiments, the affinity reagent is an antibody, an antibody fragment, or biotin, or wherein the detection reagent is a fluorescent 9MOFO-358156824Docket No: 197102019040 marker. In some embodiments, the capture nucleic acid molecule comprises a DNA, RNA, or mixed DNA / RNA molecule. In some embodiments, selectively enriching comprises amplifying the one or more nucleic acid molecules comprising nucleotide sequences corresponding to the mutant ALK nucleic acid molecule using a polymerase chain reaction (PCR) to produce an enriched sample. In some embodiments, the mutant ALK polypeptide is detected in the sample by one or more of: immunoblotting, enzyme linked immunosorbent assay (ELISA), immunohistochemistry, or mass spectrometry.

[0024] In some embodiments according to any one of the embodiments disclosed herein, the methods further comprise obtaining the sample from the individual. In some embodiments, the sample is obtained from the cancer. In some embodiments, the sample comprises a tissue biopsy sample or a liquid biopsy sample. In some embodiments, the sample is from a tumor biopsy or tumor specimen. In some embodiments, the sample is a liquid biopsy sample and comprises circulating tumor cells (CTCs). In some embodiments, the sample is a liquid biopsy sample and comprises cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof. In some embodiments, the sample is a liquid biopsy sample and comprises blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some embodiments, the sample comprises cells and / or nucleic acids from the cancer. In some embodiments, the sample comprises mRNA, DNA, circulating tumor DNA (ctDNA), cell- free DNA, or cell-free RNA from the cancer.

[0025] In another aspect, provided herein is a system for monitoring or screening for resistance of a cancer in an individual to a treatment comprising an ALK-targeted therapy, comprising: a memory configured to store one or more program instructions; and one or more processors configured to execute the one or more program instructions, the one or more program instructions when executed by the one or more processors are configured to: (a) obtain a plurality of sequence reads of one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are derived from one or more samples obtained from an individual having a cancer; (b) analyze the plurality of sequence reads for presence of a mutant ALK nucleic acid molecule encoding an ALK polypeptide that comprises a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; and (c) detect, based on the analyzing, presence of the mutant ALK nucleic acid molecule in the one or more samples; wherein presence of the mutant ALK nucleic acid molecule indicates resistance to a treatment comprising an ALK-targeted therapy. In another aspect, provided herein is a system for predicting resistance of a cancer in an individual to a treatment 10MOFO-358156824Docket No: 197102019040 comprising an ALK-targeted therapy, comprising: a memory configured to store one or more program instructions; and one or more processors configured to execute the one or more program instructions, the one or more program instructions when executed by the one or more processors are configured to: (a) obtain a plurality of sequence reads of one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are derived from one or more samples obtained from an individual having a cancer; (b) analyze the plurality of sequence reads for presence of a mutant ALK nucleic acid molecule encoding an ALK polypeptide that comprises a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; and (c) detect, based on the analyzing, presence of the mutant ALK nucleic acid molecule in the one or more samples; wherein, based at least in part on the presence of the mutant ALK nucleic acid molecule, the cancer is predicted as more likely to be resistant to a treatment comprising an ALK-targeted therapy, as compared to a cancer that does not comprise the mutant ALK nucleic acid molecule.

[0026] In some embodiments according to any one of the embodiments disclosed herein, the one or more program instructions when executed by the one or more processors are further configured to: analyze the plurality of sequence reads for presence of an ALK nucleic acid molecule that encodes an ALK polypeptide comprising a second ALK mutation associated with acquired resistance to an ALK-targeted therapy; and detect, based on the analyzing, presence of the second ALK mutation associated with acquired resistance to an ALK-targeted therapy in the one or more samples. In some embodiments, the second ALK mutation associated with acquired resistance to an ALK-targeted therapy is selected from the group consisting of T1151M, T1151R, C1156Y, I1171N, I1171T, I1171S, I1171H, I1171V, F1174X, V1180L, R1192P, L1196M, L1198F, G1202X, G1202del, D1203N, S1206Y, E1210K, and G1269A, amino acid numbering based on SEQ ID NO:2. In some embodiments, the one or more program instructions when executed by the one or more processors are further configured to: analyze the plurality of sequence reads for presence of a mutation in one or more genes selected from the group consisting of AXL, BRAF, MAP2K1, MET, NF2, PIK3CA, and NRAS; and detect, based on the analyzing, presence of the mutation in one or more genes selected from the group consisting of AXL, BRAF, MAP2K1, MET, NF2, PIK3CA, and NRAS in the one or more samples. In some embodiments, the one or more program instructions when executed by the one or more processors are further configured to: analyze the plurality of sequence reads for presence of a gene fusion, gene rearrangement, gene amplification, or activating mutation in an ALK gene; and detect, based 11MOFO-358156824Docket No: 197102019040 on the analyzing, presence of the gene fusion, gene rearrangement, gene amplification, or activating mutation in an ALK gene in the one or more samples.

[0027] In another aspect, provided herein is non-transitory computer readable storage medium comprising one or more programs executable by one or more computer processors for performing a method for monitoring or screening for resistance of a cancer in an individual to a treatment comprising an ALK-targeted therapy, the method comprising: (a) obtaining, using the one or more processors, a plurality of sequence reads of one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are derived from one or more samples obtained from an individual having a cancer; (b) analyzing, using the one or more processors, the plurality of sequence reads for presence of a mutant ALK nucleic acid molecule encoding an ALK polypeptide that comprises a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; and (c) detecting, using the one or more processors and based on the analyzing, the mutant ALK nucleic acid molecule in the one or more samples; wherein presence of the mutant ALK nucleic acid molecule or polypeptide indicates resistance to a treatment comprising an ALK-targeted therapy. In another aspect, provided herein is non-transitory computer readable storage medium comprising one or more programs executable by one or more computer processors for performing a method for predicting resistance of a cancer in an individual to a treatment comprising an ALK-targeted therapy, the method comprising: (a) obtaining, using the one or more processors, a plurality of sequence reads of one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are derived from one or more samples obtained from an individual having a cancer; (b) analyzing, using the one or more processors, the plurality of sequence reads for presence of a mutant ALK nucleic acid molecule encoding an ALK polypeptide that comprises a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; and (c) detecting, using the one or more processors and based on the analyzing, the mutant ALK nucleic acid molecule in the one or more samples; wherein, based at least in part on the presence of the mutant ALK nucleic acid molecule, the cancer is predicted as more likely to be resistant to a treatment comprising an ALK-targeted therapy, as compared to a cancer that does not comprise the mutant ALK nucleic acid molecule.

[0028] In some embodiments according to any one of the embodiments disclosed herein, the method further comprises: analyzing, using the one or more processors, the plurality of sequence reads for presence of an ALK nucleic acid molecule that encodes an ALK 12MOFO-358156824Docket No: 197102019040 polypeptide comprising a second ALK mutation associated with acquired resistance to an ALK-targeted therapy; and detecting, using the one or more processors and based on the analyzing, presence of the second ALK mutation associated with acquired resistance to an ALK-targeted therapy in the one or more samples. In some embodiments, the second ALK mutation associated with acquired resistance to an ALK-targeted therapy is selected from the group consisting of T1151M, T1151R, C1156Y, I1171N, I1171T, I1171S, I1171H, I1171V, F1174X, V1180L, R1192P, L1196M, L1198F, G1202X, G1202del, D1203N, S1206Y, E1210K, and G1269A, amino acid numbering based on SEQ ID NO:2. In some embodiments, the method further comprises: analyzing, using the one or more processors, the plurality of sequence reads for presence of a mutation in one or more genes selected from the group consisting of AXL, BRAF, MAP2K1, MET, NF2, PIK3CA, and NRAS; and detecting, using the one or more processors and based on the analyzing, presence of the mutation in one or more genes selected from the group consisting of AXL, BRAF, MAP2K1, MET, NF2, PIK3CA, and NRAS in the one or more samples. In some embodiments, the method further comprises: analyzing, using the one or more processors, the plurality of sequence reads for presence of a gene fusion, gene rearrangement, gene amplification, or activating mutation in an ALK gene; and detecting, using the one or more processors and based on the analyzing, presence of the gene fusion, gene rearrangement, gene amplification, or activating mutation in an ALK gene in the one or more samples.

[0029] In some embodiments according to any one of the embodiments disclosed herein, the cancer is lung cancer, anaplastic large cell lymphoma (ALCL), neuroblastoma, an inflammatory myofibroblastic tumor (IMT), spitzoid tumor, colorectal cancer, B-cell lymphoma, ovarian cancer, non-Hodgkin’s lymphoma (NHL), or thyroid cancer. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the one or more samples are obtained or derived from the cancer. In some embodiments, the one or more samples comprise a tissue biopsy sample or a liquid biopsy sample. In some embodiments, the sample is from a tumor biopsy or tumor specimen. In some embodiments, the sample is a liquid biopsy sample and comprises circulating tumor cells (CTCs). In some embodiments, the sample is a liquid biopsy sample and comprises cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof. In some embodiments, the sample is a liquid biopsy sample and comprises blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some embodiments, the sample comprises cells and / or nucleic acids from the cancer. In some embodiments, the sample comprises mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, or cell-free RNA from the cancer. In some 13MOFO-358156824Docket No: 197102019040 embodiments, the plurality of sequence reads is obtained by sequencing, whole exome sequencing, whole genome sequencing, gene-targeted sequencing, or next-generation sequencing.

[0030] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art. These and other embodiments of the invention are further described by the detailed description that follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 depicts an exemplary device, in accordance with some embodiments.

[0032] FIG. 2 depicts an exemplary system, in accordance with some embodiments.

[0033] FIG. 3 depicts a block diagram of an exemplary process for detecting a mutant ALK nucleic acid molecule, in accordance with some embodiments.

[0034] FIG. 4 shows a consort diagram for study cohorts. The genomic database contains 116,288 tissue and 18,660 liquid samples from NSCLC patients. ALK rearrangements were detected in 2,957 tissue and 387 liquid samples. Of these, 198 tissue and 71 liquid samples had secondary ALK mutations present. CGDB contains 24,313 NSCLC patients with CGP samples via FMI. ALK rearrangements were detected in 510 patients and of these, 412 received an ALK TKI and 105 had a CGP specimen collected post-ALK TKI initiation.

[0035] FIGS. 5A-6 show the landscape of secondary ALK mutations associated with ALK TKI acquired resistance and co-occurrence patterns in tissue and liquid samples with secondary ALK mutations. FIG. 5A shows prevalence of secondary ALK mutations in ALK+ samples. Bars are shaded based on sample type (top: tissue; bottom: liquid). Number of samples is indicated in each bar. ALK+, ALK rearrangement present. FIG. 5B shows pairwise co-occurrence of secondary ALK mutations. FIG. 5C shows distribution of secondary ALK mutations in tissue (left) versus liquid (right) NSCLC samples. Mutations are categorized based on previously reported associations with resistance to respective ALK TKI generations. FIG. 6 shows co-occurrence of secondary ALK mutations and off-target mutations in 269 ALK+ samples. Selected off-target mutations (AXL, BRAF, MAP2K1, MET, NF2, NRAS) have previously reported associations with ALK TKI resistance.

[0036] FIGS. 7A-7C show real-world treatment patterns and outcomes post-ALK TKI therapy. FIG. 7A shows distribution of post-ALK TKI lines of therapy between patients who received a CGP prior next line treatment (N = 18) and who did not (N = 15). FIG. 7B shows 14MOFO-358156824Docket No: 197102019040 real-world progression free survival post-ALK TKI for 33 patients with secondary ALK mutations. FIG. 7C shows real-world progression free survival post-ALK TKI for 11 patients with off-target mutations. Asterisks denote that the patient has both secondary ALK mutations and off-target mutations.

[0037] FIG. 8 shows proportion of tissue and liquid -samples harboring secondary ALK mutations associated with acquired resistance to 1st, 2nd, and 3rdgeneration ALK TKI.

[0038] FIG. 9 shows real-world genomic profiling of post-ALK TKI therapy NSCLC samples. DETAILED DESCRIPTION

[0039] The present disclosure relates generally to detecting mutant ALK nucleic acid molecules and / or polypeptides in cancer, as well as methods of treatment, and uses related thereto. In particular, the present disclosure describes ALK mutations (e.g., mutant ALK nucleic acid molecules of the present disclosure) observed in ALK-rearrangement positive (ALK+) cancers that occur along with known ALK mutations associated with acquired resistance (ALK AR) to ALK-targeted therapies such as ALK-targeting tyrosine kinase inhibitors (TKIs). As such, an ALK mutation of the present disclosure may indicate potential resistance to an ALK-targeted therapy such as a TKI, and thus detection of these mutations may potentially influence treatment decisions for these patients.

[0040] In some embodiments, provided herein are methods of predicting resistance of a cancer in an individual to a treatment comprising an ALK-targeted therapy, the methods comprising acquiring knowledge of, or detecting, a mutant ALK nucleic acid molecule or mutant ALK polypeptide in a sample obtained from an individual, wherein the mutant ALK nucleic acid molecule encodes an ALK polypeptide that comprises, or the mutant ALK polypeptide comprises, a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; wherein responsive to the acquisition of said knowledge or detection, the cancer is predicted as more likely to be resistant to a treatment comprising an ALK-targeted therapy, as compared to a cancer that does not comprise the mutant ALK nucleic acid molecule or polypeptide. In some embodiments, the individual has received a prior treatment comprising an ALK-targeted therapy.

[0041] In some embodiments, provided herein are methods of monitoring resistance of a cancer in an individual to a treatment comprising an ALK-targeted therapy, the methods comprising acquiring knowledge of, or detecting, a mutant ALK nucleic acid molecule or 15MOFO-358156824Docket No: 197102019040 mutant ALK polypeptide in a sample obtained from an individual, wherein the mutant ALK nucleic acid molecule encodes an ALK polypeptide that comprises, or the mutant ALK polypeptide comprises, a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; wherein presence of the mutant ALK nucleic acid molecule or polypeptide indicates resistance to a treatment comprising an ALK-targeted therapy. In some embodiments, provided herein are methods of screening for resistance of a cancer in an individual to a treatment comprising an ALK-targeted therapy, the methods comprising acquiring knowledge of, or detecting, a mutant ALK nucleic acid molecule or mutant ALK polypeptide in a sample obtained from an individual, wherein the mutant ALK nucleic acid molecule encodes an ALK polypeptide that comprises, or the mutant ALK polypeptide comprises, a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; wherein presence of the mutant ALK nucleic acid molecule or polypeptide indicates resistance to a treatment comprising an ALK-targeted therapy. In some embodiments, the individual has received a prior treatment comprising an ALK-targeted therapy.

[0042] In some embodiments, provided herein are methods of treating or delaying progression of cancer, comprising acquiring knowledge of, or detecting, a mutant ALK nucleic acid molecule or mutant ALK polypeptide in a sample obtained from an individual, wherein the mutant ALK nucleic acid molecule encodes an ALK polypeptide that comprises, or the mutant ALK polypeptide comprises, a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; and administering to the individual a treatment comprising an ALK-targeted therapy. In some embodiments, the individual has received a prior treatment comprising a different ALK-targeted therapy. In some embodiments, provided herein are methods of treating or delaying progression of cancer, comprising administering to the individual a treatment comprising a first ALK-targeted therapy; acquiring knowledge of, or detecting, a mutant ALK nucleic acid molecule or mutant ALK polypeptide in a sample obtained from an individual (e.g., after administration of the treatment comprising the first ALK-targeted therapy), wherein the mutant ALK nucleic acid molecule encodes an ALK polypeptide that comprises, or the mutant ALK polypeptide comprises, a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID 16MOFO-358156824Docket No: 197102019040 NO:2; and administering (e.g., after the acquisition of knowledge or detection) to the individual a treatment comprising a second ALK-targeted therapy different from the first ALK-targeted therapy. In some embodiments, the methods further comprise ceasing administration of the first ALK-targeted therapy. I. General Techniques

[0043] The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993). II. Definitions

[0044] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for 17MOFO-358156824Docket No: 197102019040 example, reference to “a molecule” optionally includes a combination of two or more such molecules, and the like.

[0045] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0046] It is understood that aspects and embodiments of the invention described herein include “comprising,” “consisting,” and “consisting essentially of” aspects and embodiments.

[0047] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers.

[0048] The term “tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” and “tumor” are not mutually exclusive as referred to herein.

[0049] “Polynucleotide,” “nucleic acid,” or “nucleic acid molecule” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. Thus, for instance, polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or include single- and double-stranded regions. In addition, the term “polynucleotide” as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. The term “polynucleotide” specifically includes cDNAs.

[0050] A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non- nucleotide components. A polynucleotide may be further modified after synthesis, such as by 18MOFO-358156824Docket No: 197102019040 conjugation with a label. Other types of modifications include, for example, “caps,” substitution of one or more of the naturally-occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, and the like) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, and the like), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, and the like), those with intercalators (e.g., acridine, psoralen, and the like), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, and the like), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-0-methyl-, 2'-0-allyl-, 2'-fluoro-, or 2'- azido-ribose, carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S ("thioate"), P(S)S ("dithioate"), "(0)NR2("amidate"), P(0)R, P(0)OR', CO or CH2 ("formacetal"), in which each R or R' is independently H or substituted or unsubstituted alkyl (1 -20 C) optionally containing an ether (-0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. A polynucleotide can contain one or more different types of modifications as described herein and / or multiple modifications of the same type. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0051] “Oligonucleotide,” as used herein, generally refers to short, single stranded, polynucleotides that are, but not necessarily, less than about 250 nucleotides in length. Oligonucleotides may be synthetic. The terms “oligonucleotide” and “polynucleotide” are not 19MOFO-358156824Docket No: 197102019040 mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.

[0052] The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0053] An “isolated” antibody is one which has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with research, diagnostic, and / or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, an antibody is purified (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of, for example, a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using, for example, Coomassie blue or silver stain. An isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0054] “Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.

[0055] The “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa (“κ”) and lambda (“λ”), based on the amino acid sequences of their constant domains. 20MOFO-358156824Docket No: 197102019040

[0056] The term “constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain contains the CH1, CH2, and CH3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.

[0057] The “variable region” or “variable domain” of an antibody refers to the amino- terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as “VH.” The variable domain of the light chain may be referred to as “VL.” These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[0058] The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs) both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991 )). The constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0059] The term “hypervariable region,” “HVR,” or “HV,” as used herein, refers to the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). In native antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1 -25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are 21MOFO-358156824Docket No: 197102019040 functional and stable in the absence of light chain. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0060] A number of HVR delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991 )). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901 -917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The “contact” HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below. Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101

[0061] HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56 or 50- 56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[0062] “Framework” or “FR” residues are those variable domain residues other than the HVR residues as herein defined.

[0063] The term “variable domain residue numbering as in Kabat” or “amino acid position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. 22MOFO-358156824Docket No: 197102019040

[0064] The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1 -107 of the light chain and residues 1 -113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991 )). The “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The “EU index as in Kabat” refers to the residue numbering of the human lgG1 EU antibody.

[0065] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain an Fc region.

[0066] “Antibody fragments” comprise a portion of an intact antibody comprising the antigen-binding region thereof. In some embodiments, the antibody fragment described herein is an antigen-binding fragment. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0067] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that a selected target-binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target-binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target-binding sequence is also a monoclonal antibody of this invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In 23MOFO-358156824Docket No: 197102019040 addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

[0068] The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature 256:495-97 (1975); Hongo et al., Hybridoma 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981 )), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g., Clackson et al., Nature, 352: 624-628 (1991 ); Marks et al., J. Mol. Biol. 222: 581 -597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101 (34): 12467- 12472 (2004); and Lee et al., J. Immunol. Methods 284(1 -2): 119-132 (2004)), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741 ; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg et al., Intern. Rev. Immunol. 13: 65-93 (1995)).

[0069] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody- encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0070] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human framework regions (FRs). In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs 24MOFO-358156824Docket No: 197102019040 (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.

[0071] A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0072] A “blocking” antibody or an “antagonist” antibody is one which inhibits or reduces biological activity of the antigen it binds. For example, blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0073] As used herein, the term “binds”, “specifically binds to” or is “specific for” refers to measurable and reproducible interactions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antibody that binds to or specifically binds to a target (which can be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and / or with greater duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of < 1 μΜ, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among the protein from different species. In another embodiment, specific binding can include, but does not require exclusive binding.

[0074] “Percent (%) amino acid sequence identity” or “homology” with respect to the polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.

[0075] The term “detection” includes any means of detecting, including direct and indirect detection. The term “biomarker” as used herein (e.g., a “biomarker” such as a mutant nucleic 25MOFO-358156824Docket No: 197102019040 acid molecule or polypeptide described herein) refers to an indicator, e.g., predictive, diagnostic, and / or prognostic, which can be detected in a sample. The biomarker may serve as an indicator of a particular subtype of a disease or disorder (e.g., cancer) characterized by certain, molecular, pathological, histological, and / or clinical features (e.g., responsiveness to therapy including a checkpoint inhibitor). In some embodiments, a biomarker is a collection of genes or a collective number of mutations / alterations (e.g., somatic mutations) in a collection of genes. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and / or RNA), polynucleotide alterations (e.g., polynucleotide copy number alterations, e.g., DNA copy number alterations), polypeptides, polypeptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrates, and / or glycolipid-based molecular markers.

[0076] “Amplification,” as used herein generally refers to the process of producing multiple copies of a desired sequence. “Multiple copies” mean at least two copies. A “copy” does not necessarily mean perfect sequence complementarity or identity to the template sequence. For example, copies can include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced through a primer comprising a sequence that is hybridizable, but not complementary, to the template), and / or sequence errors that occur during amplification.

[0077] The technique of “polymerase chain reaction” or “PCR” as used herein generally refers to a procedure wherein minute amounts of a specific piece of nucleic acid, RNA and / or DNA, are amplified as described, for example, in U.S. Pat. No. 4,683,195. Generally, sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage, or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263 (1987) and Erlich, ed., PCR Technology (Stockton Press, NY, 1989). As used herein, PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, comprising the use of a known nucleic acid (DNA or RNA) as a primer and utilizes a nucleic acid polymerase to amplify or generate a specific piece of nucleic acid or to amplify or generate a specific piece of nucleic acid which is complementary to a particular nucleic acid. 26MOFO-358156824Docket No: 197102019040

[0078] The term “diagnosis” is used herein to refer to the identification or classification of a molecular or pathological state, disease or condition (e.g., cancer). For example, “diagnosis” may refer to identification of a particular type of cancer. “Diagnosis” may also refer to the classification of a particular subtype of cancer, for instance, by histopathological criteria, or by molecular features (e.g., a subtype characterized by expression of one or a combination of biomarkers (e.g., particular genes or proteins encoded by said genes)).

[0079] The term “aiding diagnosis” is used herein to refer to methods that assist in making a clinical determination regarding the presence, or nature, of a particular type of symptom or condition of a disease or disorder (e.g., cancer). For example, a method of aiding diagnosis of a disease or condition (e.g., cancer) can comprise measuring certain somatic mutations in a biological sample from an individual.

[0080] The term “sample,” as used herein, refers to a composition that is obtained or derived from a subject and / or individual of interest that contains a cellular and / or other molecular entity that is to be characterized and / or identified, for example, based on physical, biochemical, chemical, and / or physiological characteristics. For example, the phrase “disease sample” and variations thereof refers to any sample obtained from a subject of interest that would be expected or is known to contain the cellular and / or molecular entity that is to be characterized. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, plasma, serum, blood-derived cells, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, tumor tissue, cellular extracts, and combinations thereof. In some instances, the sample is a whole blood sample, a plasma sample, a serum sample, or a combination thereof. In some embodiments, the sample is from a tumor (e.g., a “tumor sample”), such as from a biopsy. In some embodiments, the sample is a formalin-fixed paraffin-embedded (FFPE) sample.

[0081] A “tumor cell” as used herein, refers to any tumor cell present in a tumor or a sample thereof. Tumor cells may be distinguished from other cells that may be present in a tumor sample, for example, stromal cells and tumor-infiltrating immune cells, using methods known in the art and / or described herein.

[0082] A “reference sample,” “reference cell,” “reference tissue,” “control sample,” “control cell,” or “control tissue,” as used herein, refer to a sample, cell, tissue, standard, or level that is used for comparison purposes. 27MOFO-358156824Docket No: 197102019040

[0083] By “correlate” or “correlating” is meant comparing, in any way, the performance and / or results of a first analysis or protocol with the performance and / or results of a second analysis or protocol. For example, one may use the results of a first analysis or protocol in carrying out a second protocol and / or one may use the results of a first analysis or protocol to determine whether a second analysis or protocol should be performed. With respect to the embodiment of polypeptide analysis or protocol, one may use the results of the polypeptide expression analysis or protocol to determine whether a specific therapeutic regimen should be performed. With respect to the embodiment of polynucleotide analysis or protocol, one may use the results of the polynucleotide expression analysis or protocol to determine whether a specific therapeutic regimen should be performed.

[0084] “Individual response” or “response” can be assessed using any endpoint indicating a benefit to the individual, including, without limitation, (1) inhibition, to some extent, of disease progression (e.g., cancer progression), including slowing down or complete arrest; (2) a reduction in tumor size; (3) inhibition (i.e., reduction, slowing down, or complete stopping) of cancer cell infiltration into adjacent peripheral organs and / or tissues; (4) inhibition (i.e. reduction, slowing down, or complete stopping) of metastasis; (5) relief, to some extent, of one or more symptoms associated with the disease or disorder (e.g., cancer); (6) increase or extension in the length of survival, including overall survival and progression free survival; and / or (7) decreased mortality at a given point of time following treatment.

[0085] An “effective response” of a patient or a patient's “responsiveness” to treatment with a medicament and similar wording refers to the clinical or therapeutic benefit imparted to a patient at risk for, or suffering from, a disease or disorder, such as cancer. In one embodiment, such benefit includes any one or more of: extending survival (including overall survival and / or progression-free survival); resulting in an objective response (including a complete response or a partial response); or improving signs or symptoms of cancer.

[0086] An “effective amount” refers to an amount of a therapeutic agent to treat or prevent a disease or disorder in a mammal. In the case of cancers, the therapeutically effective amount of the therapeutic agent may reduce the number of cancer cells; reduce the primary tumor size; inhibit (i.e., slow to some extent and in some embodiments stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and in some embodiments stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with the disorder. To the extent the drug may prevent growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. For cancer therapy, efficacy in vivo can, for example, be measured by assessing the duration of survival, 28MOFO-358156824Docket No: 197102019040 time to disease progression (TTP), response rates (e.g., CR and PR), duration of response, and / or quality of life.

[0087] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0088] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0089] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0090] As used herein, the terms “individual,” “patient,” or “subject” are used interchangeably and refer to any single animal, e.g., a mammal (including such non-human animals as, for example, dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and non- human primates) for which treatment is desired. In particular embodiments, the patient herein is a human.

[0091] As used herein, “administering” is meant a method of giving a dosage of an agent or a pharmaceutical composition (e.g., a pharmaceutical composition including the agent) to a subject (e.g., a patient). Administering can be by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein. 29MOFO-358156824Docket No: 197102019040

[0092] The term “concurrently” is used herein to refer to administration of two or more therapeutic agents, where at least part of the administration overlaps in time. Accordingly, concurrent administration includes a dosing regimen when the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s).

[0093] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings concerning the use of such therapeutic products.

[0094] An “article of manufacture” is any manufacture (e.g., a package or container) or kit comprising at least one reagent, e.g., a medicament for treatment of a disease or disorder (e.g., cancer), or a reagent for specifically detecting a biomarker (e.g., a kinase fusion or a fusion nucleic acid molecule or polypeptide described herein) described herein. In certain embodiments, the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0095] The phrase “based on” when used herein means that the information about one or more biomarkers (e.g., a kinase fusion or a fusion nucleic acid molecule or polypeptide described herein) is used to inform a treatment decision, information provided on a package insert, or marketing / promotional guidance, etc. III. Methods, Systems, and Devices

[0096] Certain aspects of the present disclosure relate to methods for predicting resistance of a cancer to a treatment comprising an ALK-targeted therapy; monitoring for resistance of a cancer to a treatment comprising an ALK-targeted therapy; screening for resistance of a cancer to a treatment comprising an ALK-targeted therapy; selecting a treatment for an individual having a cancer; identifying one or more treatment options for an individual having a cancer; predicting survival of an individual having a cancer; treating or delaying progression of cancer; monitoring, evaluating or screening an individual having a cancer; or assessing a mutant ALK nucleic acid molecule or polypeptide in a cancer in an individual. In some embodiments, the methods comprise acquiring knowledge of a mutant ALK nucleic acid molecule or mutant ALK polypeptide in a sample obtained from an individual, wherein the mutant ALK nucleic acid molecule encodes an ALK polypeptide that comprises, or the mutant ALK polypeptide comprises, a mutation selected from the group consisting of 30MOFO-358156824Docket No: 197102019040 G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2.

[0097] In some embodiments of any of the methods provided herein, the methods comprise acquiring knowledge of or detecting in a sample from an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer, a mutant ALK nucleic acid molecule or mutant ALK polypeptide of the disclosure. For example, in some embodiments, the methods comprise acquiring knowledge of or detecting in a sample from an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer, a mutant ALK nucleic acid molecule or mutant ALK polypeptide in a sample obtained from an individual, wherein the mutant ALK nucleic acid molecule encodes an ALK polypeptide that comprises, or the mutant ALK polypeptide comprises, a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2.

[0098] In some embodiments of any of the methods provided herein, knowledge or detection of a mutant ALK nucleic acid molecule or mutant ALK polypeptide of the disclosure in a sample from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer) identifies the individual as one who may benefit from a treatment comprising an anti-cancer therapy, such as an anti-cancer therapy provided herein, e.g., an ALK-targeted therapy (in some embodiments, an ALK-targeted therapy other than a previous ALK-targeted therapy administered to the individual). In some embodiments, knowledge or detection of a mutant ALK nucleic acid molecule or mutant ALK polypeptide of the disclosure in a sample from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer) indicates that a cancer of the individual is more likely to be resistant to a treatment comprising an ALK-targeted therapy, e.g., as compared to a cancer that does not comprise the mutant ALK nucleic acid molecule or polypeptide. In some embodiments, knowledge or detection of a mutant ALK nucleic acid molecule or mutant ALK polypeptide of the disclosure in a sample from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer) indicates resistance or potential resistance to a treatment comprising an ALK-targeted therapy. In some embodiments, knowledge or detection of a mutant ALK nucleic acid molecule or mutant ALK polypeptide of the disclosure in a sample from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer) indicates that the individual should be treated with a different ALK-targeted therapy, e.g., 31MOFO-358156824Docket No: 197102019040 other than a previous ALK-targeted therapy administered to the individual. In some embodiments, knowledge or detection of a mutant ALK nucleic acid molecule or mutant ALK polypeptide of the disclosure in a sample from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer) indicates that the individual should be treated with a subsequent line of treatment comprising an ALK-targeted therapy, e.g., as compared to a line of treatment comprising a previous ALK-targeted therapy administered to the individual. In some embodiments, knowledge or detection of a mutant ALK nucleic acid molecule or mutant ALK polypeptide of the disclosure in a sample from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer) indicates that the individual should be treated with a later-generation ALK-targeted therapy (such as a TKI), e.g., as compared to a previous ALK-targeted therapy (such as a TKI) administered to the individual.

[0099] In other aspects, provided herein are systems and non-transitory computer readable storage media. In some embodiments, a system of the disclosure comprises a memory configured to store one or more program instructions; and one or more processors configured to execute the one or more program instructions, the one or more program instructions when executed by the one or more processors are configured to: (a) obtain a plurality of sequence reads of one or more nucleic acids, wherein the one or more nucleic acids are derived from a sample obtained from an individual; (b) analyze the plurality of sequence reads for the presence of a mutant ALK nucleic acid molecule provided herein; and (c) detect, based on the analyzing, the mutant ALK nucleic acid molecule in the sample. In some embodiments, a non-transitory computer readable storage medium of the disclosure comprises one or more programs executable by one or more computer processors for performing a method, comprising: (a) obtaining, using the one or more processors, a plurality of sequence reads of one or more nucleic acids, wherein the one or more nucleic acids are derived from a sample obtained from an individual; (b) analyzing, using the one or more processors, the plurality of sequence reads for the presence of a mutant ALK nucleic acid molecule provided herein; and (c) detecting, using the one or more processors and based on the analyzing, the mutant ALK nucleic acid molecule in the sample. 32MOFO-358156824Docket No: 197102019040 A. Mutant ALK nucleic acid molecules and polypeptides

[0100] Certain aspects of the present disclosure relate to mutant ALK nucleic acid molecules. Accordingly, certain aspects of the present disclosure relate to mutant ALK nucleic acid molecules, as well as to mutant ALK polypeptides encoded by such mutant ALK nucleic acid molecules.

[0101] As used herein “anaplastic lymphoma kinase” or “ALK” refer to a gene encoding an ALK mRNA or polypeptide. The ALK gene encodes the ALK receptor tyrosine kinase protein. ALK is also known as CD246, NBLST3, anaplastic lymphoma receptor tyrosine kinase, and ALK receptor tyrosine kinase. In some embodiments, an ALK gene is a human ALK gene. An exemplary ALK gene is represented by NCBI Gene ID No. 238. Exemplary ALK transcript sequences are represented by NCBI Ref. Seq. NM_004304 and provided below as SEQ ID NO: 1. Exemplary amino acid sequences of an ALK polypeptide are represented by NCBI Ref. Seq. NP_004295 and provided below as SEQ ID NO: 2. ATGGGAGCCATCGGGCTCCTGTGGCTCCTGCCGCTGCTGCTTTCCACGGCAGCTG TGGGCTCCGGGATGGGGACCGGCCAGCGCGCGGGCTCCCCAGCTGCGGGGCCGC CGCTGCAGCCCCGGGAGCCACTCAGCTACTCGCGCCTGCAGAGGAAGAGTCTGG CAGTTGACTTCGTGGTGCCCTCGCTCTTCCGTGTCTACGCCCGGGACCTACTGCTG CCACCATCCTCCTCGGAGCTGAAGGCTGGCAGGCCCGAGGCCCGCGGCTCGCTA GCTCTGGACTGCGCCCCGCTGCTCAGGTTGCTGGGGCCGGCGCCGGGGGTCTCCT GGACCGCCGGTTCACCAGCCCCGGCAGAGGCCCGGACGCTGTCCAGGGTGCTGA AGGGCGGCTCCGTGCGCAAGCTCCGGCGTGCCAAGCAGTTGGTGCTGGAGCTGG GCGAGGAGGCGATCTTGGAGGGTTGCGTCGGGCCCCCCGGGGAGGCGGCTGTGG GGCTGCTCCAGTTCAATCTCAGCGAGCTGTTCAGTTGGTGGATTCGCCAAGGCGA AGGGCGACTGAGGATCCGCCTGATGCCCGAGAAGAAGGCGTCGGAAGTGGGCA GAGAGGGAAGGCTGTCCGCGGCAATTCGCGCCTCCCAGCCCCGCCTTCTCTTCCA GATCTTCGGGACTGGTCATAGCTCCTTGGAATCACCAACAAACATGCCTTCTCCT TCTCCTGATTATTTTACATGGAATCTCACCTGGATAATGAAAGACTCCTTCCCTTT CCTGTCTCATCGCAGCCGATATGGTCTGGAGTGCAGCTTTGACTTCCCCTGTGAG CTGGAGTATTCCCCTCCACTGCATGACCTCAGGAACCAGAGCTGGTCCTGGCGCC GCATCCCCTCCGAGGAGGCCTCCCAGATGGACTTGCTGGATGGGCCTGGGGCAG AGCGTTCTAAGGAGATGCCCAGAGGCTCCTTTCTCCTTCTCAACACCTCAGCTGA CTCCAAGCACACCATCCTGAGTCCGTGGATGAGGAGCAGCAGTGAGCACTGCAC ACTGGCCGTCTCGGTGCACAGGCACCTGCAGCCCTCTGGAAGGTACATTGCCCAG CTGCTGCCCCACAACGAGGCTGCAAGAGAGATCCTCCTGATGCCCACTCCAGGG AAGCATGGTTGGACAGTGCTCCAGGGAAGAATCGGGCGTCCAGACAACCCATTT CGAGTGGCCCTGGAATACATCTCCAGTGGAAACCGCAGCTTGTCTGCAGTGGACT TCTTTGCCCTGAAGAACTGCAGTGAAGGAACATCCCCAGGCTCCAAGATGGCCCT GCAGAGCTCCTTCACTTGTTGGAATGGGACAGTCCTCCAGCTTGGGCAGGCCTGT GACTTCCACCAGGACTGTGCCCAGGGAGAAGATGAGAGCCAGATGTGCCGGAAA CTGCCTGTGGGTTTTTACTGCAACTTTGAAGATGGCTTCTGTGGCTGGACCCAAG GCACACTGTCACCCCACACTCCTCAATGGCAGGTCAGGACCCTAAAGGATGCCC GGTTCCAGGACCACCAAGACCATGCTCTATTGCTCAGTACCACTGATGTCCCCGC TTCTGAAAGTGCTACAGTGACCAGTGCTACGTTTCCTGCACCGATCAAGAGCTCT CCATGTGAGCTCCGAATGTCCTGGCTCATTCGTGGAGTCTTGAGGGGAAACGTGT 33MOFO-358156824Docket No: 197102019040 CCTTGGTGCTAGTGGAGAACAAAACCGGGAAGGAGCAAGGCAGGATGGTCTGGC ATGTCGCCGCCTATGAAGGCTTGAGCCTGTGGCAGTGGATGGTGTTGCCTCTCCT CGATGTGTCTGACAGGTTCTGGCTGCAGATGGTCGCATGGTGGGGACAAGGATC CAGAGCCATCGTGGCTTTTGACAATATCTCCATCAGCCTGGACTGCTACCTCACC ATTAGCGGAGAGGACAAGATCCTGCAGAATACAGCACCCAAATCAAGAAACCTG TTTGAGAGAAACCCAAACAAGGAGCTGAAACCCGGGGAAAATTCACCAAGACA GACCCCCATCTTTGACCCTACAGTTCATTGGCTGTTCACCACATGTGGGGCCAGC GGGCCCCATGGCCCCACCCAGGCACAGTGCAACAACGCCTACCAGAACTCCAAC CTGAGCGTGGAGGTGGGGAGCGAGGGCCCCCTGAAAGGCATCCAGATCTGGAAG GTGCCAGCCACCGACACCTACAGCATCTCGGGCTACGGAGCTGCTGGCGGGAAA GGCGGGAAGAACACCATGATGCGGTCCCACGGCGTGTCTGTGCTGGGCATCTTC AACCTGGAGAAGGATGACATGCTGTACATCCTGGTTGGGCAGCAGGGAGAGGAC GCCTGCCCCAGTACAAACCAGTTAATCCAGAAAGTCTGCATTGGAGAGAACAAT GTGATAGAAGAAGAAATCCGTGTGAACAGAAGCGTGCATGAGTGGGCAGGAGG CGGAGGAGGAGGGGGTGGAGCCACCTACGTATTTAAGATGAAGGATGGAGTGCC GGTGCCCCTGATCATTGCAGCCGGAGGTGGTGGCAGGGCCTACGGGGCCAAGAC AGACACGTTCCACCCAGAGAGACTGGAGAATAACTCCTCGGTTCTAGGGCTAAA CGGCAATTCCGGAGCCGCAGGTGGTGGAGGTGGCTGGAATGATAACACTTCCTT GCTCTGGGCCGGAAAATCTTTGCAGGAGGGTGCCACCGGAGGACATTCCTGCCC CCAGGCCATGAAGAAGTGGGGGTGGGAGACAAGAGGGGGTTTCGGAGGGGGTG GAGGGGGGTGCTCCTCAGGTGGAGGAGGCGGAGGATATATAGGCGGCAATGCA GCCTCAAACAATGACCCCGAAATGGATGGGGAAGATGGGGTTTCCTTCATCAGT CCACTGGGCATCCTGTACACCCCAGCTTTAAAAGTGATGGAAGGCCACGGGGAA GTGAATATTAAGCATTATCTAAACTGCAGTCACTGTGAGGTAGACGAATGTCACA TGGACCCTGAAAGCCACAAGGTCATCTGCTTCTGTGACCACGGGACGGTGCTGG CTGAGGATGGCGTCTCCTGCATTGTGTCACCCACCCCGGAGCCACACCTGCCACT CTCGCTGATCCTCTCTGTGGTGACCTCTGCCCTCGTGGCCGCCCTGGTCCTGGCTT TCTCCGGCATCATGATTGTGTACCGCCGGAAGCACCAGGAGCTGCAAGCCATGC AGATGGAGCTGCAGAGCCCTGAGTACAAGCTGAGCAAGCTCCGCACCTCGACCA TCATGACCGACTACAACCCCAACTACTGCTTTGCTGGCAAGACCTCCTCCATCAG TGACCTGAAGGAGGTGCCGCGGAAAAACATCACCCTCATTCGGGGTCTGGGCCA TGGCGCCTTTGGGGAGGTGTATGAAGGCCAGGTGTCCGGAATGCCCAACGACCC AAGCCCCCTGCAAGTGGCTGTGAAGACGCTGCCTGAAGTGTGCTCTGAACAGGA CGAACTGGATTTCCTCATGGAAGCCCTGATCATCAGCAAATTCAACCACCAGAAC ATTGTTCGCTGCATTGGGGTGAGCCTGCAATCCCTGCCCCGGTTCATCCTGCTGG AGCTCATGGCGGGGGGAGACCTCAAGTCCTTCCTCCGAGAGACCCGCCCTCGCC CGAGCCAGCCCTCCTCCCTGGCCATGCTGGACCTTCTGCACGTGGCTCGGGACAT TGCCTGTGGCTGTCAGTATTTGGAGGAAAACCACTTCATCCACCGAGACATTGCT GCCAGAAACTGCCTCTTGACCTGTCCAGGCCCTGGAAGAGTGGCCAAGATTGGA GACTTCGGGATGGCCCGAGACATCTACAGGGCGAGCTACTATAGAAAGGGAGGC TGTGCCATGCTGCCAGTTAAGTGGATGCCCCCAGAGGCCTTCATGGAAGGAATAT TCACTTCTAAAACAGACACATGGTCCTTTGGAGTGCTGCTATGGGAAATCTTTTC TCTTGGATATATGCCATACCCCAGCAAAAGCAACCAGGAAGTTCTGGAGTTTGTC ACCAGTGGAGGCCGGATGGACCCACCCAAGAACTGCCCTGGGCCTGTATACCGG ATAATGACTCAGTGCTGGCAACATCAGCCTGAAGACAGGCCCAACTTTGCCATC ATTTTGGAGAGGATTGAATACTGCACCCAGGACCCGGATGTAATCAACACCGCTT TGCCGATAGAATATGGTCCACTTGTGGAAGAGGAAGAGAAAGTGCCTGTGAGGC CCAAGGACCCTGAGGGGGTTCCTCCTCTCCTGGTCTCTCAACAGGCAAAACGGG AGGAGGAGCGCAGCCCAGCTGCCCCACCACCTCTGCCTACCACCTCCTCTGGCAA GGCTGCAAAGAAACCCACAGCTGCAGAGATCTCTGTTCGAGTCCCTAGAGGGCC 34MOFO-358156824Docket No: 197102019040 GGCCGTGGAAGGGGGACACGTGAATATGGCATTCTCTCAGTCCAACCCTCCTTCG GAGTTGCACAAGGTCCACGGATCCAGAAACAAGCCCACCAGCTTGTGGAACCCA ACGTACGGCTCCTGGTTTACAGAGAAACCCACCAAAAAGAATAATCCTATAGCA AAGAAGGAGCCACACGACAGGGGTAACCTGGGGCTGGAGGGAAGCTGTACTGT CCCACCTAACGTTGCAACTGGGAGACTTCCGGGGGCCTCACTGCTCCTAGAGCCC TCTTCGCTGACTGCCAATATGAAGGAGGTACCTCTGTTCAGGCTACGTCACTTCC CTTGTGGGAATGTCAATTACGGCTACCAGCAACAGGGCTTGCCCTTAGAAGCCGC TACTGCCCCTGGAGCTGGTCATTACGAGGATACCATTCTGAAAAGCAAGAATAG CATGAACCAGCCTGGGCCCTGA (SEQ ID NO: 1)

[0102] Exemplary amino acid sequences of an ALK polypeptide are represented by NCBI Ref. Seq. NP_004295 and provided below as SEQ ID NO: 2. MGAIGLLWLLPLLLSTAAVGSGMGTGQRAGSPAAGPPLQPREPLSYSRLQRKSLAVD FVVPSLFRVYARDLLLPPSSSELKAGRPEARGSLALDCAPLLRLLGPAPGVSWTAGSP APAEARTLSRVLKGGSVRKLRRAKQLVLELGEEAILEGCVGPPGEAAVGLLQFNLSE LFSWWIRQGEGRLRIRLMPEKKASEVGREGRLSAAIRASQPRLLFQIFGTGHSSLESPT NMPSPSPDYFTWNLTWIMKDSFPFLSHRSRYGLECSFDFPCELEYSPPLHDLRNQSWS WRRIPSEEASQMDLLDGPGAERSKEMPRGSFLLLNTSADSKHTILSPWMRSSSEHCTL AVSVHRHLQPSGRYIAQLLPHNEAAREILLMPTPGKHGWTVLQGRIGRPDNPFRVAL EYISSGNRSLSAVDFFALKNCSEGTSPGSKMALQSSFTCWNGTVLQLGQACDFHQDC AQGEDESQMCRKLPVGFYCNFEDGFCGWTQGTLSPHTPQWQVRTLKDARFQDHQD HALLLSTTDVPASESATVTSATFPAPIKSSPCELRMSWLIRGVLRGNVSLVLVENKTG KEQGRMVWHVAAYEGLSLWQWMVLPLLDVSDRFWLQMVAWWGQGSRAIVAFD NISISLDCYLTISGEDKILQNTAPKSRNLFERNPNKELKPGENSPRQTPIFDPTVHWLFT TCGASGPHGPTQAQCNNAYQNSNLSVEVGSEGPLKGIQIWKVPATDTYSISGYGAAG GKGGKNTMMRSHGVSVLGIFNLEKDDMLYILVGQQGEDACPSTNQLIQKVCIGENN VIEEEIRVNRSVHEWAGGGGGGGGATYVFKMKDGVPVPLIIAAGGGGRAYGAKTDT FHPERLENNSSVLGLNGNSGAAGGGGGWNDNTSLLWAGKSLQEGATGGHSCPQAM KKWGWETRGGFGGGGGGCSSGGGGGGYIGGNAASNNDPEMDGEDGVSFISPLGIL YTPALKVMEGHGEVNIKHYLNCSHCEVDECHMDPESHKVICFCDHGTVLAEDGVSC IVSPTPEPHLPLSLILSVVTSALVAALVLAFSGIMIVYRRKHQELQAMQMELQSPEYK LSKLRTSTIMTDYNPNYCFAGKTSSISDLKEVPRKNITLIRGLGHGAFGEVYEGQVSG MPNDPSPLQVAVKTLPEVCSEQDELDFLMEALIISKFNHQNIVRCIGVSLQSLPRFILL ELMAGGDLKSFLRETRPRPSQPSSLAMLDLLHVARDIACGCQYLEENHFIHRDIAAR NCLLTCPGPGRVAKIGDFGMARDIYRASYYRKGGCAMLPVKWMPPEAFMEGIFTSK TDTWSFGVLLWEIFSLGYMPYPSKSNQEVLEFVTSGGRMDPPKNCPGPVYRIMTQC WQHQPEDRPNFAIILERIEYCTQDPDVINTALPIEYGPLVEEEEKVPVRPKDPEGVPPL LVSQQAKREEERSPAAPPPLPTTSSGKAAKKPTAAEISVRVPRGPAVEGGHVNMAFS QSNPPSELHKVHGSRNKPTSLWNPTYGSWFTEKPTKKNNPIAKKEPHDRGNLGLEGS CTVPPNVATGRLPGASLLLEPSSLTANMKEVPLFRLRHFPCGNVNYGYQQQGLPLEA ATAPGAGHYEDTILKSKNSMNQPGP(SEQ ID NO: 2)

[0103] In some embodiments, a mutant ALK nucleic acid molecule of the present disclosure encodes a G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, or L1196Q ALK mutation, amino acid numbering based on SEQ ID NO:2. In some embodiments, a mutant ALK polypeptide of the present disclosure comprises a G1123A, T1151_L1152insT, 35MOFO-358156824Docket No: 197102019040 I1171M, G1202del, I1268V, E1129V, or L1196Q mutation, amino acid numbering based on SEQ ID NO:2.

[0104] In some embodiments, a cancer of the present disclosure comprising a mutant ALK nucleic acid molecule or polypeptide of the present disclosure further comprises an ALK mutation associated with resistance (e.g., acquired resistance) to an ALK-targeted therapy and / or comprises or expresses a mutant ALK polypeptide associated with resistance (e.g., acquired resistance) to an ALK-targeted therapy. In some embodiments, the methods of the present disclosure further comprise acquiring knowledge of, or detecting, an ALK mutation or mutant ALK polypeptide associated with resistance (e.g., acquired resistance) to an ALK- targeted therapy. For example, in some embodiments, the ALK mutation (e.g., a second ALK mutation of the present disclosure) encodes a T1151M, T1151R, C1156Y, I1171N, I1171T, I1171S, I1171H, I1171V, F1174X, V1180L, R1192P, L1196M, L1198F, G1202X, G1202del, D1203N, S1206Y, E1210K, or G1269A ALK mutation, amino acid numbering based on SEQ ID NO:2. In some embodiments, the mutant ALK polypeptide associated with resistance (e.g., acquired resistance) to an ALK-targeted therapy comprises a T1151M, T1151R, C1156Y, I1171N, I1171T, I1171S, I1171H, I1171V, F1174X, V1180L, R1192P, L1196M, L1198F, G1202X, G1202del, D1203N, S1206Y, E1210K, or G1269A ALK mutation, amino acid numbering based on SEQ ID NO:2. In some embodiments, a G1202X refers to an alteration at the codon encoding G1202, such as a mutation of G1202 to any other amino acid, or an insertion affecting G1202. In certain embodiments, G1202X includes, without limitation, G1202R. In some embodiments, a F1174X refers to an alteration at the codon encoding F1174, such as a mutation of F1174 to any other amino acid or an insertion or deletion affecting F1174. In some embodiments,

[0105] In some embodiments, the ALK mutation (e.g., a second ALK mutation of the present disclosure) encodes a T1151M, L1152R, C1156Y, I1171T, F1174 (any missense), R1192P, L1196M, G1202R, G1269A, S1206Y, or E1210K ALK mutation, amino acid numbering based on SEQ ID NO:2. In some embodiments, the mutant ALK polypeptide associated with resistance (e.g., acquired resistance) to an ALK-targeted therapy comprises a L1152R, C1156Y, I1171T, F1174 (any missense), R1192P, L1196M, G1202R, G1269A, S1206Y, or E1210K ALK mutation, amino acid numbering based on SEQ ID NO:2. In some embodiments, the ALK mutation (e.g., a second ALK mutation of the present disclosure) or mutant ALK polypeptide is associated with resistance to a first-generation ALK TKI, e.g., crizotinib. 36MOFO-358156824Docket No: 197102019040

[0106] In some embodiments, the ALK mutation (e.g., a second ALK mutation of the present disclosure) encodes a T1151R, I1171N, I1171T, I1171S, I1171H, I1171V, F1174C, V1180L, G1202 (any missense), G1202R, G1202del, or D1203N ALK mutation, amino acid numbering based on SEQ ID NO:2. In some embodiments, the mutant ALK polypeptide associated with resistance (e.g., acquired resistance) to an ALK-targeted therapy comprises a T1151R, I1171N, I1171T, I1171S, I1171H, I1171V, F1174C, V1180L, G1202 (any missense), G1202R, G1202del, or D1203N ALK mutation, amino acid numbering based on SEQ ID NO:2. In some embodiments, the ALK mutation (e.g., a second ALK mutation of the present disclosure) or mutant ALK polypeptide is associated with resistance to a second- generation ALK TKI, e.g., ceritinib, brigatinib, or alectinib.

[0107] In some embodiments, the ALK mutations (e.g., second ALK mutations of the present disclosure) encode C1156Y + L1198F, L1196M + D1203N, L1196M + G1202R, G1202R + G1269A, G1202R + F1174L, L1196M + F1174L ALK mutations, amino acid numbering based on SEQ ID NO:2. In some embodiments, the mutant ALK polypeptide associated with resistance (e.g., acquired resistance) to an ALK-targeted therapy comprises C1156Y + L1198F, L1196M + D1203N, L1196M + G1202R, G1202R + G1269A, G1202R + F1174L, L1196M + F1174L ALK mutations, amino acid numbering based on SEQ ID NO:2. In some embodiments, the ALK mutation (e.g., a second ALK mutation of the present disclosure) or mutant ALK polypeptide is associated with resistance to a third-generation ALK TKI, e.g., lorlatinib.

[0108] In some embodiments, the mutant ALK nucleic acid molecule or polypeptide of the present disclosure is expressed by the same cell (e.g., of a cancer of the present disclosure) as the ALK mutation (e.g., a second ALK mutation of the present disclosure) associated with resistance (e.g., acquired resistance) to an ALK-targeted therapy. In other embodiments, the mutant ALK nucleic acid molecule or polypeptide of the present disclosure is expressed by different cells (e.g., of a cancer of the present disclosure) as the ALK mutation (e.g., a second ALK mutation of the present disclosure) associated with resistance (e.g., acquired resistance) to an ALK-targeted therapy. For example, the mutant ALK nucleic acid molecule or polypeptide of the present disclosure can be expressed by the same or different clones of the cancer, or can be expressed by the same or different tumors of the cancer (e.g., the primary tumor vs. a metastatic site, or different metastatic sites).

[0109] In some embodiments, a cancer of the present disclosure comprising a mutant ALK nucleic acid molecule or polypeptide of the present disclosure further comprises an off-target mutation (i.e., in a gene other than ALK) associated with resistance (e.g., acquired resistance) 37MOFO-358156824Docket No: 197102019040 to an ALK-targeted therapy and / or comprises or expresses an off-target mutant polypeptide associated with resistance (e.g., acquired resistance) to an ALK-targeted therapy. In some embodiments, the methods of the present disclosure further comprise acquiring knowledge of, or detecting, an off-target mutation (i.e., in a gene other than ALK) or off-target mutant polypeptide associated with resistance (e.g., acquired resistance) to an ALK-targeted therapy. For example, in some embodiments, the off-target mutation includes a mutation in one or more of the following genes: AXL, BRAF, MAP2K1, MET, NF2, PIK3CA, and / or NRAS. In some embodiments, the off-target mutant polypeptide includes a mutation in one or more of the following genes: AXL, BRAF, MAP2K1, MET, NF2, PIK3CA, and / or NRAS.

[0110] In some embodiments, the mutant ALK nucleic acid molecule or polypeptide of the present disclosure is expressed by the same cell (e.g., of a cancer of the present disclosure) as the off-target mutation associated with resistance (e.g., acquired resistance) to an ALK- targeted therapy. In other embodiments, the mutant ALK nucleic acid molecule or polypeptide of the present disclosure is expressed by different cells (e.g., of a cancer of the present disclosure) as the off-target mutation associated with resistance (e.g., acquired resistance) to an ALK-targeted therapy. For example, the mutant ALK nucleic acid molecule or polypeptide of the present disclosure can be expressed by the same or different clones of the cancer, or can be expressed by the same or different tumors of the cancer (e.g., the primary tumor vs. a metastatic site, or different metastatic sites).

[0111] In some embodiments, the methods comprise detecting, in a first sample obtained from the individual at a first time point, the presence or absence of a mutant ALK nucleic acid molecule or polypeptide of the disclosure. In some embodiments, the methods further comprise detecting, in a second sample obtained from the individual at a second time point after the first time point, the presence or absence of a mutant ALK nucleic acid molecule or polypeptide of the disclosure. In some embodiments, the methods further comprise providing an assessment of resistance to an ALK-targeted therapy in the cancer of the individual based, at least in part, on the presence or absence of the mutant ALK nucleic acid molecule or polypeptide in the first sample and / or in the second sample. In some embodiments, the presence of the mutant ALK nucleic acid molecule or polypeptide in the first sample and / or in the second sample identifies the cancer of the individual as having increased risk of resistance to an ALK-targeted therapy. In some embodiments, the methods further comprise selecting a treatment, changing a treatment or therapeutic agent, administering a treatment, adjusting a treatment, adjusting the dose of a treatment, or applying a treatment to the individual based, at least in part, on detecting the presence of the mutant ALK nucleic acid molecule or 38MOFO-358156824Docket No: 197102019040 polypeptide in the first sample and / or in the second sample, wherein the treatment comprises an anti-cancer therapy, such as an anti-cancer therapy provided herein, e.g., an ALK-targeted therapy such as a TKI.

[0112] In some embodiments, the methods comprise performing DNA sequencing on a sample obtained from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer) to determine a sequencing mutation profile on a group of genes comprising ALK and optionally one or more of AXL, BRAF, MAP2K1, MET, NF2, PIK3CA, and NRAS, wherein the sequencing mutation profile identifies the presence or absence of a mutant ALK nucleic acid molecule of the disclosure. In some embodiments, the methods further comprise identifying a candidate treatment for a cancer in an individual, based at least in part on the sequencing mutation profile. In some embodiments, the candidate treatment comprises an anti-cancer therapy, such as an anti- cancer therapy provided herein, e.g., an ALK-targeted therapy.

[0113] In some embodiments of any of the methods provided herein, the methods further comprise generating a report comprising one or more treatment options identified for the individual based at least in part on detection of the mutant ALK nucleic acid molecule or polypeptide in the sample, wherein the one or more treatment options comprise an anti-cancer therapy, such as an anti-cancer therapy provided herein, e.g., an ALK-targeted therapy.

[0114] In some embodiments of any of the methods provided herein, responsive to acquisition of knowledge of a mutant ALK nucleic acid molecule or polypeptide of the disclosure in a sample from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer): (i) the individual is classified as a candidate to receive a treatment comprising an anti-cancer therapy, such as an anti-cancer therapy provided herein, e.g., an ALK-targeted therapy; (ii) the individual is identified as likely to respond to a treatment that comprises an anti-cancer therapy, such as an anti-cancer therapy provided herein, e.g., an ALK-targeted therapy; (iii) the individual is identified as unlikely to respond to a treatment that comprises a previous ALK-targeted therapy; and / or (iv) the cancer of the individual is identified as likely resistant to a treatment that comprises a previous ALK-targeted therapy. In some embodiments, responsive to acquisition of knowledge of the mutant ALK nucleic acid molecule or polypeptide in a sample from the individual, the individual is predicted to have resistance to an anti-cancer therapy (e.g., a previous ALK-targeted therapy administered to the individual), the individual is predicted to respond to an anti-cancer therapy (e.g., an anti-cancer therapy provided herein, such as an ALK-targeted therapy), and / or the individual is predicted to have poor prognosis, 39MOFO-358156824Docket No: 197102019040 e.g., when treated with a previous ALK-targeted therapy administered to the individual, as compared to an individual whose cancer does not comprise the mutant ALK nucleic acid molecule or polypeptide.

[0115] In some embodiments, responsive to acquisition of knowledge of a mutant ALK nucleic acid molecule or polypeptide of the disclosure in a sample from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer), the methods comprise administering to the individual an effective amount of a treatment that comprises an anti-cancer therapy, such as an anti-cancer therapy provided herein, e.g., an ALK-targeted therapy. In some embodiments, the ALK-targeted therapy is different from a previous ALK-targeted therapy administered to the individual.

[0116] In some embodiments of any of the methods provided herein, the methods further comprise generating a report comprising one or more treatment options identified for the individual based, at least in part, on knowledge of a mutant ALK nucleic acid molecule or polypeptide of the disclosure in a sample from the individual, wherein the one or more treatment options comprise an anti-cancer therapy, such as an anti-cancer therapy provided herein, e.g., an ALK targeted therapy.

[0117] In some embodiments, acquiring knowledge of a mutant ALK nucleic acid molecule or polypeptide of the disclosure in a sample comprises detecting the mutant ALK nucleic acid molecule or polypeptide, or fragment thereof, in the sample.

[0118] In some embodiments, the methods of the disclosure further comprise providing an assessment of the mutant ALK nucleic acid molecule or polypeptide of the disclosure. ALK-targeted therapies

[0119] In some embodiments of any of the methods provided herein, the anti-cancer therapy is an ALK-targeted therapy.

[0120] In some embodiments, the ALK-targeted therapy is a small molecule inhibitor, an antibody, a cellular therapy, a nucleic acid, a virus-based therapy, an antibody-drug conjugate, a recombinant protein, a fusion protein, a natural compound, a peptide, a PROteolysis-TArgeting Chimera (PROTAC), a treatment for ALK-positive or ALK- rearranged cancer, an ALK-targeted therapy being tested in a clinical trial, a treatment for cancer being tested in a clinical trial, a targeted therapy, a treatment being tested in a clinical trial for cancer comprising a mutant ALK nucleic acid molecule or polypeptide, or any combination thereof, e.g., a described in further detail below. 40MOFO-358156824Docket No: 197102019040

[0121] In some embodiments, the ALK-targeted therapy is a kinase inhibitor, such as a kinase inhibitor described herein or known in the art. In some embodiments, the ALK- targeted therapy is a tyrosine kinase inhibitor (TKI) described herein or known in the art. In some embodiments, the ALK-targeted therapy is a kinase inhibitor (e.g., a TKI) that inhibits the kinase activity of an ALK polypeptide. In some embodiments, the ALK-targeted therapy is a multi-kinase inhibitor or an ALK-specific inhibitor. In some embodiments, the ALK- targeted therapy is a kinase inhibitor (e.g., a TKI) that inhibits signaling downstream of an ALK polypeptide.

[0122] In some embodiments, the ALK-targeted therapy comprises one or more of crizotinib, alectinib, ceritinib, lorlatinib, brigatinib, ensartinib (X-396), repotrectinib (TPX-005), entrectinib (RXDX-101), AZD3463, CEP-37440, belizatinib (TSR-011), gilteritinib, XMU- MP-5, NVL-655, ASP3026, KRCA-0008, TQ-B3139, TPX-0131, TAE684 (NVP-TAE684), CT-707 (see, e.g., clinicaltrials.gov / ct2 / show / NCT02695550), WX-0593 (see, e.g., clinicaltrials.gov / ct2 / show / NCT04641754), alkotinib (see, e.g., clinicaltrials.gov / ct2 / show / NCT04211922), SIM1803-1A (see, e.g., clinicaltrials.gov / ct2 / show / NCT04671849), PLB1003 (see, e.g., clinicaltrials.gov / ct2 / show / NCT03130881), SAF-189s (see, e.g., clinicaltrials.gov / ct2 / show / NCT04237805), PF03446962 (see, e.g., clinicaltrials.gov / ct2 / show / NCT01620970), TQ-B3101 (see, e.g., clinicaltrials.gov / ct2 / show / NCT04412564), APG-2449 (see, e.g., clinicaltrials.gov / ct2 / show / NCT03917043), X-376 (see, e.g., Awad et al., Clinical advances in hematology & oncology : H&O vol. 12,7 (2014):429-39), CEP-28122 (see, e.g., Cheng et al., Molecular cancer therapeutics vol. 11,3 (2012): 670-9; and Spagnuolo et al., Expert opinion on emerging drugs vol. 23,3 (2018): 231-241), and GSK1838705A (see, e.g., Sabbatini, Peter et al., Molecular cancer therapeutics vol. 8,10 (2009): 2811-20).

[0123] In some embodiments, the ALK-targeted therapy inhibits the expression of a mutant ALK nucleic acid molecule or polypeptide of the disclosure. In some embodiments, the cellular therapy is an adoptive therapy, a T cell-based therapy, a natural killer (NK) cell- based therapy, a chimeric antigen receptor (CAR)-T cell therapy, a recombinant T cell receptor (TCR) T cell therapy, a macrophage-based therapy, an induced pluripotent stem cell- based therapy, a B cell-based therapy, or a dendritic cell (DC)-based therapy. In some embodiments, the nucleic acid inhibits the expression of a mutant ALK nucleic acid molecule or polypeptide of the disclosure. In some embodiments, the nucleic acid comprises a double- stranded RNA (dsRNA), a small interfering RNA (siRNA), or a small hairpin RNA (shRNA), 41MOFO-358156824Docket No: 197102019040 e.g., as described herein. In some embodiments, the ALK-targeted therapy comprises an ALK proteolysis-targeting chimeric (PROTAC) degrader. In some embodiments, the ALK- targeted therapy is a first-line or front-line treatment for cancer.

[0124] In some embodiments of any of the methods provided herein, the individual to be treated has received a prior treatment with an ALK-targeted therapy. In some embodiments, the methods comprise administering to the individual a treatment comprising a first ALK- targeted therapy; acquiring knowledge of or detecting a mutant ALK nucleic acid molecule or polypeptide of the present disclosure in a sample obtained from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer, in which the sample is obtained during or after treatment with the first ALK- targeted therapy); and administering to the individual a treatment comprising a second ALK- targeted therapy different from the first ALK-targeted therapy. In some embodiments, the methods comprise administering to the individual a treatment comprising a first ALK- targeted therapy; acquiring knowledge of or detecting a mutant ALK nucleic acid molecule or polypeptide of the present disclosure in a sample obtained from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer, in which the sample is obtained during or after treatment with the first ALK- targeted therapy); and recommending a treatment comprising a second ALK-targeted therapy different from the first ALK-targeted therapy to the individual. In some embodiments, the methods comprise administering to the individual a treatment comprising a first ALK- targeted therapy; acquiring knowledge of or detecting a mutant ALK nucleic acid molecule or polypeptide of the present disclosure in a sample obtained from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer, in which the sample is obtained during or after treatment with the first ALK- targeted therapy); and generating a report recommending a treatment for the individual comprising a second ALK-targeted therapy different from the first ALK-targeted therapy. In some embodiments, the methods further comprise ceasing administration of the first ALK- targeted therapy. In some embodiments, the methods comprise administering to an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer) a treatment comprising an ALK-targeted therapy, wherein a mutant ALK nucleic acid molecule or polypeptide of the present disclosure has been detected in a sample obtained from the individual, optionally wherein the individual has received a prior treatment with an ALK-targeted therapy. In some embodiments, the ALK-targeted therapy 42MOFO-358156824Docket No: 197102019040 previously administered to the individual is different from the ALK-targeted therapy administered to the individual.

[0125] In some embodiments of any of the methods provided herein, an ALK-targeted therapy of the present disclosure administered or recommended to an individual is different from a previous ALK-targeted therapy administered to the individual. For example, detection of a mutant ALK nucleic acid molecule or polypeptide of the present disclosure in a cancer can indicate resistance to an ALK-targeted therapy previous administered to treat the cancer, and the patient may benefit from administration of a different ALK-targeted therapy in order to avoid potential resistance. In some embodiments, a mutant ALK nucleic acid molecule or polypeptide of the present disclosure (e.g., detected as described herein) is associated with resistance to a previous ALK-targeted therapy, and the ALK-targeted therapy of the present disclosure administered or recommended to an individual is a different ALK-targeted therapy, e.g., lacking an association between resistance to the therapy and the mutant ALK nucleic acid molecule or polypeptide.

[0126] In some embodiments, an ALK-targeted therapy of the present disclosure administered or recommended to an individual is a subsequent line of treatment, e.g., as compared to a previous ALK-targeted therapy administered to the individual. For example, the individual may have previously been treated with a first-line treatment comprising an ALK-targeted therapy, and a second- or subsequent-line treatment comprising a different ALK-targeted therapy may be administered or recommended to an individual, e.g., based at least in part on acquiring knowledge of or detecting a mutant ALK nucleic acid molecule or polypeptide of the present disclosure in a sample obtained from the individual.

[0127] In some embodiments, an ALK-targeted therapy of the present disclosure administered or recommended to an individual is a subsequent generation of ALK-targeted therapy (such as a TKI), e.g., as compared to a previous ALK-targeted therapy administered to the individual. For example, in some embodiments, an ALK-targeted therapy of the present disclosure administered or recommended to an individual comprises alectinib, brigatinib, ceritinib, ensartinib, or lorlatinib, and the previous ALK-targeted therapy comprises crizotinib. In some embodiments, an ALK-targeted therapy of the present disclosure administered or recommended to an individual comprises lorlatinib, and the previous ALK-targeted therapy comprises crizotinib, alectinib, brigatinib, ceritinib, or ensartinib. Crizotinib is known as a first-generation ALK-targeted TKI, while alectinib, ceritinib, ensartinib, and brigatinib are known as second-generation ALK-targeted TKIs, and lorlatinib is known as a third-generation ALK-targeted TKI. See, e.g., Peng, L. et al. (2022) 43MOFO-358156824Docket No: 197102019040 Front Oncol. 12:863461. In some embodiments, an ALK-targeted therapy of the present disclosure administered or recommended to an individual is an ALK-targeted therapy (such as a TKI) recommended or indicated for treatment of a cancer with resistance (e.g., acquired resistance) to another ALK-targeted therapy, e.g., an ALK-targeted therapy previously administered to the individual.

[0128] In some embodiments of any of the methods provided herein, the methods further comprise acquiring knowledge of or detecting in a sample from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer) a base substitution, a short insertion / deletion (indel), a copy number alteration, or a genomic rearrangement in one or more genes. In some embodiments, a cancer to be treated using the methods of the present disclosure comprises a gene fusion, gene rearrangement, gene amplification, or activating mutation in an ALK gene, i.e., an ALK+ cancer of the present disclosure. In some embodiments, the methods further comprise acquiring knowledge of or detecting in a sample from an individual (e.g., an individual having cancer, suspected of having cancer, being tested for cancer, or being treated for cancer) a gene fusion, gene rearrangement, gene amplification, or activating mutation in an ALK gene.

[0129] In some embodiments of any of the methods provided herein, the treatment or the one or more treatment options, e.g., the ALK-targeted therapy, further comprise an additional anti-cancer therapy, e.g., an ALK-targeted therapy in combination with an additional anti- cancer therapy. In some embodiments of any of the methods provided herein, the treatment or the one or more treatment options, e.g., the ALK-targeted therapy, further comprise administering an additional anti-cancer therapy to the individual, e.g., administering an ALK- targeted therapy in combination with an additional anti-cancer therapy. In some embodiments, the additional anti-cancer therapy is any anti-cancer therapy known in the art or described herein. In some embodiments, the additional anti-cancer therapy comprises one or more of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti-angiogenic therapy, an anti-DNA repair therapy, an anti-inflammatory therapy, an anti-neoplastic agent, a growth inhibitory agent, a cytotoxic agent, a vaccine, a small molecule agonist, a virus- based therapy, an antibody-drug conjugate, a recombinant protein, a fusion protein, a natural compound, a peptide, a PROteolysis-TArgeting Chimera (PROTAC), or any combination thereof. In some embodiments, the additional anti-cancer therapy is an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the additional anti-cancer therapy is a PD-1-, a CTLA4-, or a PD-L1-targeted agent. In some embodiments, the additional anti- 44MOFO-358156824Docket No: 197102019040 cancer therapy is a heat shock protein 90 inhibitor (Golding et al., Molecular cancer vol. 17,1 52, 2018; Pall, Current opinion in oncology vol. 27,2 (2015):118-24), an EGFR inhibitor (Golding et al., Molecular cancer vol. 17,152, 2018), a SHP2 inhibitor (Dardaei et al., Nature medicine vol. 24,4 (2018): 512-517), a MEK inhibitor (Shrestha et al., Scientific reports vol. 9,118842, 2019; Shrestha et al., The Journal of pharmacology and experimental therapeutics vol. 374,1 (2020):134-140), an IGF-1R inhibitor (George, Journal of hematology & oncology vol. 12,180, 2019), a vascular endothelial growth factor (VEGF)-targeted therapy (Makimoto et al., Acta medica Okayama vol. 74,5 (2020): 371-379; Gristina et al., Pharmaceuticals (Basel, Switzerland) vol. 13,12474, 2020), an mTOR inhibitor (Kim et al., Anticancer research vol. 40,3 (2020): 1395-1403), or any combination thereof.

[0130] In some embodiments, the individual has been previously treated, or is being treated, for cancer with a treatment for cancer, e.g., an anti-cancer therapy described herein or any other anti-cancer therapy or treatment known in the art. In some embodiments, the individual has been previously treated, or is being treated, for cancer with a kinase inhibitor. In some embodiments, a mutant ALK nucleic acid molecule and / or a mutant ALK polypeptide of the disclosure confer resistance of a cancer to a treatment for cancer, e.g., a prior treatment for cancer comprising an ALK-targeted therapy. In some embodiments, the cancer progressed on a prior treatment, such as an ALK-targeted therapy or TKI.

[0131] In some embodiments of any of the methods provided herein, the cancer is a carcinoma, a sarcoma, a lymphoma, a leukemia, a myeloma, a germ cell cancer, or a blastoma. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer is lung cancer, anaplastic large cell lymphoma (ALCL), neuroblastoma, an inflammatory myofibroblastic tumor (IMT), spitzoid tumor, colorectal cancer, B-cell lymphoma, ovarian cancer, non- Hodgkin’s lymphoma (NHL), or thyroid cancer. In some embodiments, the cancer is non- small cell lung cancer (NSCLC). In some embodiments, the cancer comprises a gene fusion, gene rearrangement, gene amplification, or activating mutation in an ALK gene. In some embodiments, the cancer is an ALK+ cancer, e.g., an ALK-rearrangement positive (ALK+) cancer.

[0132] In some embodiments, the methods further comprise detecting the presence or absence of a cancer in a sample from the individual. In some embodiments, the methods further comprise administering an effective amount of anti-cancer therapy to the individual, e.g., an anti-cancer therapy described herein, such as an ALK-targeted therapy. 45MOFO-358156824Docket No: 197102019040

[0133] In some embodiments of any of the methods provided herein, the sample is a sample described below. In some embodiments, the sample is obtained from the individual or from the cancer. In some embodiments, the methods further comprise obtaining the sample, e.g., from the individual or from the cancer. In some embodiments, the sample comprises a tissue biopsy sample, a liquid biopsy sample, or a normal control. In some embodiments, the sample is from a tumor biopsy, tumor specimen, or circulating tumor cell. In some embodiments, the sample is a liquid biopsy sample and comprises blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some embodiments, the sample comprises cells and / or nucleic acids from the cancer. In some embodiments, the sample comprises mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, or cell-free RNA from the cancer. In some embodiments, the sample is a liquid biopsy sample and comprises circulating tumor cells (CTCs). In some embodiments, the sample is a liquid biopsy sample and comprises cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof. In some embodiments, the fusion nucleic acid molecule or polypeptide is detected in a tissue biopsy sample, in a liquid biopsy sample, or in both a tissue biopsy sample and a liquid biopsy sample, from the individual. B. Detection of Mutant ALK Nucleic Acid Molecules and Polypeptides

[0134] Certain aspects of the present disclosure relate to detection of mutant ALK nucleic acid molecules and / or mutant ALK polypeptides of the present disclosure, e.g., in a patient sample. In some embodiments, the mutant ALK nucleic acid molecule or polypeptide is detected in vitro. (i) Detection of mutant ALK Nucleic Acid Molecules

[0135] Methods for detecting a mutant ALK nucleic acid molecule of the disclosure (e.g., a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2), are known in the art. For example, in some embodiments, a mutant ALK nucleic acid molecule is detected by sequencing part or all of an ALK gene by next-generation or other sequencing of DNA, RNA, or cDNA. In some embodiments, a mutant ALK nucleic acid molecule of the disclosure is detected by PCR amplification of DNA, RNA, or cDNA. In some embodiments, a mutant ALK nucleic acid molecule of the disclosure is detected in a cancer or tumor cell, e.g., using tumor tissue, such as from a tumor biopsy or other tumor specimen; in a circulating cancer or tumor cell, e.g., using a liquid biopsy, such as from 46MOFO-358156824Docket No: 197102019040 blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva; or in circulating tumor DNA (ctDNA), e.g., using a liquid biopsy, such as from blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva.

[0136] Exemplary and non-limiting methods for detecting a mutant ALK nucleic acid molecule of the disclosure are provided below.

[0137] In some embodiments, a mutant ALK nucleic acid molecule of the disclosure is detected using any suitable method known in the art, such as a nucleic acid hybridization assay, an amplification-based assay (e.g., polymerase chain reaction, PCR), a PCR-RFLP assay, real-time PCR, sequencing (e.g., Sanger sequencing or next-generation sequencing), a screening analysis (e.g., using karyotype methods), fluorescence in situ hybridization (FISH), break away FISH, spectral karyotyping, multiplex-FISH, comparative genomic hybridization, in situ hybridization, single specific primer-polymerase chain reaction (SSP-PCR), high performance liquid chromatography (HPLC), or mass-spectrometric genotyping. Methods of analyzing samples, e.g., to detect a nucleic acid molecule, are described in U.S. Patent No. 9,340,830 and in WO2012092426A1, which are hereby incorporated by reference in their entirety. In some embodiments, a mutant ALK nucleic acid molecule of the disclosure is detected by sequencing. In some embodiments, the sequencing comprises a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique. In some embodiments, the massively parallel sequencing (MPS) technique comprises next- generation sequencing (NGS).

[0138] In some embodiments, a mutant ALK nucleic acid molecule of the disclosure is detected using an in situ hybridization method, such as a fluorescence in situ hybridization (FISH) method.

[0139] In some embodiments, a mutant ALK nucleic acid molecule of the disclosure is detected using an array-based method, such as array-based comparative genomic hybridization (CGH) methods. In array-based CGH methods, a first sample of nucleic acids (e.g., from a sample, such as from a tumor, or a tissue or liquid biopsy) is labeled with a first label, while a second sample of nucleic acids (e.g., a control, such as from a healthy cell / tissue) is labeled with a second label. In some embodiments, equal quantities of the two samples are mixed and co-hybridized to a DNA microarray of several thousand evenly spaced cloned DNA fragments or oligonucleotides, which have been spotted in triplicate on the array. After hybridization, digital imaging systems are used to capture and quantify the relative fluorescence intensities of each of the hybridized fluorophores. The resulting ratio of 47MOFO-358156824Docket No: 197102019040 the fluorescence intensities is proportional to the ratio of the copy numbers of DNA sequences in the two samples. In some embodiments, where there are chromosomal deletions or multiplications, differences in the ratio of the signals from the two labels are detected and the ratio provides a measure of the copy number. Array-based CGH can also be performed with single-color labeling. In single color CGH, a control (e.g., control nucleic acid sample, such as from a healthy cell / tissue) is labeled and hybridized to one array and absolute signals are read, and a test sample (e.g., a nucleic acid sample obtained from an individual or from a tumor, or a tissue or liquid biopsy) is labeled and hybridized to a second array (with identical content) and absolute signals are read. Copy number differences are calculated based on absolute signals from the two arrays.

[0140] In some embodiments, a mutant ALK nucleic acid molecule of the disclosure is detected using an amplification-based method. As is known in the art, in such amplification- based methods, a sample of nucleic acids, such as a sample obtained from an individual, a tumor or a tissue or liquid biopsy, is used as a template in an amplification reaction (e.g., Polymerase Chain Reaction (PCR)) using one or more oligonucleotides or primers, e.g., such as one or more oligonucleotides or primers provided herein. The presence of a mutant ALK nucleic acid molecule of the disclosure in the sample can be determined based on the presence or absence of an amplification product. Quantitative amplification methods are also known in the art and may be used according to the methods provided herein. Methods of measurement of DNA copy number at microsatellite loci using quantitative PCR analysis are known in the art. The known nucleotide sequence for genes is sufficient to enable one of skill in the art to routinely select primers to amplify any portion of the gene. Fluorogenic quantitative PCR can also be used. In fluorogenic quantitative PCR, quantitation is based on the amount of fluorescence signals, e.g., TaqMan and Sybr green.

[0141] Other amplification methods suitable for use according to the methods provided herein include, e.g., ligase chain reaction (LCR), transcription amplification, self-sustained sequence replication, dot PCR, and linker adapter PCR.

[0142] In some embodiments, a mutant ALK nucleic acid molecule of the disclosure is detected using a sequencing method. Any method of sequencing known in the art can be used to detect a mutant ALK nucleic acid molecule provided herein. Exemplary sequencing methods that may be used to detect a mutant ALK nucleic acid molecule provided herein include those based on techniques developed by Maxam and Gilbert or Sanger. Automated sequencing procedures may also be used, e.g., including sequencing by mass spectrometry. 48MOFO-358156824Docket No: 197102019040

[0143] In some embodiments, a mutant ALK nucleic acid molecule of the disclosure is detected using hybrid capture-based sequencing (hybrid capture-based NGS), e.g., using adaptor ligation-based libraries. See, e.g., Frampton, G.M. et al. (2013) Nat. Biotech. 31:1023-1031, which is hereby incorporated by reference. In some embodiments, a mutant ALK nucleic acid molecule of the disclosure is detected using next-generation sequencing (NGS). Next-generation sequencing includes any sequencing method that determines the nucleotide sequence of either individual nucleic acid molecules or clonally expanded proxies for individual nucleic acid molecules in a highly parallel fashion (e.g., greater than 105molecules may be sequenced simultaneously). Next generation sequencing methods suitable for use according to the methods provided herein are known in the art and include, without limitation, massively parallel short-read sequencing, template-based sequencing, pyrosequencing, real-time sequencing comprising imaging the continuous incorporation of dye-labeling nucleotides during DNA synthesis, nanopore sequencing, sequencing by hybridization, nano-transistor array based sequencing, polony sequencing, scanning tunneling microscopy (STM)-based sequencing, or nanowire-molecule sensor based sequencing. See, e.g., Metzker, M. (2010) Nature Biotechnology Reviews 11:31-46, which is hereby incorporated by reference. Exemplary NGS methods and platforms that may be used to detect a mutant ALK nucleic acid molecule provided herein include, without limitation, the HeliScope Gene Sequencing system from Helicos BioSciences (Cambridge, MA., USA), the PacBio RS system from Pacific Biosciences (Menlo Park, CA, USA), massively parallel short-read sequencing such as the Solexa sequencer and other methods and platforms from Illumina Inc. (San Diego, CA, USA), 454 sequencing from 454 LifeSciences (Branford, CT, USA), Ion Torrent sequencing from ThermoFisher (Waltham, MA, USA), or the SOLiD sequencer from Applied Biosystems (Foster City, CA, USA). Additional exemplary methods and platforms that may be used to detect a mutant ALK nucleic acid molecule provided herein include, without limitation, the Genome Sequencer (GS) FLX System from Roche (Basel, CHE), the G.007 polonator system, the Solexa Genome Analyzer, HiSeq 2500, HiSeq3000, HiSeq 4000, and NovaSeq 6000 platforms from Illumina Inc. (San Diego, CA, USA).

[0144] In some embodiments of any of the methods provided herein, the methods may comprise one or more of the steps of: (i) obtaining a sample from an individual (e.g., an individual having, suspected of having, or determined to have cancer), (ii) extracting nucleic acid molecules (e.g., a mixture of tumor or cancer nucleic acid molecules and non-tumor or non-cancer nucleic acid molecules) from the sample, (iii) ligating one or more adapters to the nucleic acid molecules extracted from the sample (e.g., one or more amplification primers, 49MOFO-358156824Docket No: 197102019040 flow cell adapter sequences, substrate adapter sequences, sample index sequences, or unique molecular identifier (UMI) sequences), (iv) amplifying the nucleic acid molecules (e.g., using a polymerase chain reaction (PCR) amplification technique, a non-PCR amplification technique, or an isothermal amplification technique), (v) capturing nucleic acid molecules from the amplified nucleic acid molecules (e.g., by hybridization to one or more bait molecules, where the bait molecules each comprise one or more nucleic acid molecules (e.g., capture nucleic acid molecules) that each comprise a region that is complementary to a region of a captured nucleic acid molecule), (vi) sequencing the nucleic acid molecules extracted from the sample (or library proxies derived therefrom) using, e.g., a next-generation (massively parallel) sequencing technique, a whole genome sequencing (WGS) technique, a whole exome sequencing technique, a targeted sequencing technique, a direct sequencing technique, or a Sanger sequencing technique) using, e.g., a next-generation (massively parallel) sequencer, and (vii) generating, displaying, transmitting, and / or delivering a report (e.g., an electronic, web-based, or paper report) to the individual (or patient), a caregiver, a healthcare provider, a physician, an oncologist, an electronic medical record system, a hospital, a clinic, a third-party payer, an insurance company, or a government office. In some instances, the report comprises output from the methods described herein. In some instances, all or a portion of the report may be displayed in a graphical user interface of an online or web-based healthcare portal. In some instances, the report is transmitted via a computer network or peer-to-peer connection.

[0145] In some embodiments of any of the methods provided herein, the methods may comprise one or more of the steps of: (a) providing a plurality of nucleic acid molecules obtained from a sample from an individual (e.g., an individual having, suspected of having or determined to have cancer), wherein the plurality of nucleic acid molecules comprises nucleic acid molecules corresponding to a mutant ALK nucleic acid molecule of the disclosure; (b) ligating one or more adapters onto one or more nucleic acid molecules from the plurality of nucleic acid molecules; (c) amplifying the one or more ligated nucleic acid molecules from the plurality of nucleic acid molecules; (d) capturing amplified nucleic acid molecules from the amplified nucleic acid molecules; (e) sequencing, e.g., by a sequencer, the captured nucleic acid molecules to obtain a plurality of sequence reads that represent the captured nucleic acid molecules, wherein one or more of the plurality of sequence reads correspond to the mutant ALK nucleic acid molecule; (f) analyzing the plurality of sequence reads; and (g) based on the analysis, detecting the presence or absence of the mutant ALK nucleic acid molecule in the sample. In some embodiments, the methods further comprise 50MOFO-358156824Docket No: 197102019040 receiving, e.g., at one or more processors, sequence read data for the plurality of sequence reads. In some embodiments, the analyzing the plurality of sequence reads comprises identifying, e.g., using the one or more processors, the presence or absence of sequence reads corresponding to the fusion nucleic acid molecule. In some embodiments, the amplified nucleic acid molecules are captured by hybridization with one or more bait molecules.

[0146] In some embodiments of any of the methods provided herein, the methods may comprise one or more of the steps of: (a) providing a sample from an individual (e.g., an individual having, suspected of having or determined to have cancer), wherein the sample comprises a plurality of nucleic acid molecules; (b) preparing a nucleic acid sequencing library from the plurality of nucleic acid molecules in the sample; (c) amplifying said library; (d) selectively enriching for one or more nucleic acid molecules comprising nucleotide sequences corresponding to a mutant ALK nucleic acid molecule of the disclosure in said library to produce an enriched sample; (e) sequencing the enriched sample, thereby producing a plurality of sequence reads; (f) analyzing the plurality of sequence reads for the presence of the mutant ALK nucleic acid molecule; (g) detecting, based on the analyzing step, the presence or absence of the mutant ALK nucleic acid molecule in the sample from the individual.

[0147] In some embodiments of any of the methods provided herein, the plurality of nucleic acid molecules comprises a mixture of cancer nucleic acid molecules and non-cancer nucleic acid molecules. In some embodiments, the cancer nucleic acid molecules are derived from a tumor portion of a heterogeneous tissue biopsy sample, and the non-cancer nucleic acid molecules are derived from a normal portion of the heterogeneous tissue biopsy sample. In some embodiments, the sample comprises a liquid biopsy sample, and the cancer nucleic acid molecules are derived from a circulating tumor DNA (ctDNA) fraction of the liquid biopsy sample; and the non-cancer nucleic acid molecules are derived from a non-tumor fraction of the liquid biopsy sample or a cell-free DNA (cfDNA) fraction of the liquid biopsy sample.

[0148] In some embodiments of any of the methods, the one or more adapters comprise amplification primers, flow cell adaptor sequences, substrate adapter sequences, sample index sequences, or unique molecular identifier (UMI) sequences. In some embodiments, the one or more adapters comprise one or more sample index sequences. As is known in the art, sample indexes allow the sequencing of multiple samples on the same instrument flow cell or chip (i.e., multiplexing). Sample indexes are typically between about 8 and about 10 bases in length, and comprise a nucleotide sequence specific to a sample that is used to assign sequence reads to the correct sample during data analysis. In some embodiments, the one or 51MOFO-358156824Docket No: 197102019040 more adapters comprise one or more unique molecule identifiers (UMIs). As is known in the art, UMIs comprise short nucleotide sequences that include a unique barcode that is incorporated into each molecule in a given sample library. UMIs are useful for identifying PCR duplicates created during library amplification steps, and / or for reducing the rate of false-positive variant calls and increasing variant detection, since variant alleles present in the original sample (true variants) can be distinguished from errors introduced during library preparation, target enrichment, or sequencing.

[0149] Methods for mutation calling can include one or more of the following: making independent calls based on the information at each position or certain positions in the reference sequence (e.g., examining the sequence reads; examining the base calls and quality scores; calculating the probability of observed bases and quality scores given a potential genotype; and assigning genotypes (e.g., using Bayes’ rule)); removing false positives (e.g., using depth thresholds to reject SNPs with read depth much lower or higher than expected; local realignment to remove false positives due to small indels); and performing linkage disequilibrium (LD) / imputation-based analysis to refine the calls.

[0150] After alignment, detection of substitutions can be performed using a mutation calling method (e.g., a Bayesian mutation calling method) which is applied to selected bases or at each base in each of the subject intervals, e.g., exons of a gene or other locus to be evaluated, where presence of alternate alleles is observed. This method will compare the probability of observing the read data in the presence of a mutation with the probability of observing the read data in the presence of base-calling error alone. Mutations can be called if this comparison is sufficiently strongly supportive of the presence of a mutation.

[0151] In some embodiments, the selectively enriching comprises: (a) combining one or more bait molecules with the library, thereby hybridizing the one or more bait molecules to one or more nucleic acid molecules comprising nucleotide sequences corresponding to the mutant ALK nucleic acid molecule (or wild-type version thereof) and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids to produce the enriched sample. In some embodiments, the captured nucleic acid molecules are captured from the amplified nucleic acid molecules by hybridization to one or more bait molecules. In some embodiments, the amplifying comprises performing a polymerase chain reaction (PCR) amplification technique, a non-PCR amplification technique, or an isothermal amplification technique. In some embodiments, the sequencing comprises use of a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique. In some embodiments, the sequencing 52MOFO-358156824Docket No: 197102019040 comprises a massively parallel sequencing technique, and the massively parallel sequencing technique comprises next generation sequencing (NGS). In some embodiments, the sequencer comprises a next generation sequencer.

[0152] In some embodiments of any of the methods provided herein, the methods further comprise selectively enriching for one or more nucleic acids in the sample comprising nucleotide sequences corresponding to the mutant ALK nucleic acid molecule of the disclosure. In some embodiments, the selectively enriching produces an enriched sample. In some embodiments, the selectively enriching comprises: (a) combining one or more bait molecules with the sample, thereby hybridizing the one or more bait molecules to one or more nucleic acids in the sample comprising nucleotide sequences corresponding to the mutant ALK nucleic acid molecule and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids to produce the enriched sample. In some embodiments, the selectively enriching comprises amplifying the one or more nucleic acids comprising nucleotide sequences corresponding to the mutant ALK nucleic acid molecule using a polymerase chain reaction (PCR) to produce an enriched sample. In some embodiments, the methods further comprise sequencing the enriched sample.

[0153] In some embodiments of any of the methods provided herein, the methods further comprise generating a molecular profile for the individual or the sample, based, at least in part, on detecting the presence or absence of the mutant ALK nucleic acid molecule. In some embodiments, the molecular profile for the individual or sample further comprises results from a comprehensive genomic profiling (CGP) test, a gene expression profiling test, a cancer hotspot panel test, a DNA methylation test, a DNA fragmentation test, an RNA fragmentation test, or any combination thereof. In some embodiments, the molecular profile further comprises results from a nucleic acid sequencing-based test.

[0154] In some embodiments of any of the methods provided herein, the methods further comprise selecting a treatment, administering a treatment, or applying a treatment to the individual based on the generated molecular profile, wherein the treatment comprises an anti- cancer therapy, e.g., as described herein, e.g., an ALK-targeted therapy. In some embodiments, the methods further comprise ceasing administration of a treatment (e.g., a previous ALK-targeted therapy) based on the generated molecular profile.

[0155] In some embodiments of any of the methods provided herein, the methods further comprise generating a report indicating the presence or absence of the mutant ALK nucleic acid molecule in the sample. In some embodiments, the methods further comprise generating, by the one or more processors, a report indicating the presence or absence of the mutant ALK 53MOFO-358156824Docket No: 197102019040 nucleic acid molecule in the sample. In some embodiments, the methods further comprise transmitting the report to a healthcare provider. In some embodiments, the report is transmitted via a computer network or a peer-to-peer connection.

[0156] In some embodiments of any of the methods provided herein, the methods further comprise acquiring knowledge of or detecting in a sample from the individual an ALK mutation associated with acquired resistance to an ALK-targeted therapy. Exemplary ALK mutations associated with acquired resistance to an ALK-targeted therapy include, without limitation, T1151M, T1151R, C1156Y, I1171N, I1171T, I1171S, I1171H, I1171V, F1174X, V1180L, R1192P, L1196M, L1198F, G1202X, G1202del, D1203N, S1206Y, E1210K, and G1269A, amino acid numbering based on SEQ ID NO:2.

[0157] In some embodiments of any of the methods provided herein, the methods further comprise acquiring knowledge of or detecting in a sample from the individual a gene fusion, gene rearrangement, gene amplification, or activating mutation in an ALK gene. In some embodiments of any of the methods provided herein, the methods further comprise acquiring knowledge of or detecting in a sample from the individual an ALK+ cancer or cancer cell.

[0158] In some embodiments of any of the methods provided herein, the methods further comprise acquiring knowledge of or detecting in a sample from the individual a base substitution, a short insertion / deletion (indel), a copy number alteration, or a genomic rearrangement in one or more genes. In some embodiments, the one or more genes comprise one or more of AXL, BRAF, MAP2K1, MET, NF2, PIK3CA, and NRAS, or a panel of known / suspected oncogenes and / or tumor suppressors, or any combination thereof.

[0159] The disclosed methods may be used with any of a variety of samples, e.g., as described in further detail below. For example, in some instances, the sample may comprise a tissue biopsy sample, a liquid biopsy sample, or a normal control. In some instances, the sample may be a liquid biopsy sample and may comprise blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some instances, the sample may be a liquid biopsy sample and may comprise circulating tumor cells (CTCs). In some instances, the sample may be a liquid biopsy sample and may comprise cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof.

[0160] In some instances, the nucleic acid molecules extracted from a sample may comprise a mixture of tumor or cancer nucleic acid molecules and non-tumor or non-cancer nucleic acid molecules. In some instances, the tumor nucleic acid molecules may be derived from a tumor portion of a heterogeneous tissue biopsy sample, and the non-tumor nucleic acid molecules may be derived from a normal portion of the heterogeneous tissue biopsy sample. 54MOFO-358156824Docket No: 197102019040 In some instances, the sample may comprise a liquid biopsy sample, and the tumor or cancer nucleic acid molecules may be derived from a circulating tumor DNA (ctDNA) fraction of the liquid biopsy sample while the non-tumor or non-cancer nucleic acid molecules may be derived from a non-tumor or non-cancer, cell-free DNA (cfDNA) fraction of the liquid biopsy sample. In some embodiments of any of the methods provided herein, the method further comprises determining the circulating tumor DNA (ctDNA) fraction of a liquid biopsy sample. (ii) Detection of Mutant Polypeptides

[0161] Also provided herein are methods of detecting a mutant ALK polypeptide of the disclosure (e.g., comprising a G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, or L1196Q mutation, amino acid numbering based on SEQ ID NO:2), or a fragment thereof.

[0162] A mutant ALK polypeptide provided herein, or a fragment thereof, may be detected or measured, e.g., in a sample obtained from an individual, using any method known in the art, such as using antibodies (e.g., an antibody described herein), mass spectrometry (e.g., tandem mass spectrometry), a reporter assay (e.g., a fluorescence-based assay), immunoblots such as a Western blot, immunoassays such as enzyme-linked immunosorbent assays (ELISA), immunohistochemistry, other immunological assays (e.g., fluid or gel precipitin reactions, immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), immunofluorescent assays), and analytic biochemical methods (e.g., electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography).

[0163] In some embodiments, a mutant ALK polypeptide provided herein, or a fragment thereof, can be distinguished from a reference polypeptide, e.g., a non-mutant or wild type protein or polypeptide, with an antibody or antibody fragment that reacts differentially with a mutant protein or polypeptide (e.g., a mutant ALK polypeptide provided herein or a fragment thereof) as compared to a reference protein or polypeptide. In some embodiments, a mutant ALK polypeptide of the disclosure, or a fragment thereof, can be distinguished from a reference polypeptide, e.g., a non-mutant or wild type protein or polypeptide, by reaction with a detection reagent, e.g., a substrate, e.g., a substrate for catalytic activity, e.g., phosphorylation.

[0164] In some aspects, methods of detection of a mutant ALK polypeptide of the disclosure, or a fragment thereof, are provided, comprising contacting a sample, e.g., a sample described 55MOFO-358156824Docket No: 197102019040 herein, comprising a mutant ALK polypeptide described herein, with a detection reagent provided herein (e.g., an antibody of the disclosure), and determining if the mutant ALK polypeptide is present in the sample. (iii) Detection Reagents

[0165] In some aspects, provided herein are reagents for detecting a mutant ALK nucleic acid molecule of the disclosure (e.g., encoding a G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, or L1196Q mutation, amino acid numbering based on SEQ ID NO:2), or a fragment thereof, e.g., according to the methods of detection provided herein. In some embodiments, a detection reagent provided herein comprises a nucleic acid molecule, e.g., a DNA, RNA, or mixed DNA / RNA molecule, comprising a nucleotide sequence that is complementary to a nucleotide sequence on a target nucleic acid molecule, e.g., a nucleic acid molecule that is or comprises a mutant ALK nucleic acid molecule described herein or a fragment or portion thereof.

[0166] In other aspects, provided herein are reagents for detecting a mutant ALK polypeptide of the disclosure (e.g., comprising a G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, or L1196Q mutation, amino acid numbering based on SEQ ID NO:2), or a fragment thereof, e.g., according to the methods of detection provided herein. In some embodiments, a detection reagent provided herein comprises an antibody or antibody fragment that specifically binds to a mutant ALK polypeptide of the disclosure, or to a fragment thereof. Baits

[0167] In some embodiments, nucleic acids corresponding to a mutant ALK nucleic acid molecule described herein are captured (e.g., from amplified nucleic acids) by hybridization with a bait molecule. Provided herein are bait molecules suitable for the detection of a mutant ALK nucleic acid molecule of the disclosure (e.g., encoding a G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, or L1196Q mutation, amino acid numbering based on SEQ ID NO:2).

[0168] In some embodiments, a bait molecule comprises a capture nucleic acid molecule configured to hybridize to a target nucleic acid molecule comprising a mutant ALK nucleic acid molecule of the disclosure, or a fragment or portion thereof. In some embodiments, the capture nucleic acid molecule is configured to hybridize to the mutant ALK nucleic acid molecule of the target nucleic acid molecule. 56MOFO-358156824Docket No: 197102019040

[0169] In some embodiments, the capture nucleic acid molecule is configured to hybridize to a fragment of a mutant ALK nucleic acid molecule of the disclosure. In some embodiments, the fragment comprises (or is) between about 5 and about 25 nucleotides, between about 5 and about 300 nucleotides, between about 100 and about 300 nucleotides, between about 130 and about 230 nucleotides, or between about 150 and about 200 nucleotides. In some embodiments, the fragment comprises (or is) about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, about 250 nucleotides, about 275 nucleotides, or about 300 nucleotides in length.

[0170] In some embodiments, the capture nucleic acid molecule comprises (or is) between about 5 and about 25 nucleotides, between about 5 and about 300 nucleotides, between about 100 and about 300 nucleotides, between about 130 and about 230 nucleotides, or between about 150 and about 200 nucleotides. In some embodiments, the capture nucleic acid molecule comprises (or is) about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, about 250 nucleotides, about 275 nucleotides, or about 300 nucleotides in length.

[0171] In some embodiments, the capture nucleic acid molecule is configured to hybridize to a nucleotide sequence in an intron or an exon of an ALK gene, or in a breakpoint joining the introns or exons of an ALK gene (e.g., plus or minus any of between about 10 and about 20, about 20 and about 30, about 30 and about 40, about 40 and about 50, about 50 and about 60, about 60 and about 70, about 70 and about 80, about 80 and about 90, or about 90 and about 100, or more nucleotides).

[0172] In some embodiments, the capture nucleic acid molecule is a DNA, RNA, or a DNA / RNA molecule. In some embodiments, the capture nucleic acid molecule comprises any of between about 50 and about 1000 nucleotides, between about 50 and about 500 nucleotides, between about 100 and about 500 nucleotides, between about 100 and about 300 nucleotides, between about 130 and about 230 nucleotides, or between about 150 and about 200 nucleotides. In some embodiments, the capture nucleic acid molecule comprises any of between about 50 nucleotides and about 100 nucleotides, about 100 nucleotides and about 150 nucleotides, about 150 nucleotides and about 200 nucleotides, about 200 nucleotides and about 250 nucleotides, about 250 nucleotides and about 300 nucleotides, about 300 nucleotides and about 350 nucleotides, about 350 nucleotides and about 400 nucleotides, about 400 nucleotides and about 450 nucleotides, about 450 nucleotides and about 500 nucleotides, about 500 nucleotides and about 550 nucleotides, about 550 nucleotides and about 600 nucleotides, about 600 nucleotides and about 650 nucleotides, about 650 57MOFO-358156824Docket No: 197102019040 nucleotides and about 700 nucleotides, about 700 nucleotides and about 750 nucleotides, about 750 nucleotides and about 800 nucleotides, about 800 nucleotides and about 850 nucleotides, about 850 nucleotides and about 900 nucleotides, about 900 nucleotides and about 950 nucleotides, or about 950 nucleotides and about 1000 nucleotides. In some embodiments, the capture nucleic acid molecule comprises between about 10 and about 30 nucleotides, between about 50 and about 1000 nucleotides, between about 100 and about 500 nucleotides, between about 100 and about 300 nucleotides, or between about 100 and about 200 nucleotides. In some embodiments, the capture nucleic acid molecule comprises about 150 nucleotides. In some embodiments, the capture nucleic acid molecule is about 150 nucleotides. In some embodiments, the capture nucleic acid molecule comprises about 170 nucleotides. In some embodiments, the capture nucleic acid molecule is about 170 nucleotides.

[0173] In some embodiments, a bait provided herein comprises a DNA, RNA, or a DNA / RNA molecule. In some embodiments, a bait provided herein includes a label, a tag or detection reagent. In some embodiments, the label, tag or detection reagent is a radiolabel, a fluorescent label, an enzymatic label, a sequence tag, biotin, or another ligand. In some embodiments, a bait provided herein includes a detection reagent such as a fluorescent marker. In some embodiments, a bait provided herein includes (e.g., is conjugated to) an affinity tag or reagent, e.g., that allows capture and isolation of a hybrid formed by a bait and a nucleic acid molecule hybridized to the bait. In some embodiments, the affinity tag or reagent is an antibody, an antibody fragment, biotin, or any other suitable affinity tag or reagent known in the art. In some embodiments, a bait is suitable for solution phase hybridization.

[0174] Baits can be produced and used according to methods known in the art, e.g., as described in WO2012092426A1 and / or or in Frampton et al (2013) Nat Biotechnol, 31:1023- 1031, incorporated herein by reference. For example, biotinylated baits (e.g., RNA baits) can be produced by obtaining a pool of synthetic long oligonucleotides, originally synthesized on a microarray, and amplifying the oligonucleotides to produce the bait sequences. In some embodiments, the baits are produced by adding an RNA polymerase promoter sequence at one end of the bait sequences, and synthesizing RNA sequences using RNA polymerase. In one embodiment, libraries of synthetic oligodeoxynucleotides can be obtained from commercial suppliers, such as Agilent Technologies, Inc., and amplified using known nucleic acid amplification methods. 58MOFO-358156824Docket No: 197102019040

[0175] In some embodiments, a bait provided herein is between about 100 nucleotides and about 300 nucleotides. In some embodiments, a bait provided herein is between about 130 nucleotides and about 230 nucleotides. In some embodiments, a bait provided herein is between about 150 nucleotides and about 200 nucleotides. In some embodiments, a bait provided herein comprises a target-specific bait sequence (e.g., a capture nucleic acid molecule described herein) and universal tails on each end. In some embodiments, the target- specific sequence, e.g., a capture nucleic acid molecule described herein, is between about 40 nucleotides and about 300 nucleotides. In some embodiments, the target-specific sequence, e.g., a capture nucleic acid molecule described herein, is between about 100 nucleotides and about 200 nucleotides. In some embodiments, the target-specific sequence, e.g., a capture nucleic acid molecule described herein, is between about 120 nucleotides and about 170 nucleotides. In some embodiments, the target-specific sequence, e.g., a capture nucleic acid molecule described herein, is about 150 nucleotides or about 170 nucleotides. In some embodiments, a bait provided herein comprises an oligonucleotide comprising about 200 nucleotides, of which about 150 nucleotides or about 170 nucleotides are target-specific (e.g., a capture nucleic acid molecule described herein), and the other 50 nucleotides or 30 nucleotides (e.g., 25 or 15 nucleotides on each end of the bait) are universal arbitrary tails, e.g., suitable for PCR amplification.

[0176] In some embodiments, a bait provided herein hybridizes to a nucleotide sequence corresponding to an intron or an exon of a mutant ALK molecule described herein, and / or a breakpoint joining the introns and / or exons.

[0177] The baits described herein can be used for selection of exons and short target sequences.

[0178] In some embodiments, a bait of the disclosure distinguishes a nucleic acid molecule, e.g., a genomic or transcribed nucleic acid molecule, e.g., a cDNA or RNA, having a mutation of a mutant ALK nucleic acid molecule described herein from a reference nucleotide sequence, e.g., a nucleotide sequence not having the mutation. Probes

[0179] Also provided herein are probes, e.g., nucleic acid molecules, suitable for the detection of a mutant ALK nucleic acid molecule of the disclosure (e.g., encoding a G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, or L1196Q mutation, amino acid numbering based on SEQ ID NO:2). In some embodiments, a probe provided herein comprises a nucleic acid sequence configured to hybridize to a target nucleic acid molecule 59MOFO-358156824Docket No: 197102019040 that is or comprises a mutant ALK nucleic acid molecule of the disclosure, or a fragment or portion thereof. In some embodiments, the probe comprises a nucleic acid sequence configured to hybridize to the mutant ALK nucleic acid molecule of the disclosure, or the fragment or portion thereof, of the target nucleic acid molecule. In some embodiments, the probe comprises a nucleic acid sequence configured to hybridize to a fragment or portion of the mutant ALK nucleic acid molecule of the target nucleic acid molecule. In some embodiments, the fragment or portion comprises between about 5 and about 25 nucleotides, between about 5 and about 300 nucleotides, between about 100 and about 300 nucleotides, between about 130 and about 230 nucleotides, or between about 150 and about 200 nucleotides.

[0180] In some embodiments, the probe comprises a nucleotide sequence configured to hybridize to a nucleotide sequence in an intron or an exon of a gene involved in a mutant ALK nucleic acid molecule described herein, e.g., an ALK gene, or in a breakpoint joining the introns or exons of the gene (e.g., plus or minus any of between about 10 and about 20, about 20 and about 30, about 30 and about 40, about 40 and about 50, about 50 and about 60, about 60 and about 70, about 70 and about 80, about 80 and about 90, or about 90 and about 100, or more nucleotides).

[0181] In some embodiments, the probe comprises a nucleic acid molecule which is a DNA, RNA, or a DNA / RNA molecule. In some embodiments, the probe comprises a nucleic acid molecule comprising any of between about 10 and about 20 nucleotides, between about 12 and about 20 nucleotides, between about 10 and about 1000 nucleotides, between about 50 and about 500 nucleotides, between about 100 and about 500 nucleotides, between about 100 and about 300 nucleotides, between about 130 and about 230 nucleotides, or between about 150 and about 200 nucleotides. In some embodiments, the probe comprises a nucleic acid molecule comprising any of 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides. In some embodiments, the probe comprises a nucleic acid molecule comprising any of between about 40 nucleotides and about 50 nucleotides, about 50 nucleotides and about 100 nucleotides, about 100 nucleotides and about 150 nucleotides, about 150 nucleotides and about 200 nucleotides, about 200 nucleotides and about 250 nucleotides, about 250 nucleotides and about 300 nucleotides, about 300 nucleotides and about 350 nucleotides, about 350 nucleotides and about 400 nucleotides, about 400 nucleotides and about 450 nucleotides, 60MOFO-358156824Docket No: 197102019040 about 450 nucleotides and about 500 nucleotides, about 500 nucleotides and about 550 nucleotides, about 550 nucleotides and about 600 nucleotides, about 600 nucleotides and about 650 nucleotides, about 650 nucleotides and about 700 nucleotides, about 700 nucleotides and about 750 nucleotides, about 750 nucleotides and about 800 nucleotides, about 800 nucleotides and about 850 nucleotides, about 850 nucleotides and about 900 nucleotides, about 900 nucleotides and about 950 nucleotides, or about 950 nucleotides and about 1000 nucleotides. In some embodiments, the probe comprises a nucleic acid molecule comprising between about 12 and about 20 nucleotides.

[0182] In some embodiments, a probe provided herein comprises a DNA, RNA, or a DNA / RNA molecule. In some embodiments, a probe provided herein includes a label or a tag. In some embodiments, the label or tag is a radiolabel (e.g., a radioisotope), a fluorescent label (e.g., a fluorescent compound), an enzymatic label, an enzyme co-factor, a sequence tag, biotin, or another ligand. In some embodiments, a probe provided herein includes a detection reagent such as a fluorescent marker. In some embodiments, a probe provided herein includes (e.g., is conjugated to) an affinity tag, e.g., that allows capture and isolation of a hybrid formed by a probe and a nucleic acid molecule hybridized to the probe. In some embodiments, the affinity tag is an antibody, an antibody fragment, biotin, or any other suitable affinity tag or reagent known in the art. In some embodiments, a probe is suitable for solution phase hybridization.

[0183] In some embodiments, probes provided herein may be used according to the methods of detection of mutant ALK nucleic acid molecules provided herein. For example, a probe provided herein may be used for detecting a mutant ALK nucleic acid molecule of the disclosure in a sample, e.g., a sample obtained from an individual. In some embodiments, the probe may be used for identifying cells or tissues that express a mutant ALK nucleic acid molecule of the disclosure, e.g., by measuring levels of the mutant ALK nucleic acid molecule. In some embodiments, the probe may be used for detecting levels of a mutant ALK nucleic acid molecule of the disclosure, e.g., mRNA levels, in a sample of cells from an individual.

[0184] In some embodiments, a probe of the disclosure distinguishes a nucleic acid, e.g., a genomic or transcribed nucleic acid, e.g., a cDNA or RNA, having a mutation of a mutant ALK nucleic acid molecule of the disclosure, from a reference nucleotide sequence, e.g., a nucleotide sequence not having the mutation.

[0185] Also provided herein are isolated pairs of allele-specific probes, wherein, for example, the first probe of the pair specifically hybridizes to a mutant ALK nucleic acid molecule of the 61MOFO-358156824Docket No: 197102019040 disclosure, and the second probe of the pair specifically hybridizes to a corresponding wild type sequence. Probe pairs can be designed and produced for any of the mutant ALK nucleic acid molecules described herein and are useful in detecting a somatic mutation in a sample. In some embodiments, a first probe of a pair specifically hybridizes to a mutation (e.g., the breakpoint of an alteration, rearrangement, inversion, duplication, deletion, insertion or translocation resulting in a mutant ALK nucleic acid molecule described herein), and a second probe of a pair specifically hybridizes to a sequence upstream or downstream of the mutation.

[0186] In some embodiments, one or more probes provided herein are suitable for use in in situ hybridization methods, e.g., as described above, such as FISH.

[0187] Chromosomal probes, e.g., for use in the FISH methods described herein, are typically about 50 to about 105nucleotides in length. Longer probes typically comprise smaller fragments of about 100 to about 500 nucleotides. Probes that hybridize with centromeric DNA and locus-specific DNA are available commercially, for example, from Vysis, Inc. (Downers Grove, Ill.), Molecular Probes, Inc. (Eugene, Oreg.) or from Cytocell (Oxfordshire, UK). Alternatively, probes can be made non-commercially from chromosomal or genomic DNA through standard techniques. For example, sources of DNA that can be used include genomic DNA, cloned DNA sequences, somatic cell hybrids that contain one, or a part of one, chromosome (e.g., human chromosome) along with the normal chromosome complement of the host, and chromosomes purified by flow cytometry or microdissection. The region of interest can be isolated through cloning, or by site-specific amplification via the polymerase chain reaction (PCR). Probes of the disclosure may also hybridize to RNA molecules, e.g., mRNA, such as an RNA that is or comprises a mutant ALK nucleic acid molecule of the disclosure.

[0188] In some embodiments, probes, such as probes for use in the FISH methods described herein, are labeled such that a chromosomal region or a region on an RNA to which the probes hybridize can be detected. Probes typically are directly labeled with a fluorophore, allowing the probe to be visualized without a secondary detection molecule. Probes can also be labeled by nick translation, random primer labeling or PCR labeling. Labeling may be accomplished using fluorescent (direct)-or haptene (indirect)-labeled nucleotides. Representative, non-limiting examples of labels include: AMCA-6-dUTP, CascadeBlue-4- dUTP, Fluorescein-12-dUTP, Rhodamine-6-dUTP, TexasRed-6-dUTP, Cy3-6-dUTP, Cy5- dUTP, Biotin(BIO)-11-dUTP, Digoxygenin(DIG)-11-dUTP and Dinitrophenyl (DNP)-11- dUTP. Probes can also be indirectly labeled with biotin or digoxygenin, or labeled with radioactive isotopes such as32P and3H, and secondary detection molecules may be used, or 62MOFO-358156824Docket No: 197102019040 further processing may be performed, to visualize the probes. For example, a probe labeled with biotin can be detected by avidin conjugated to a detectable marker, e.g., avidin can be conjugated to an enzymatic marker such as alkaline phosphatase or horseradish peroxidase. Enzymatic markers can be detected in standard colorimetric reactions using a substrate and / or a catalyst for the enzyme. Catalysts for alkaline phosphatase include 5-bromo-4-chloro-3- indolylphosphate and nitro blue tetrazolium. Diaminobenzoate can be used as a catalyst for horseradish peroxidase. Probes can also be prepared such that a fluorescent or other label is added after hybridization of the probe to its target to detect that the probe hybridized to the target. For example, probes can be used that have antigenic molecules incorporated into the nucleotide sequence. After hybridization, these antigenic molecules are detected, for example, using specific antibodies reactive with the antigenic molecules. Such antibodies can, for example, themselves incorporate a fluorochrome, or can be detected using a second antibody with a bound fluorochrome. For fluorescent probes, e.g., used in FISH techniques, fluorescence can be viewed with a fluorescence microscope equipped with an appropriate filter for each fluorophore, or by using dual or triple band-pass filter sets to observe multiple fluorophores. Alternatively, techniques such as flow cytometry can be used to examine the hybridization pattern of the chromosomal probes. Oligonucleotides

[0189] In some aspects, provided herein are oligonucleotides, e.g., useful as primers. In some embodiments, an oligonucleotide, e.g., a primer, provided herein comprises a nucleotide sequence configured to hybridize to a target nucleic acid molecule that is or comprises a mutant ALK nucleic acid molecule of the disclosure (e.g., encoding a G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, or L1196Q mutation, amino acid numbering based on SEQ ID NO:2), or a fragment or portion thereof. In some embodiments, the oligonucleotide comprises a nucleotide sequence configured to hybridize to the mutant ALK nucleic acid molecule of the target nucleic acid molecule. In some embodiments, the oligonucleotide comprises a nucleotide sequence configured to hybridize to a fragment or portion of the mutant ALK nucleic acid molecule of the target nucleic acid molecule.

[0190] In some embodiments, the oligonucleotide, e.g., the primer, comprises a nucleotide sequence configured to hybridize to a nucleotide sequence in an intron or an exon of a gene involved in a mutant ALK nucleic acid molecule of the disclosure (e.g., an ALK gene).

[0191] In some embodiments, the oligonucleotide comprises a nucleotide sequence corresponding to a mutant ALK nucleic acid molecule of the disclosure. In some 63MOFO-358156824Docket No: 197102019040 embodiments, the oligonucleotide comprises a nucleotide sequence corresponding to a fragment or a portion of the mutant ALK nucleic acid molecule. In some embodiments, the fragment or portion comprises between about 10 and about 30 nucleotides, between about 12 and about 20 nucleotides, or between about 12 and about 17 nucleotides. In some embodiments, the oligonucleotide comprises a nucleotide sequence complementary to a mutant ALK nucleic acid molecule provided herein. In some embodiments, the oligonucleotide comprises a nucleotide sequence complementary to a fragment or a portion of the mutant ALK nucleic acid molecule provided herein. In some embodiments, the fragment or portion comprises between about 10 and about 30 nucleotides, between about 12 and about 20 nucleotides, or between about 12 and about 17 nucleotides.

[0192] In some embodiments, an oligonucleotide, e.g., a primer, provided herein comprises a nucleotide sequence that is sufficiently complementary to its target nucleotide sequence such that the oligonucleotide specifically hybridizes to a nucleic acid molecule comprising the target nucleotide sequence, e.g., under high stringency conditions. In some embodiments, an oligonucleotide, e.g., a primer, provided herein comprises a nucleotide sequence that is sufficiently complementary to its target nucleotide sequence such that the oligonucleotide specifically hybridizes to a nucleic acid molecule comprising the target nucleotide sequence under conditions that allow a polymerization reaction (e.g., PCR) to occur.

[0193] In some embodiments, an oligonucleotide, e.g., a primer, provided herein may be useful for initiating DNA synthesis via PCR (polymerase chain reaction) or a sequencing method. In some embodiments, the oligonucleotide may be used to amplify a nucleic acid molecule that is or comprises a mutant ALK nucleic acid molecule of the disclosure, or a fragment thereof, e.g., using PCR. In some embodiments, the oligonucleotide may be used to sequence a nucleic acid molecule that is or comprises a mutant ALK nucleic acid molecule provided herein, or a fragment thereof. In some embodiments, the oligonucleotide may be used to amplify a nucleic acid molecule comprising a breakpoint of a mutant ALK nucleic acid molecule described herein, e.g., using PCR. In some embodiments, the oligonucleotide may be used to sequence a nucleic acid molecule comprising a breakpoint of a mutant ALK nucleic acid molecule described herein.

[0194] In some embodiments, pairs of oligonucleotides, e.g., pairs of primers, are provided herein, which are configured to hybridize to a nucleic acid molecule that is or comprises a mutant ALK nucleic acid molecule of the disclosure, or a fragment thereof. In some embodiments, a pair of oligonucleotides of the disclosure may be used for directing amplification of the mutant ALK nucleic acid molecule or fragment thereof, e.g., using a PCR 64MOFO-358156824Docket No: 197102019040 reaction. In some embodiments, pairs of oligonucleotides, e.g., pairs of primers, are provided herein, which are configured to hybridize to a nucleic acid molecule comprising a breakpoint of a mutant ALK nucleic acid molecule described herein, e.g., for use in directing amplification of the corresponding fusion nucleic acid molecule or fragment thereof, e.g., using a PCR reaction.

[0195] In some embodiments, an oligonucleotide, e.g., a primer, provided herein is a single stranded nucleic acid molecule, e.g., for use in sequencing or amplification methods. In some embodiments, an oligonucleotide provided herein is a double stranded nucleic acid molecule. In some embodiments, a double stranded oligonucleotide is treated, e.g., denatured, to separate its two strands prior to use, e.g., in sequencing or amplification methods. Oligonucleotides provided herein comprise a nucleotide sequence of sufficient length to hybridize to their target, e.g., a mutant ALK nucleic acid molecule of the disclosure, or a fragment thereof, and to prime the synthesis of extension products, e.g., during PCR or sequencing.

[0196] In some embodiments, an oligonucleotide, e.g., a primer, provided herein comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises at least about 8 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises at least about 10 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises at least about 12 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises at least about 15 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises at least about 20 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises at least about 30 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises between about 10 and about 30 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises between about 10 and about 25 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises between about 10 and about 20 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises 65MOFO-358156824Docket No: 197102019040 between about 10 and about 15 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises between about 12 and about 20 deoxyribonucleotides or ribonucleotides. In some embodiments, an oligonucleotide provided herein comprises between about 17 and about 20 deoxyribonucleotides or ribonucleotides. In some embodiments, the length and nucleotide sequence of an oligonucleotide provided herein is determined according to methods known in the art, e.g., based on factors such as the specific application (e.g., PCR, sequencing library preparation, sequencing), reaction conditions (e.g., buffers, temperature), and the nucleotide composition of the nucleotide sequence of the oligonucleotide or of its target complementary sequence.

[0197] In some embodiments, an oligonucleotide, e.g., a primer, of the disclosure distinguishes a nucleic acid, e.g., a genomic or transcribed nucleic acid, e.g., a cDNA or RNA, having a breakpoint of a mutant ALK nucleic acid molecule described herein, from a reference nucleotide sequence, e.g., a nucleotide sequence not having the breakpoint.

[0198] In another aspect, provided herein is a primer or primer set for amplifying a nucleic acid molecule comprising an alteration, rearrangement, chromosomal inversion, insertion, deletion, translocation, duplication or other rearrangement resulting in a mutant ALK nucleic acid molecule of the disclosure. In certain aspects, provided herein are allele-specific oligonucleotides, e.g., primers, wherein a first oligonucleotide of a pair specifically hybridizes to a mutation (e.g., of a mutant ALK nucleic acid molecule described herein), and a second oligonucleotide of a pair specifically hybridizes to a sequence upstream or downstream of the mutation. In certain aspects, provided herein are pairs of oligonucleotides, e.g., primers, wherein a first oligonucleotide of a pair specifically hybridizes to a sequence upstream of a mutation (e.g., of a mutant ALK nucleic acid molecule described herein), and a second oligonucleotide of the pair specifically hybridizes to a sequence downstream of the mutation. Antibodies

[0199] Provided herein are antibodies or antibody fragments that specifically bind to a mutant ALK polypeptide of the disclosure (e.g., comprising a G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, or L1196Q mutation, amino acid numbering based on SEQ ID NO:2), or a fragment thereof.

[0200] The antibody may be of any suitable type of antibody, including, but not limited to, a monoclonal antibody, a polyclonal antibody, a multi-specific antibody (e.g., a bispecific antibody), or an antibody fragment, so long as the antibody or antibody fragment exhibits a 66MOFO-358156824Docket No: 197102019040 specific antigen binding activity, e.g., binding to a mutant ALK polypeptide of the disclosure, or a fragment thereof.

[0201] In some embodiments, a fusion polypeptide of the disclosure, or a fragment thereof, is used as an immunogen to generate one or more antibodies of the disclosure, e.g., using standard techniques for polyclonal and monoclonal antibody preparation. In some embodiments, a mutant ALK polypeptide provided herein, is used to provide antigenic peptide fragments (e.g., comprising any of at least about 8, at least about 10, at least about 15, at least about 20, at least about 30 or more amino acids) for use as immunogens to generate one or more antibodies of the disclosure, e.g., using standard techniques for polyclonal and monoclonal antibody preparation. As is known in the art, an antibody of the disclosure may be prepared by immunizing a suitable (i.e., immunocompetent) subject such as a rabbit, goat, mouse, or other mammal or vertebrate. An appropriate immunogenic preparation can contain, for example, recombinantly-expressed or chemically-synthesized polypeptides, e.g., a mutant ALK polypeptide of the disclosure, or a fragment thereof. The preparation can further include an adjuvant, such as Freund’s complete or incomplete adjuvant, or a similar immunostimulatory agent.

[0202] In some embodiments, an antibody provided herein is a polyclonal antibody. Methods of producing polyclonal antibodies are known in the art. In some embodiments, an antibody provided herein is a monoclonal antibody, wherein a population of the antibody molecules contain only one species of an antigen binding site capable of immunoreacting or binding with a particular epitope, e.g., an epitope on a mutant ALK polypeptide provided herein. Methods of preparation of monoclonal antibodies are known in the art, e.g., using standard hybridoma techniques originally described by Kohler and Milstein (1975) Nature 256:495- 497, human B cell hybridoma techniques (see Kozbor et al., 1983, Immunol. Today 4:72), EBV-hybridoma techniques (see Cole et al., pp. 77-96 In Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985), or trioma techniques. The technology for producing hybridomas is well known (see generally Current Protocols in Immunology, Coligan et al. ed., John Wiley & Sons, New York, 1994). A monoclonal antibody of the disclosure may also be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide of interest, e.g., a mutant ALK polypeptide provided herein or a fragment thereof. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable 67MOFO-358156824Docket No: 197102019040 for use in generating and screening antibody display libraries can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No. WO 92 / 18619; PCT Publication No. WO 91 / 17271; PCT Publication No. WO 92 / 20791; PCT Publication No. WO 92 / 15679; PCT Publication No. WO 93 / 01288; PCT Publication No. WO 92 / 01047; PCT Publication No. WO 92 / 09690; PCT Publication No. WO 90 / 02809; Fuchs et al. (1991) Bio / Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275- 1281; and Griffiths et al. (1993) EMBO J. 12:725-734. In some embodiments, monoclonal antibodies of the disclosure are recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions. Such chimeric and / or humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example, using methods described in PCT Publication No. WO 87 / 02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86 / 01533; U.S. Patent No. 4,816,567; European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521- 3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559; Morrison (1985) Science 229:1202-1207; Oi et al. (1986) Bio / Techniques 4:214; U.S. Patent 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060. In some embodiments, a monoclonal antibody of the disclosure is a human monoclonal antibody. In some embodiments, human monoclonal antibodies are prepared using methods known in the art, e.g., using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies, and protocols for producing such antibodies, see, e.g., U.S. Patent 5,625,126; U.S. Patent 5,633,425; U.S. Patent 5,569,825; U.S. Patent 5,661,016; and U.S. Patent 5,545,806.

[0203] In some embodiments, the antibody or antibody fragment of the disclosure is an isolated antibody or antibody fragment, which has been separated from a component of its natural environment or a cell culture used to produce the antibody or antibody fragment. In some embodiments, an antibody of the disclosure is purified to greater than 95% or 99% 68MOFO-358156824Docket No: 197102019040 purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods.

[0204] In some embodiments, an antibody of the disclosure can be used to isolate a mutant ALK polypeptide provided herein, or a fragment thereof, by standard techniques, such as affinity chromatography or immunoprecipitation. In some embodiments, an antibody of the disclosure can be used to detect a mutant ALK polypeptide provided herein, or a fragment thereof, e.g., in a tissue sample, cellular lysate, or cell supernatant, in order to evaluate the level and / or pattern of expression of the fusion polypeptide. Detection can be facilitated by coupling the antibody to a detectable substance. Thus, in some embodiments, an antibody of the disclosure is coupled to a detectable substance, such as enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Non-limiting examples of suitable enzymes include, e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include, e.g., streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include, e.g., umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes, but is not limited to, luminol; examples of bioluminescent materials include, e.g., luciferase, luciferin, and aequorin; and examples of suitable 125 131 35 3 radioactive materials include, e.g., I, I, S or H.

[0205] An antibody or antibody fragment of the disclosure may also be used diagnostically, e.g., to detect and / or monitor protein levels (e.g., protein levels of a mutant ALK polypeptide provided herein) in tissues or body fluids (e.g., in a tumor cell-containing tissue or body fluid), e.g., according to the methods provided herein.

[0206] In certain embodiments, an antibody provided herein has a dissociation constant (Kd) of ≤ 1μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10-8M or less, e.g., from 10-8M to 10-13M, e.g., from 10-9M to 10-13M). Methods of measuring antibody affinity (e.g., Kd) are known in the art, and include, without limitation, a radiolabeled antigen binding assay (RIA) and a BIACORE®surface plasmon resonance assay. In some embodiments, antibody affinity (e.g., Kd) is determined using the Fab version of an antibody of the disclosure and its antigen (e.g., a mutant ALK polypeptide provided herein). In some embodiments, a RIA is performed with the Fab version of an antibody of the disclosure and its antigen (e.g., a mutant ALK polypeptide provided herein). 69MOFO-358156824Docket No: 197102019040

[0207] In certain embodiments, an antibody provided herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, and single-chain antibody molecule (e.g., scFv) fragments, and other fragments described herein or known in the art.

[0208] In certain embodiments, an antibody provided herein is a diabody. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. In certain embodiments, an antibody provided herein is a triabody or a tetrabody.

[0209] In certain embodiments, an antibody provided herein is a single-domain antibody. Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody.

[0210] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody, as well as production by recombinant host cells (e.g., E. coli or phage), as known in the art and as described herein.

[0211] In certain embodiments, an antibody provided herein is a chimeric antibody. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey), and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody, in which the class or subclass of the antibody has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0212] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof), are derived from a non-human antibody, and framework regions (FRs) (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non- human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity. Humanized antibodies and methods of making them are known in the art. Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the "best-fit" method; framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions; human mature (somatically mutated) 70MOFO-358156824Docket No: 197102019040 framework regions or human germline framework regions; and framework regions derived from screening FR libraries.

[0213] In certain embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. For example, human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic animals, e.g., mice, the endogenous immunoglobulin loci have generally been inactivated. Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region. Human antibodies can also be made by hybridoma-based methods known in the art, e.g., using known human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies. Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are known in the art and described herein.

[0214] Antibodies of the disclosure may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage. Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, a naive antibody repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization. Naive libraries can also be made synthetically by cloning un-rearranged V- gene segments from stem cells, and using PCR primers containing random sequences to amplify the highly variable CDR3 regions and to accomplish rearrangement in vitro. 71MOFO-358156824Docket No: 197102019040 Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0215] In certain embodiments, an antibody provided herein is a multispecific antibody, e.g., a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites or at least two different antigens. For example, one of the binding specificities can be to a mutant ALK polypeptide of the disclosure, and the other can be to any other antigen. Multispecific antibodies can be prepared as full length antibodies or as antibody fragments. Techniques for making multispecific antibodies are known in the art and include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities, and “knob-in- hole” engineering. Multispecific antibodies may also be made by engineering electrostatic steering effects (e.g., by introducing mutations in the constant region) for making heterodimeric Fcs; cross-linking two or more antibodies or fragments; using leucine zippers to produce bispecific antibodies; using “diabody” technology for making bispecific antibody fragments; using single-chain Fv (scFv) dimers; and preparing trispecific antibodies. Engineered antibodies with three or more functional antigen binding sites, including “Octopus antibodies,” are also included in the disclosure. Antibodies or antibody fragments of the disclosure also include “Dual Acting FAbs” or “DAF,” e.g., comprising an antigen binding site that binds to a mutant ALK polypeptide of the disclosure as well as another, different antigen.

[0216] In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody of the disclosure may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, and / or insertions, and / or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final antibody, provided that the final antibody possesses the desired characteristics, e.g., antigen-binding.

[0217] In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Amino acid substitutions may be introduced into an antibody of interest, and the products may be screened for a desired activity, e.g., retained / improved antigen binding, 72MOFO-358156824Docket No: 197102019040 decreased immunogenicity, or improved or reduced antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0218] In certain embodiments, an antibody of the present disclosure is altered to increase or to decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence of the antibody, such that one or more glycosylation sites is created or removed. Antibody variants having bisected oligosaccharides are further provided, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. In some embodiments, antibody variants of the disclosure may have increased fucosylation. In some embodiments, antibody variants of the disclosure may have reduced fucosylation. In some embodiments, antibody variants of the disclosure may have improved ADCC function. In some embodiments, antibody variants of the disclosure may have decreased ADCC function. Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. In some embodiments, antibody variants of the disclosure may have increased CDC function. In some embodiments, antibody variants of the disclosure may have decreased CDC function.

[0219] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody of the present disclosure, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.

[0220] In certain embodiments, the present disclosure contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important, yet certain effector functions (such as CDC and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks Fc-gamma-R binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells that mediate ADCC, e.g., NK cells, express Fc-gamma-RIII only, whereas monocytes express Fc-gamma-RI, Fc-gamma-RII and Fc- gamma-RIII. Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329. Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 73MOFO-358156824Docket No: 197102019040 327, including the so-called “DANA” Fc mutant with substitutions of residues 265 and 297 to alanine. Antibody variants with improved or diminished binding to FcRs are also included in the disclosure. In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region. In some embodiments, numbering of Fc region residues is according to EU numbering of residues. In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) C1q binding and / or CDC. In some embodiments, antibodies of the disclosure include antibodies with increased half- lives and improved binding to the neonatal Fc receptor (FcRn), e.g., comprising one or more substitutions that improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434. See, also, Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0221] In certain embodiments, an antibody provided herein is a cysteine-engineered antibody, e.g., “thioMAb,” in which one or more residues of the antibody are substituted with cysteine residues. In some embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody, and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, e.g., to create an immunoconjugate, as described further herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies may be generated using any suitable method known in the art.

[0222] In some embodiments, an antibody or antibody fragment provided herein comprises a label or a tag. In some embodiments, the label or tag is a radiolabel, a fluorescent label, an enzymatic label, a sequence tag, biotin, or other ligands. Examples of labels or tags include, but are not limited to, 6xHis-tag, biotin-tag, Glutathione-S-transferase (GST)-tag, green fluorescent protein (GFP)-tag, c-myc-tag, FLAG-tag, Thioredoxin-tag, Glu-tag, Nus-tag, V5- tag, calmodulin-binding protein (CBP)-tag, Maltose binding protein (MBP)-tag, Chitin-tag, alkaline phosphatase (AP)-tag, HRP-tag, Biotin Caboxyl Carrier Protein (BCCP)-tag, Calmodulin-tag, S-tag, Strep-tag, haemoglutinin (HA)-tag, digoxigenin (DIG)-tag, DsRed, 74MOFO-358156824Docket No: 197102019040 RFP, Luciferase, Short Tetracysteine Tags, Halo-tag, and Nus-tag. In some embodiments, the label or tag comprises a detection agent, such as a fluorescent molecule or an affinity reagent or tag.

[0223] In some embodiments, an antibody or antibody fragment provided herein is conjugated to a drug molecule, e.g., an anti-cancer agent described herein, or a cytotoxic agent such as mertansine or monomethyl auristatin E (MMAE).

[0224] In certain embodiments, an antibody or antibody fragment provided herein may be further modified to contain additional nonproteinaceous moieties. Such moieties may be suitable for derivatization of the antibody, e.g., including but not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3- dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide / ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, polyethylene glycol propionaldehyde, and mixtures thereof. The polymers may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, the polymers can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, or whether the antibody derivative will be used in a therapy under defined conditions. In some embodiments, provided herein are antibodies conjugated to carbon nanotubes, e.g., for use in methods to selectively heat the antibody using radiation to a temperature at which cells proximal to the antibody are killed. (iv) Samples

[0225] A variety of materials can be the source of, or serve as, samples for use in any of the methods of the disclosure, such as the methods for detection of a mutant ALK nucleic acid molecule or polypeptide of the disclosure, or fragments thereof.

[0226] For example, the sample can be, or be derived from: solid tissue such as from a fresh, frozen and / or preserved organ, tissue sample, biopsy (e.g., tumor, tissue or liquid biopsy), resection, smear, or aspirate; scrapings; bone marrow or bone marrow specimens; a bone 75MOFO-358156824Docket No: 197102019040 marrow aspirate; blood or any blood constituents; blood cells; bodily fluids such as cerebrospinal fluid, amniotic fluid, urine, saliva, sputum, peritoneal fluid or interstitial fluid; pleural fluid; ascites; tissue or fine needle biopsy samples; surgical specimens; cell- containing body fluids; free-floating nucleic acids; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as ductal lavages or bronchoalveolar lavages; cells from any time in gestation or development of an individual; cells from a cancer or tumor; other body fluids, secretions, and / or excretions, and / or cells therefrom. In some embodiments, a sample is or comprises cells obtained from an individual. In some embodiments, the sample is or is derived from blood or blood constituents, e.g., obtained from a liquid biopsy. In some embodiments, the sample is or is derived from a tumor sample. In some embodiments, the sample is or comprises biological tissue or fluid. In some embodiments, the sample can contain compounds that are not naturally intermixed with the source of the sample in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics or the like. In some embodiments, the sample is preserved as a frozen sample or as a formaldehyde- or paraformaldehyde-fixed paraffin-embedded (FFPE) tissue preparation. In some embodiments, the sample comprises circulating tumor cells (CTCs).

[0227] In one embodiment, the sample comprises one or more cells associated with a tumor, e.g., tumor cells or tumor-infiltrating lymphocytes (TIL). In one embodiment, the sample includes one or more premalignant or malignant cells. In one embodiment, the sample is acquired from a hematologic malignancy (or pre-malignancy), e.g., a hematologic malignancy (or pre-malignancy) described herein. In one embodiment, the sample is acquired from a cancer, such as a cancer described herein. In some embodiments, the sample is acquired from a solid tumor, a soft tissue tumor or a metastatic lesion. In other embodiments, the sample includes tissue or cells from a surgical margin. In one embodiment, the sample is or is acquired from a liquid biopsy of blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some embodiments, the sample includes cell-free DNA (cfDNA) and / or circulating tumor DNA (ctDNA), e.g., from a biopsy of blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In another embodiment, the sample includes one or more circulating tumor cells (CTCs) (e.g., a CTC acquired from a blood sample). In one embodiment, the sample is a cell not associated with a tumor or cancer, e.g., a non-tumor or non-cancer cell or a peripheral blood lymphocyte.

[0228] In some embodiments, a sample is a primary sample obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by a method chosen from biopsy (e.g., fine needle aspiration or 76MOFO-358156824Docket No: 197102019040 tissue biopsy), surgery, or collection of body fluid (e.g., blood, lymph, or feces). In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. Such a processed sample may comprise, for example, nucleic acids (e.g., for use in any of the methods for detection of mutant ALK nucleic acid molecules provided herein) or proteins (e.g., for use in any of the methods for detection of mutant ALK polypeptides provided herein) extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification methods, reverse transcription of mRNA, or isolation and / or purification of certain components such as nucleic acids and / or proteins.

[0229] In some embodiments, the sample comprises nucleic acids, e.g., genomic DNA, cDNA, or mRNA. In some embodiments, the sample comprises cell-free DNA (cfDNA). In some embodiments, the sample comprises cell-free RNA (cfRNA). In some embodiments, the sample comprises circulating tumor DNA (ctDNA). In certain embodiments, the nucleic acids are purified or isolated (e.g., removed from their natural state). In some embodiments, the sample comprises tumor or cancer nucleic acids, such as nucleic acids from a tumor or cancer sample, e.g., genomic DNA, RNA, or cDNA derived from RNA, or from a liquid biopsy, e.g., ctDNA from blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In certain embodiments, a tumor or cancer nucleic acid sample, or a ctDNA sample, is purified or isolated (e.g., it is removed from its natural state).

[0230] In some embodiments, the sample comprises tumor or cancer proteins or polypeptides, such as proteins or polypeptides from a tumor or a cancer sample, or from a liquid biopsy, e.g., from blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In certain embodiments, the proteins or polypeptides are purified or isolated (e.g., removed from their natural state).

[0231] In some embodiments, the sample is obtained from an individual having a cancer, such as a cancer described herein. In some embodiments, the sample comprises a mutant ALK nucleic acid molecule or polypeptide of the disclosure.

[0232] In some embodiments, the sample is a control sample or a reference sample, e.g., not containing a mutant ALK nucleic acid molecule or polypeptide described herein. In certain embodiments, the reference sample is purified or isolated (e.g., it is removed from its natural state). In certain embodiments, the reference or control sample comprises a wild type or a non-mutated nucleic acid molecule or polypeptide counterpart to any of the mutant ALK nucleic acid molecules or polypeptides described herein. In other embodiments, the 77MOFO-358156824Docket No: 197102019040 reference sample is from a non-tumor or cancer sample, e.g., a blood control, a normal adjacent tumor (NAT), or any other non-cancerous sample from the same or a different individual.

[0233] In some embodiments, a mutant ALK nucleic acid molecule of the disclosure is detected in a sample comprising genomic or subgenomic DNA fragments, or RNA (e.g., mRNA), isolated from a sample, e.g., a tumor or cancer sample, a normal adjacent tissue (NAT) sample, a tissue sample, or a blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva sample obtained from an individual. In some embodiments, the sample comprises cDNA derived from an mRNA sample or from a sample comprising mRNA. In some embodiments, a mutant ALK nucleic acid molecule of the disclosure is detected in a sample comprising cell-free DNA (cfDNA), cell-free RNA, and / or circulating tumor DNA (ctDNA). In some embodiments, a mutant ALK nucleic acid molecule of the disclosure is detected in a sample comprising cell-free DNA (cfDNA) and / or circulating tumor DNA (ctDNA). In some embodiments, a mutant ALK nucleic acid molecule of the disclosure is detected in a sample comprising circulating tumor DNA (ctDNA). C. Anti-Cancer Therapies

[0234] Certain aspects of the present disclosure relate to anti-cancer therapies, as well as methods for identifying an individual having a cancer who may benefit from a treatment comprising an anti-cancer therapy; selecting a treatment for an individual having a cancer; identifying one or more treatment options for an individual having a cancer; predicting survival of an individual having a cancer; treating or delaying progression of cancer; monitoring, evaluating or screening an individual having a cancer; detecting the presence or absence of a cancer in an individual; monitoring progression or recurrence of a cancer in an individual; or identifying a candidate treatment for a cancer in an individual in need thereof. The present disclosure also provides uses for anti-cancer therapies (e.g., in methods of treating or delaying progression of cancer in an individual, or in methods for manufacturing a medicament for treating or delaying progression of cancer). In some instances, the methods of the disclosure can include administering an anti-cancer therapy or applying an anti-cancer therapy to an individual based on a generated genomic and / or sequencing mutation profile. An anti-cancer therapy can refer to a compound that is effective in the treatment of cancer cells. Examples of anti-cancer agents or anti-cancer therapies include, but not limited to, ALK-targeted therapies (e.g., TKIs) alkylating agents, antimetabolites, natural products, hormones, chemotherapy, radiation therapy, immunotherapy, surgery, or a therapy configured 78MOFO-358156824Docket No: 197102019040 to target a defect in a specific cell signaling pathway, e.g., a defect in a DNA mismatch repair (MMR) pathway.

[0235] In some embodiments, an anti-cancer therapy of the disclosure is a small molecule inhibitor, an antibody, a cellular therapy, a nucleic acid, a virus-based therapy, an antibody- drug conjugate, a recombinant protein, a fusion protein, a natural compound, a peptide, a PROteolysis-TArgeting Chimera (PROTAC), a treatment for cancer comprising a mutant ALK nucleic acid molecule or polypeptide of the disclosure, a treatment for cancer being tested in a clinical trial, a targeted therapy, a treatment being tested in a clinical trial for cancer comprising a mutant ALK nucleic acid molecule or polypeptide of the disclosure, or any combination thereof, e.g., a described in further detail below. In some embodiments, the anti-cancer therapy is an ALK-targeted therapy. In some embodiments, the anti-cancer therapy is a kinase inhibitor, such as a kinase inhibitor described herein or known in the art. In some embodiments, the kinase inhibitor is a multi-kinase inhibitor or an ALK-specific inhibitor known in the art or described herein. In some embodiments, the nucleic acid inhibits the expression of a mutant ALK nucleic acid molecule or polypeptide of the disclosure.

[0236] In some embodiments, an anti-cancer therapy of the disclosure is an ALK-targeted therapy, e.g., as described herein or known in the art. In some embodiments, the ALK- targeted therapy is a small molecule inhibitor, an antibody, a cellular therapy, a nucleic acid, a virus-based therapy, an antibody-drug conjugate, a recombinant protein, a fusion protein, a natural compound, a peptide, a PROteolysis-TArgeting Chimera (PROTAC), a treatment for ALK-positive or ALK-rearranged cancer, an ALK-targeted therapy being tested in a clinical trial, a treatment for ALK-positive or ALK-rearranged cancer being tested in a clinical trial, or any combination thereof. In some embodiments, the ALK-targeted therapy is a kinase inhibitor known in the art or described herein. In some embodiments, the ALK-targeted therapy is a tyrosine kinase inhibitor known in the art or described herein. In some embodiments, the ALK-targeted therapy is a multi-kinase inhibitor or an ALK-specific inhibitor known in the art or described herein. In some embodiments, the kinase inhibitor inhibits the kinase activity of an ALK polypeptide. In some embodiments, the ALK-targeted therapy comprises one or more of crizotinib, alectinib, ceritinib, lorlatinib, brigatinib, ensartinib (X-396), repotrectinib (TPX-005), entrectinib (RXDX-101), AZD3463, CEP- 37440, belizatinib (TSR-011), gilteritinib, XMU-MP-5, NVL-655, ASP3026, KRCA-0008, TQ-B3139, TPX-0131, TAE684 (NVP-TAE684), CT-707, WX-0593, alkotinib, SIM1803- 1A, PLB1003, SAF-189s, PF03446962, TQ-B3101, APG-2449, X-376, CEP-28122, and 79MOFO-358156824Docket No: 197102019040 GSK1838705A. In some embodiments, the nucleic acid inhibits the expression of a mutant ALK nucleic acid molecule or polypeptide of the disclosure.

[0237] In some embodiments, an anti-cancer therapy of the disclosure (e.g., an ALK-targeted therapy) is administered in combination with an additional anti-cancer therapy. In some embodiments, the additional anti-cancer therapy is any anti-cancer therapy known in the art or described herein. In some embodiments, the additional anti-cancer therapy comprises one or more of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti-angiogenic therapy, an anti-DNA repair therapy, an anti-inflammatory therapy, an anti-neoplastic agent, a growth inhibitory agent, a cytotoxic agent, a vaccine, a small molecule agonist, a virus- based therapy, an antibody-drug conjugate, a recombinant protein, a fusion protein, a natural compound, a peptide, a PROteolysis-TArgeting Chimera (PROTAC), or any combination thereof.

[0238] In some embodiments, an anti-cancer therapy of the disclosure comprises a cyclin- dependent kinase (CDK) inhibitor, e.g., alone or in combination with an ALK-targeted therapy. In some embodiments, the CDK inhibitor inhibits CDK4. In some embodiments, the CDK inhibitor inhibits Cyclin D / CDK4. In some embodiments, the CDK inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of CDK4, (b) an antibody that inhibits one or more activities of CDK4 (e.g., by binding to and inhibiting one or more activities of CDK4, binding to and inhibiting expression of CDK4, and / or binding to and inhibiting one or more activities of a cell expressing CDK4, such as by inducing antibody- dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of CDK4 (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapeutic, and the like). In some embodiments, the CDK inhibitor inhibits CDK4 and CDK6. In some embodiments, the CDK inhibitor is a small molecule inhibitor of CDK4 (e.g., a competitive or non-competitive inhibitor). Non-limiting examples of CDK inhibitors include palbociclib, ribociclib, and abemaciclib, as well as pharmaceutically acceptable salts thereof.

[0239] In some embodiments, an anti-cancer therapy of the disclosure comprises a murine double minute 2 homolog (MDM2) inhibitor, e.g., alone or in combination with an ALK- targeted therapy. In some embodiments, the MDM2 inhibitor is (a) a small molecule that inhibits one or more activities of MDM2 (e.g., binding to p53), (b) an antibody that inhibits one or more activities of MDM2 (e.g., by binding to and inhibiting one or more activities of MDM2, binding to and inhibiting expression of MDM2, and / or binding to and inhibiting one 80MOFO-358156824Docket No: 197102019040 or more activities of a cell expressing MDM2, such as by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of MDM2 (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapeutic, and the like). In some embodiments, the MDM2 inhibitor is a small molecule inhibitor of MDM2 (e.g., a competitive or non-competitive inhibitor). Non- limiting examples of MDM2 inhibitors include nutlin-3a, RG7112, idasanutlin (RG7388), AMG-232, MI-63, MI-291, MI-391, MI-77301 (SAR405838), APG-115, DS-3032b, NVP- CGM097, and HDM-201 (siremadlin), as well as pharmaceutically acceptable salts thereof. In some embodiments, the MDM2 inhibitor inhibits or disrupts interaction between MDM2 and p53.

[0240] In some embodiments, an anti-cancer therapy of the disclosure comprises (alone or in combination with an ALK-targeted therapy) one or more of an antimetabolite, DNA- damaging agent, or platinum-containing therapeutic (e.g., 5-azacitadine, 5-fluorouracil, acadesine, busulfan, carboplatin, cisplatin, chlorambucil, CPT-11, cytarabine, daunorubicin, decitabine, doxorubicin, etoposide, fludarabine, gemcitabine, idarubicin, radiation, oxaliplatin, temozolomide, topotecan, trabectedin, GSK2830371, or rucaparib); a pro- apoptotic agent (e.g., a BCL2 inhibitor or downregulator, SMAC mimetic, or TRAIL agonist such as ABT-263, ABT-737, oridonin, venetoclax, combination of venetoclax and an anti- CD20 antibody such as obinutuzumab or rituximab, 1396-11, ABT-10, SM-164, D269H / E195R, or rhTRAIL); a tyrosine kinase inhibitor (e.g., as described herein); an inhibitor of RAS, RAF, MEK, or the MAPK pathway (e.g., AZD6244, dabrafenib, LGX818, PD0325901, pimasertib, trametinib, or vemurafenib); an inhibitor of PI3K, mTOR, or Akt (e.g., as described herein); a CDK inhibitor (e.g., as described herein); a PKC inhibitor (e.g., LXS196 or sotrastaurin); an antibody-based therapeutic (e.g., an anti-PD-1 or anti-PDL1 antibody such as atezolizumab, pembrolizumab, nivolumab, or spartalizumab; an anti-CD20 antibody such as obinutuzumab or rituximab; or an anti-DR5 antibody such as drozitumab); a proteasome inhibitor (e.g., bortezomib, carfilzomib, ixazomib, or MG-132); an HDAC inhibitor (e.g., SAHA or VPA); an antibiotic (e.g., actinomycin D); a zinc-containing therapeutic (e.g., zinc or ZMC1); an HSP inhibitor (e.g., geldanamycin); an ATPase inhibitor (e.g., archazolid); a mitotic inhibitor (e.g., paclitaxel or vincristine); metformin; methotrexate; tanshinone IIA; and / or P5091.

[0241] In some embodiments, an anti-cancer therapy of the disclosure comprises a tyrosine kinase inhibitor, e.g., alone or in combination with an ALK-targeted therapy. In some embodiments, the tyrosine kinase inhibitor is an ALK-targeted anti-cancer therapy or 81MOFO-358156824Docket No: 197102019040 treatment. In some embodiments, the tyrosine kinase inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of a tyrosine kinase, (b) an antibody that inhibits one or more activities of a tyrosine kinase (e.g., by binding to and inhibiting one or more activities of the tyrosine kinase, binding to and inhibiting expression, such as cell surface expression, of the tyrosine kinase, and / or binding to and inhibiting one or more activities of a cell expressing the tyrosine kinase, such as by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of a tyrosine kinase (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR- based therapeutic, and the like). In some embodiments, the tyrosine kinase inhibitor is a small molecule inhibitor of a tyrosine kinase (e.g., a competitive or non-competitive inhibitor). Non-limiting examples of tyrosine kinase inhibitors include imatinib, crenolanib, linifanib, ninetedanib, axitinib, dasatinib, imetelstat, midostaurin, pazopanib, sorafenib, sunitinb, motesanib, masitinib, vatalanib, cabozanitinib, tivozanib, OSI-930, Ki8751, telatinib, dovitinib, tyrphostin AG 1296, and amuvatinib, as well as pharmaceutically acceptable salts thereof.

[0242] In some embodiments, an anti-cancer therapy of the disclosure comprises a mitogen- activated protein kinase (MEK) inhibitor, e.g., alone or in combination with an ALK-targeted therapy. In some embodiments, the MEK inhibitor inhibits one or more activities of MEK1 and / or MEK2. In some embodiments, the anti-cancer therapy / MEK inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of MEK, (b) an antibody that inhibits one or more activities of MEK (e.g., by binding to and inhibiting one or more activities of MEK, binding to and inhibiting expression of MEK, and / or binding to and inhibiting one or more activities of a cell expressing MEK, such as by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of MEK (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapeutic, and the like). In some embodiments, the MEK inhibitor is a small molecule inhibitor of MEK (e.g., a competitive or non-competitive inhibitor). Non-limiting examples of MEK inhibitors include trametinib, cobimetinib, binimetinib, CI-1040, PD0325901, selumetinib, AZD8330, TAK-733, GDC-0623, refametinib, pimasertib, RO4987655, RO5126766, WX-544, and HL-085, as well as pharmaceutically acceptable salts thereof. In some embodiments, the anti-cancer therapy inhibits one or more activities of the Raf / MEK / ERK pathway, including inhibitors of Raf, MEK, and / or ERK.

[0243] In some embodiments, an anti-cancer therapy of the disclosure comprises a mammalian target of rapamycin (mTOR) inhibitor, e.g., alone or in combination with an 82MOFO-358156824Docket No: 197102019040 ALK-targeted therapy. In some embodiments, the mTOR inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of mTOR, (b) an antibody that inhibits one or more activities of mTOR (e.g., by binding to and inhibiting one or more activities of mTOR, binding to and inhibiting expression of mTOR, and / or binding to and inhibiting one or more activities of a cell expressing mTOR, such as by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of mTOR (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapeutic, and the like). In some embodiments, the mTOR inhibitor is a small molecule inhibitor of mTOR (e.g., a competitive inhibitor, such as an ATP-competitive inhibitor, or a non-competitive inhibitor, such as a rapamycin analog). Non-limiting examples of mTOR inhibitors include temsirolimus, everolimus, ridaforolimus, dactolisib, GSK2126458, XL765, AZD8055, AZD2014, MLN128, PP242, NVP-BEZ235, LY3023414, PQR309, PKI587, and OSI027, as well as pharmaceutically acceptable salts thereof. In some embodiments, the anti- cancer therapy inhibits one or more activities of the Akt / mTOR pathway, including inhibitors of Akt and / or mTOR.

[0244] In some embodiments, an anti-cancer therapy of the disclosure comprises a PI3K inhibitor or Akt inhibitor, e.g., alone or in combination with an ALK-targeted therapy. In some embodiments, the PI3K inhibitor inhibits one or more activities of PI3K. In some embodiments, the anti-cancer therapy / PI3K inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of PI3K, (b) an antibody that inhibits one or more activities of PI3K (e.g., by binding to and inhibiting one or more activities of PI3K, binding to and inhibiting expression of PI3K, and / or binding to and inhibiting one or more activities of a cell expressing PI3K, such as by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of PI3K (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapeutic, and the like). In some embodiments, the PI3K inhibitor is a small molecule inhibitor of PI3K (e.g., a competitive or non-competitive inhibitor). Non-limiting examples of PI3K inhibitors include GSK2636771, buparlisib (BKM120), AZD8186, copanlisib (BAY80-6946), LY294002, PX- 866, TGX115, TGX126, BEZ235, SF1126, idelalisib (GS-1101, CAL-101), pictilisib (GDC- 094), GDC0032, IPI145, INK1117 (MLN1117), SAR260301, KIN-193 (AZD6482), duvelisib, GS-9820, GSK2636771, GDC-0980, AMG319, pazobanib, and alpelisib (BYL719, Piqray), as well as pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor inhibits one or more activities of AKT (e.g., AKT1). In some embodiments, the AKT inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of 83MOFO-358156824Docket No: 197102019040 AKT1, (b) an antibody that inhibits one or more activities of AKT1 (e.g., by binding to and inhibiting one or more activities of AKT1, binding to and inhibiting expression of AKT1, and / or binding to and inhibiting one or more activities of a cell expressing AKT1, such as by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of AKT1 (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapeutic, and the like). In some embodiments, the AKT1 inhibitor is a small molecule inhibitor of AKT1 (e.g., a competitive or non-competitive inhibitor). Non-limiting examples of AKT1 inhibitors include GSK690693, GSK2141795 (uprosertib), GSK2110183 (afuresertib), AZD5363, GDC-0068 (ipatasertib), AT7867, CCT128930, MK-2206, BAY 1125976, AKT1 and AKT2-IN-1, perifosine, and VIII, as well as pharmaceutically acceptable salts thereof. In some embodiments, the AKT1 inhibitor is a pan-Akt inhibitor.

[0245] In some embodiments, an anti-cancer therapy of the disclosure comprises a hedgehog (Hh) inhibitor, e.g., alone or in combination with an ALK-targeted therapy. In some embodiments, the Hh inhibitor is (a) a small molecule that inhibits one or more enzymatic activities of Hh, (b) an antibody that inhibits one or more activities of Hh (e.g., by binding to and inhibiting one or more activities of Hh, binding to and inhibiting expression of Hh, and / or binding to and inhibiting one or more activities of a cell expressing Hh, such as by inducing antibody-dependent cellular cytotoxicity, ADCC, or phagocytosis, ADCP), or (c) a nucleic acid that inhibits expression of Hh (e.g., an antisense oligonucleotide, miRNA, siRNA, morpholino, CRISPR-based therapeutic, and the like). In some embodiments, the Hh inhibitor is a small molecule inhibitor of Hh (e.g., a competitive or non-competitive inhibitor). Non-limiting examples of Hh inhibitors include sonidegib, vismodegib, erismodegib, saridegib, BMS833923, PF-04449913, and LY2940680, as well as pharmaceutically acceptable salts thereof.

[0246] In some embodiments, an anti-cancer therapy of the disclosure comprises a heat shock protein (HSP) inhibitor, a MYC inhibitor, an HDAC inhibitor, an immunotherapy, a neoantigen, a vaccine, or a cellular therapy, e.g., alone or in combination with an ALK- targeted therapy.

[0247] In some embodiments, the anti-cancer therapy comprises one or more of an immune checkpoint inhibitor, a chemotherapy, a VEGF inhibitor, an Integrin β3 inhibitor, a statin, an EGFR inhibitor, an mTOR inhibitor, a PI3K inhibitor, a MAPK inhibitor, or a CDK4 / 6 inhibitor, e.g., alone or in combination with an ALK-targeted therapy. 84MOFO-358156824Docket No: 197102019040

[0248] In some embodiments, the anti-cancer therapy comprises a kinase inhibitor, e.g., alone or in combination with an ALK-targeted therapy.

[0249] In some embodiments, the anti-cancer therapy comprises a heat shock protein (HSP) inhibitor, e.g., alone or in combination with an ALK-targeted therapy. In some embodiments, the HSP inhibitor is a Pan-HSP inhibitor, such as KNK423. In some embodiments, the HSP inhibitor is an HSP70 inhibitor, such as cmHsp70.1, quercetin, VER155008, or 17-AAD. In some embodiments, the HSP inhibitor is a HSP90 inhibitor. In some embodiments, the HSP90 inhibitor is 17-AAD, Debio0932, ganetespib (STA-9090), retaspimycin hydrochloride (retaspimycin, IPI-504), AUY922, alvespimycin (KOS-1022, 17-DMAG), tanespimycin (KOS-953, 17-AAG), DS 2248, or AT13387 (onalespib). In some embodiments, the HSP inhibitor is an HSP27 inhibitor, such as Apatorsen (OGX-427).

[0250] In some embodiments, the anti-cancer therapy comprises a MYC inhibitor, e.g., alone or in combination with an ALK-targeted therapy. In some embodiments, the MYC inhibitor is MYCi361 (NUCC-0196361), MYCi975 (NUCC-0200975), Omomyc (dominant negative peptide), ZINC16293153 (Min9), 10058-F4, JKY-2-169, 7594-0035, or inhibitors of MYC / MAX dimerization and / or MYC / MAX / DNA complex formation.

[0251] In some embodiments, the anti-cancer therapy comprises a histone deacetylase (HDAC) inhibitor, e.g., alone or in combination with an ALK-targeted therapy. In some embodiments, the HDAC inhibitor is belinostat (PXD101), SAHA (vorinostat, suberoylanilide hydroxamine), panobinostat (LBH589, LAQ-824), ACY1215 (Rocilinostat), quisinostat (JNJ-26481585), abexinostat (PCI-24781), pracinostat (SB939), givinostat (ITF2357), resminostat (4SC-201), trichostatin A (TSA), MS-275 (etinostat), Romidepsin (depsipeptide, FK228), MGCD0103 (mocetinostat), BML-210, CAY10603, valproic acid, MC1568, CUDC-907, CI-994 (Tacedinaline), Pivanex (AN-9), AR-42, Chidamide (CS055, HBI-8000), CUDC-101, CHR-3996, MPT0E028, BRD8430, MRLB-223, apicidin, RGFP966, BG45, PCI-34051, C149 (NCC149), TMP269, Cpd2, T247, T326, LMK235, C1A, HPOB, Nexturastat A, Befexamac, CBHA, Phenylbutyrate, MC1568, SNDX275, Scriptaid, Merck60, PX089344, PX105684, PX117735, PX117792, PX117245, PX105844, compound 12 as described by Li et al., Cold Spring Harb Perspect Med (2016) 6(10):a026831, or PX117445.

[0252] In some embodiments, the anti-cancer therapy comprises a VEGF inhibitor, e.g., alone or in combination with an ALK-targeted therapy. In some embodiments, the VEGF inhibitor is Bevacizumab, BMS-690514, ramucirumab, pazopanib, sorafenib, sunitinib, 85MOFO-358156824Docket No: 197102019040 golvatinib, vandetanib, cabozantinib, levantinib, axitinib, cediranib, tivozanib, lucitanib, semaxanib, nindentanib, regorafinib, or aflibercept.

[0253] In some embodiments, the anti-cancer therapy comprises an integrin β3 inhibitor, e.g., alone or in combination with an ALK-targeted therapy. In some embodiments, the integrin β3 inhibitor is anti-avb3 (clone LM609), cilengitide (EMD121974, NSC, 707544), an siRNA, GLPG0187, MK-0429, CNTO95, TN-161, etaracizumab (MEDI-522), intetumumab (CNTO95) (anti-alphaV subunit antibody), abituzumab (EMD 525797 / DI17E6) (anti-alphaV subunit antibody), JSM6427, SJ749, BCH-15046, SCH221153, or SC56631. In some embodiments, the anti-cancer therapy comprises an αIIbβ3 integrin inhibitor, e.g., alone or in combination with an ALK-targeted therapy. In some embodiments, the αIIbβ3 integrin inhibitor is abciximab, eptifibatide, or tirofiban.

[0254] In some embodiments, the anti-cancer therapy comprises an mTOR inhibitor, e.g., alone or in combination with an ALK-targeted therapy. In some embodiments, the mTOR inhibitor is temsirolimus (CCI-779), KU-006379, PP242, Torin1, Torin2, ICSN3250, Rapalink-1, CC-223, sirolimus (rapamycin), everolimus (RAD001), dactosilib (NVP- BEZ235), GSK2126458, WAY-001, WAY-600, WYE-687, WYE-354, SF1126, XL765, INK128 (MLN012), AZD8055, OSI027, AZD2014, or AP-23573.

[0255] In some embodiments, the anti-cancer therapy comprises a statin or a statin-based agent, e.g., alone or in combination with an ALK-targeted therapy. In some embodiments, the statin or statin-based agent is simvastatin, atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, or cerivastatin.

[0256] In some embodiments, the anti-cancer therapy comprises a MAPK inhibitor, e.g., alone or in combination with an ALK-targeted therapy. In some embodiments, the MAPK inhibitor is SB203580, SKF-86002, BIRB-796, SC-409, RJW-67657, BIRB-796, VX-745, RO3201195, SB-242235, or MW181.

[0257] In some embodiments, the anti-cancer therapy comprises an EGFR inhibitor, e.g., alone or in combination with an ALK-targeted therapy. In some embodiments, the EGFR inhibitor is cetuximab, panitumumab, lapatinib, gefitinib, vandetanib, dacomitinib, icotinib, osimertinib (AZD9291), afatanib, olmutinib, EGF816 (nazartinib), avitinib (AC0010), rociletinib (CO-1686), BMS-690514, YH5448, PF-06747775, ASP8273, PF299804, AP26113, necitumumab, or erlotinib. In some embodiments, the EGFR inhibitor is gefitinib or cetuximab.

[0258] In some embodiments, the anti-cancer therapy comprises a cancer immunotherapy, such as a checkpoint inhibitor, cancer vaccine, cell-based therapy, T cell receptor (TCR)- 86MOFO-358156824Docket No: 197102019040 based therapy, adjuvant immunotherapy, cytokine immunotherapy, and oncolytic virus therapy, e.g., alone or in combination with an ALK-targeted therapy. In some embodiments, the cancer immunotherapy comprises a small molecule, nucleic acid, polypeptide, carbohydrate, toxin, cell-based agent, or cell-binding agent. Examples of cancer immunotherapies are described in greater detail herein but are not intended to be limiting. In some embodiments, the cancer immunotherapy activates one or more aspects of the immune system to attack a cell (e.g., a tumor cell) that expresses a neoantigen, e.g., a neoantigen corresponding to a fusion nucleic acid molecule or polypeptide of the disclosure. The cancer immunotherapies of the present disclosure are contemplated for use as monotherapies, or in combination approaches comprising two or more in any combination or number, subject to medical judgement. Any of the cancer immunotherapies (optionally as monotherapies or in combination with another cancer immunotherapy or other therapeutic agent described herein) may find use in any of the methods described herein.

[0259] In some embodiments, the cancer immunotherapy comprises a cancer vaccine, e.g., alone or in combination with an ALK-targeted therapy. A range of cancer vaccines have been tested that employ different approaches to promoting an immune response against a cancer (see, e.g., Emens L A, Expert Opin Emerg Drugs 13(2): 295-308 (2008) and US20190367613). Approaches have been designed to enhance the response of B cells, T cells, or professional antigen-presenting cells against tumors. Exemplary types of cancer vaccines include, but are not limited to, DNA-based vaccines, RNA-based vaccines, virus transduced vaccines, peptide-based vaccines, dendritic cell vaccines, oncolytic viruses, whole tumor cell vaccines, tumor antigen vaccines, etc. In some embodiments, the cancer vaccine can be prophylactic or therapeutic. In some embodiments, the cancer vaccine is formulated as a peptide-based vaccine, a nucleic acid-based vaccine, an antibody based vaccine, or a cell based vaccine. For example, a vaccine composition can include naked cDNA in cationic lipid formulations; lipopeptides (e.g., Vitiello, A. et al, J. Clin. Invest. 95:341, 1995), naked cDNA or peptides, encapsulated e.g., in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge, et ah, Molec. Immunol. 28:287-294, 1991: Alonso et al, Vaccine 12:299- 306, 1994; Jones et al, Vaccine 13:675-681, 1995); peptide composition contained in immune stimulating complexes (ISCOMS) (e.g., Takahashi et al, Nature 344:873-875, 1990; Hu et al, Clin. Exp. Immunol. 113:235-243, 1998); or multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J.P., J. Immunol. Methods 196: 17-32, 1996). In some embodiments, a cancer vaccine is formulated as a peptide-based vaccine, or nucleic acid based vaccine in which the nucleic acid encodes the 87MOFO-358156824Docket No: 197102019040 polypeptides. In some embodiments, a cancer vaccine is formulated as an antibody- based vaccine. In some embodiments, a cancer vaccine is formulated as a cell based vaccine. In some embodiments, the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine. In some embodiments, the cancer vaccine is a multivalent long peptide, a multiple peptide, a peptide mixture, a hybrid peptide, or a peptide pulsed dendritic cell vaccine (see, e.g., Yamada et al, Cancer Sci, 104: 14-21, 2013). In some embodiments, such cancer vaccines augment the anti-cancer response.

[0260] In some embodiments, the cancer vaccine comprises a polynucleotide that encodes a neoantigen, e.g., a neoantigen corresponding to a mutant ALK nucleic acid molecule or polypeptide of the disclosure. In some embodiments, the cancer vaccine further comprises one or more additional antigens, neoantigens, or other sequences that promote antigen presentation and / or an immune response. In some embodiments, the polynucleotide is complexed with one or more additional agents, such as a liposome or lipoplex. In some embodiments, the polynucleotide(s) are taken up and translated by antigen presenting cells (APCs), which then present the neoantigen(s) via MHC class I on the APC cell surface.

[0261] In some embodiments, the cancer vaccine is selected from sipuleucel-T, which has been approved for treatment of asymptomatic, or minimally symptomatic metastatic castrate- resistant (hormone-refractory) prostate cancer; and talimogene laherparepvec, a genetically modified oncolytic viral therapy approved for treatment of unresectable cutaneous, subcutaneous and nodal lesions in melanoma. In some embodiments, the cancer vaccine is selected from an oncolytic viral therapy such as pexastimogene devacirepvec (PexaVec / JX- 594, SillaJen / formerly Jennerex Biotherapeutics), a thymidine kinase- (TK-) deficient vaccinia virus engineered to express GM-CSF, for hepatocellular carcinoma (NCT02562755) and melanoma (NCT00429312); pelareorep, a variant of respiratory enteric orphan virus (reovirus) which does not replicate in cells that are not RAS-activated, in numerous cancers, including colorectal cancer (NCT01622543), prostate cancer (NCT01619813), head and neck squamous cell cancer (NCT01166542), pancreatic adenocarcinoma (NCT00998322), and non-small cell lung cancer (NSCLC) (NCT 00861627); enadenotucirev (NG-348, PsiOxus, formerly known as ColoAdl), an adenovirus engineered to express a full length CD80 and an antibody fragment specific for the T-cell receptor CD3 protein, in ovarian cancer (NCT02028117), metastatic or advanced epithelial tumors such as in colorectal cancer, bladder cancer, head and neck squamous cell carcinoma and salivary gland cancer (NCT02636036); ONCOS-102 (Targovax / formerly Oncos), an adenovirus engineered to express GM-CSF, in melanoma (NCT03003676), and peritoneal disease, colorectal cancer or 88MOFO-358156824Docket No: 197102019040 ovarian cancer (NCT02963831); GL-ONC1 (GLV-1h68 / GLV-1h153, Genelux GmbH), vaccinia viruses engineered to express beta-galactosidase (beta-gal) / beta-glucoronidase or beta-gal / human sodium iodide symporter (hNIS), respectively, were studied in peritoneal carcinomatosis (NCT01443260), fallopian tube cancer, ovarian cancer (NCT 02759588); or CG0070 (Cold Genesys), an adenovirus engineered to express GM-CSF in bladder cancer (NCT02365818); anti-gp100; STINGVAX; GVAX; DCVaxL; and DNX-2401. In some embodiments, the cancer vaccine is selected from JX-929 (SillaJen / formerly Jennerex Biotherapeutics), a TK- and vaccinia growth factor-deficient vaccinia virus engineered to express cytosine deaminase, which is able to convert the prodrug 5-fluorocytosine to the cytotoxic drug 5-fluorouracil; TGO1 and TG02 (Targovax / formerly Oncos), peptide-based immunotherapy agents targeted for difficult-to-treat RAS mutations; and TILT-123 (TILT Biotherapeutics), an engineered adenovirus designated: Ad5 / 3-E2F-delta24-hTNFα-IRES- hIL20; and VSV-GP (ViraTherapeutics) a vesicular stomatitis virus (VSV) engineered to express the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), which can be further engineered to express antigens designed to raise an antigen-specific CD8+ T cell response. In some embodiments, the cancer vaccine comprises a vector-based tumor antigen vaccine. Vector-based tumor antigen vaccines can be used as a way to provide a steady supply of antigens to stimulate an anti-tumor immune response. In some embodiments, vectors encoding for tumor antigens are injected into an individual (possibly with pro- inflammatory or other attractants such as GM-CSF), taken up by cells in vivo to make the specific antigens, which then provoke the desired immune response. In some embodiments, vectors may be used to deliver more than one tumor antigen at a time, to increase the immune response. In addition, recombinant virus, bacteria or yeast vectors can trigger their own immune responses, which may also enhance the overall immune response.

[0262] In some embodiments, the cancer vaccine comprises a DNA-based vaccine. In some embodiments, DNA-based vaccines can be employed to stimulate an anti-tumor response. The ability of directly injected DNA that encodes an antigenic protein, to elicit a protective immune response has been demonstrated in numerous experimental systems. Vaccination through directly injecting DNA that encodes an antigenic protein, to elicit a protective immune response often produces both cell-mediated and humoral responses. Moreover, reproducible immune responses to DNA encoding various antigens have been reported in mice that last essentially for the lifetime of the animal (see, e.g., Yankauckas et al. (1993) DNA Cell Biol., 12: 771-776). In some embodiments, plasmid (or other vector) DNA that includes a sequence encoding a protein operably linked to regulatory elements required 89MOFO-358156824Docket No: 197102019040 for gene expression is administered to individuals (e.g. human patients, non-human mammals, etc.). In some embodiments, the cells of the individual take up the administered DNA and the coding sequence is expressed. In some embodiments, the antigen so produced becomes a target against which an immune response is directed.

[0263] In some embodiments, the cancer vaccine comprises an RNA-based vaccine. In some embodiments, RNA-based vaccines can be employed to stimulate an anti-tumor response. In some embodiments, RNA-based vaccines comprise a self-replicating RNA molecule. In some embodiments, the self-replicating RNA molecule may be an alphavirus-derived RNA replicon. Self-replicating RNA (or "SAM") molecules are well known in the art and can be produced by using replication elements derived from, e.g., alphaviruses, and substituting the structural viral proteins with a nucleotide sequence encoding a protein of interest. A self- replicating RNA molecule is typically a +-strand molecule which can be directly translated after delivery to a cell, and this translation provides a RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded polypeptide, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the antigen.

[0264] In some embodiments, the cancer immunotherapy comprises a cell-based therapy. In some embodiments, the cancer immunotherapy comprises a T cell-based therapy. In some embodiments, the cancer immunotherapy comprises an adoptive therapy, e.g., an adoptive T cell-based therapy. In some embodiments, the T cells are autologous or allogeneic to the recipient. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are CD4+ T cells. Adoptive immunotherapy refers to a therapeutic approach for treating cancer or infectious diseases in which immune cells are administered to a host with the aim that the cells mediate either directly or indirectly specific immunity to (i.e., mount an immune response directed against) cancer cells. In some embodiments, the immune response results in inhibition of tumor and / or metastatic cell growth and / or proliferation, and in related embodiments, results in neoplastic cell death and / or resorption. The immune cells can be derived from a different organism / host (exogenous immune cells) or can be cells obtained from the subject organism (autologous immune cells). In some embodiments, the immune cells (e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or gamma-delta T cells), NK cells, invariant NK cells, or NKT cells) can be genetically 90MOFO-358156824Docket No: 197102019040 engineered to express antigen receptors such as engineered TCRs and / or chimeric antigen receptors (CARs). For example, the host cells (e.g., autologous or allogeneic T-cells) are modified to express a T cell receptor (TCR) having antigenic specificity for a cancer antigen. In some embodiments, NK cells are engineered to express a TCR. The NK cells may be further engineered to express a CAR. Multiple CARs and / or TCRs, such as to different antigens, may be added to a single cell type, such as T cells or NK cells. In some embodiments, the cells comprise one or more nucleic acids / expression constructs / vectors introduced via genetic engineering that encode one or more antigen receptors, and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and / or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature (e.g. chimeric). In some embodiments, a population of immune cells can be obtained from a subject in need of therapy or suffering from a disease associated with reduced immune cell activity. Thus, the cells will be autologous to the subject in need of therapy. In some embodiments, a population of immune cells can be obtained from a donor, such as a histocompatibility-matched donor. In some embodiments, the immune cell population can be harvested from the peripheral blood, cord blood, bone marrow, spleen, or any other organ / tissue in which immune cells reside in said subject or donor. In some embodiments, the immune cells can be isolated from a pool of subjects and / or donors, such as from pooled cord blood. In some embodiments, when the population of immune cells is obtained from a donor distinct from the subject, the donor may be allogeneic, provided the cells obtained are subject-compatible, in that they can be introduced into the subject. In some embodiments, allogeneic donor cells may or may not be human-leukocyte-antigen (HLA)-compatible. In some embodiments, to be rendered subject-compatible, allogeneic cells can be treated to reduce immunogenicity.

[0265] In some embodiments, the cell-based therapy comprises a T cell-based therapy, such as autologous cells, e.g., tumor-infiltrating lymphocytes (TILs); T cells activated ex-vivo using autologous DCs, lymphocytes, artificial antigen-presenting cells (APCs) or beads coated with T cell ligands and activating antibodies, or cells isolated by virtue of capturing target cell membrane; allogeneic cells naturally expressing anti-host tumor T cell receptor (TCR); and non-tumor-specific autologous or allogeneic cells genetically reprogrammed or "redirected" to express tumor-reactive TCR or chimeric TCR molecules displaying antibody- 91MOFO-358156824Docket No: 197102019040 like tumor recognition capacity known as "T- bodies". Several approaches for the isolation, derivation, engineering or modification, activation, and expansion of functional anti-tumor effector cells have been described in the last two decades and may be used according to any of the methods provided herein. In some embodiments, the T cells are derived from the blood, bone marrow, lymph, umbilical cord, or lymphoid organs. In some embodiments, the cells are human cells. In some embodiments, the cells are primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and / or persistence capacities, antigen- specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. In some embodiments, the cells may be allogeneic and / or autologous. In some embodiments, such as for off-the-shelf technologies, the cells are pluripotent and / or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs).

[0266] In some embodiments, the T cell-based therapy comprises a chimeric antigen receptor (CAR)-T cell-based therapy. This approach involves engineering a CAR that specifically binds to an antigen of interest and comprises one or more intracellular signaling domains for T cell activation. The CAR is then expressed on the surface of engineered T cells (CAR-T) and administered to a patient, leading to a T-cell-specific immune response against cancer cells expressing the antigen. In some embodiments, the CAR specifically binds a neoantigen, such as a neoantigen corresponding to an a mutant ALK nucleic acid molecule or polypeptide of the disclosure.

[0267] In some embodiments, the T cell-based therapy comprises T cells expressing a recombinant T cell receptor (TCR). This approach involves identifying a TCR that specifically binds to an antigen of interest, which is then used to replace the endogenous or native TCR on the surface of engineered T cells that are administered to a patient, leading to a T-cell-specific immune response against cancer cells expressing the antigen. In some embodiments, the recombinant TCR specifically binds a neoantigen corresponding to a fusion nucleic acid molecule or polypeptide of the disclosure.

[0268] In some embodiments, the T cell-based therapy comprises tumor-infiltrating lymphocytes (TILs). For example, TILs can be isolated from a tumor or cancer of the present disclosure, then isolated and expanded in vitro. Some or all of these TILs may specifically 92MOFO-358156824Docket No: 197102019040 recognize an antigen expressed by the tumor or cancer of the present disclosure. In some embodiments, the TILs are exposed to one or more neoantigens, e.g., a neoantigen corresponding to a mutant ALK nucleic acid molecule or polypeptide of the disclosure, in vitro after isolation. TILs are then administered to the patient (optionally in combination with one or more cytokines or other immune-stimulating substances).

[0269] In some embodiments, the cell-based therapy comprises a natural killer (NK) cell- based therapy. Natural killer (NK) cells are a subpopulation of lymphocytes that have spontaneous cytotoxicity against a variety of tumor cells, virus-infected cells, and some normal cells in the bone marrow and thymus. NK cells are critical effectors of the early innate immune response toward transformed and virus-infected cells. NK cells can be detected by specific surface markers, such as CD16, CD56, and CD8 in humans. NK cells do not express T-cell antigen receptors, the pan T marker CD3, or surface immunoglobulin B cell receptors. In some embodiments, NK cells are derived from human peripheral blood mononuclear cells (PBMC), unstimulated leukapheresis products (PBSC), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood by methods well known in the art.

[0270] In some embodiments, the cell-based therapy comprises a dendritic cell (DC)-based therapy, e.g., a dendritic cell vaccine. In some embodiments, the DC vaccine comprises antigen-presenting cells that are able to induce specific T cell immunity, which are harvested from the patient or from a donor. In some embodiments, the DC vaccine can then be exposed in vitro to a peptide antigen, for which T cells are to be generated in the patient. In some embodiments, dendritic cells loaded with the antigen are then injected back into the patient. In some embodiments, immunization may be repeated multiple times if desired. Methods for harvesting, expanding, and administering dendritic cells are known in the art; see, e.g., WO2019178081. Dendritic cell vaccines (such as Sipuleucel-T, also known as APC8015) are vaccines that involve administration of dendritic cells that act as APCs to present one or more cancer-specific antigens to the patient’s immune system. In some embodiments, the dendritic cells are autologous or allogeneic to the recipient.

[0271] In some embodiments, the cancer immunotherapy comprises a TCR-based therapy. In some embodiments, the cancer immunotherapy comprises administration of one or more TCRs or TCR-based therapeutics that specifically bind an antigen expressed by a cancer of the present disclosure, e.g., a neoantigen corresponding to a mutant ALK nucleic acid molecule or polypeptide of the disclosure. In some embodiments, the TCR-based therapeutic may further include a moiety that binds an immune cell (e.g., a T cell), such as an antibody or 93MOFO-358156824Docket No: 197102019040 antibody fragment that specifically binds a T cell surface protein or receptor (e.g., an anti- CD3 antibody or antibody fragment).

[0272] In some embodiments, the immunotherapy comprises adjuvant immunotherapy. Adjuvant immunotherapy comprises the use of one or more agents that activate components of the innate immune system, e.g., imiquimod, which targets the TLR7 pathway.

[0273] In some embodiments, the immunotherapy comprises cytokine immunotherapy. Cytokine immunotherapy comprises the use of one or more cytokines that activate components of the immune system. Examples include, but are not limited to, aldesleukin (e.g., interleukin-2), interferon alfa-2a, interferon alfa-2b, and peginterferon alfa-2b.

[0274] In some embodiments, the immunotherapy comprises oncolytic virus therapy. Oncolytic virus therapy uses genetically modified viruses to replicate in and kill cancer cells, leading to the release of antigens that stimulate an immune response. In some embodiments, replication-competent oncolytic viruses expressing a tumor antigen comprise any naturally occurring (e.g., from a “field source”) or modified replication-competent oncolytic virus. In some embodiments, the oncolytic virus, in addition to expressing a tumor antigen, may be modified to increase selectivity of the virus for cancer cells. In some embodiments, replication-competent oncolytic viruses include, but are not limited to, oncolytic viruses that are a member in the family of myoviridae, siphoviridae, podpviridae, teciviridae, corticoviridae, plasmaviridae, lipothrixviridae, fuselloviridae, poxyiridae, iridoviridae, phycodnaviridae, baculoviridae, herpesviridae, adnoviridae, papovaviridae, polydnaviridae, inoviridae, microviridae, geminiviridae, circoviridae, parvoviridae, hcpadnaviridae, retroviridae, cyctoviridae, reoviridae, birnaviridae, paramyxoviridae, rhabdoviridae, filoviridae, orthomyxoviridae, bunyaviridae, arenaviridae, Leviviridae, picornaviridae, sequiviridae, comoviridae, potyviridae, caliciviridae, astroviridae, nodaviridae, tetraviridae, tombusviridae, coronaviridae, glaviviridae, togaviridae, and barnaviridae. In some embodiments, replication-competent oncolytic viruses include adenovirus, retrovirus, reovirus, rhabdovirus, Newcastle Disease virus (NDV), polyoma virus, vaccinia virus (VacV), herpes simplex virus, picornavirus, coxsackie virus and parvovirus. In some embodiments, a replicative oncolytic vaccinia virus expressing a tumor antigen may be engineered to lack one or more functional genes in order to increase the cancer selectivity of the virus. In some embodiments, an oncolytic vaccinia virus is engineered to lack thymidine kinase (TK) activity. In some embodiments, the oncolytic vaccinia virus may be engineered to lack vaccinia virus growth factor (VGF). In some embodiments, an oncolytic vaccinia virus may be engineered to lack both VGF and TK activity. In some embodiments, an 94MOFO-358156824Docket No: 197102019040 oncolytic vaccinia virus may be engineered to lack one or more genes involved in evading host interferon (IFN) response such as E3L, K3L, B18R, or B8R. In some embodiments, a replicative oncolytic vaccinia virus is a Western Reserve, Copenhagen, Lister or Wyeth strain and lacks a functional TK gene. In some embodiments, the oncolytic vaccinia virus is a Western Reserve, Copenhagen, Lister or Wyeth strain lacking a functional B18R and / or B8R gene. In some embodiments, a replicative oncolytic vaccinia virus expressing a tumor antigen may be locally or systemically administered to a subject, e.g. via intratumoral, intraperitoneal, intravenous, intra-arterial, intramuscular, intradermal, intracranial, subcutaneous, or intranasal administration.

[0275] In some embodiments, the anti-cancer therapy comprises an immune checkpoint inhibitor, e.g., alone or in combination with an ALK-targeted therapy. In some embodiments, the methods provided herein comprise administering to an individual an effective amount of an immune checkpoint inhibitor. As is known in the art, a checkpoint inhibitor targets at least one immune checkpoint protein to alter the regulation of an immune response. Immune checkpoint proteins include, e.g., CTLA4, PD-L1, PD-1, PD-L2, VISTA, B7-H2, B7-H3, B7- H4, B7-H6, 2B4, ICOS, HVEM, CEACAM, LAIR1, CD80, CD86, CD276, VTCN1, MHC class I, MHC class II, GALS, adenosine, TGFR, CSF1R, MICA / B, arginase, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, LAG-3, BTLA, IDO, OX40, and A2aR. In some embodiments, molecules involved in regulating immune checkpoints include, but are not limited to: PD-1 (CD279), PD-L1 (B7-H1, CD274), PD-L2 (B7-CD, CD273), CTLA-4 (CD152), HVEM, BTLA (CD272), a killer-cell immunoglobulin-like receptor (KIR), LAG-3 (CD223), TIM-3 (HAVCR2), CEACAM, CEACAM-1, CEACAM-3, CEACAM-5, GAL9, VISTA (PD-1H), TIGIT, LAIR1, CD160, 2B4, TGFRbeta, A2AR, GITR (CD357), CD80 (B7-1), CD86 (B7-2), CD276 (B7-H3), VTCNI (B7-H4), MHC class I, MHC class II, GALS, adenosine, TGFR, B7-H1, OX40 (CD134), CD94 (KLRD1), CD137 (4-1BB), CD137L (4-1BBL), CD40, IDO, CSF1R, CD40L, CD47, CD70 (CD27L), CD226, HHLA2, ICOS (CD278), ICOSL (CD275), LIGHT (TNFSF14, CD258), NKG2a, NKG2d, OX40L (CD134L), PVR (NECL5, CD155), SIRPa, MICA / B, and / or arginase. In some embodiments, an immune checkpoint inhibitor (i.e., a checkpoint inhibitor) decreases the activity of a checkpoint protein that negatively regulates immune cell function, e.g., in order to enhance T cell activation and / or an anti-cancer immune response. In other embodiments, a checkpoint inhibitor increases the activity of a checkpoint protein that positively regulates immune cell function, e.g., in order to enhance T cell activation and / or an anti-cancer 95MOFO-358156824Docket No: 197102019040 immune response. In some embodiments, the checkpoint inhibitor is an antibody. Examples of checkpoint inhibitors include, without limitation, a PD-1 axis binding antagonist, a PD-L1 axis binding antagonist (e.g., an anti-PD-L1 antibody, e.g., atezolizumab (MPDL3280A)), an antagonist directed against a co-inhibitory molecule (e.g., a CTLA4 antagonist (e.g., an anti- CTLA4 antibody), a TIM-3 antagonist (e.g., an anti-TIM-3 antibody), or a LAG-3 antagonist (e.g., an anti-LAG-3 antibody)), or any combination thereof. In some embodiments, the immune checkpoint inhibitors comprise drugs such as small molecules, recombinant forms of ligand or receptors, or antibodies, such as human antibodies (see, e.g., International Patent Publication W02015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012; both incorporated herein by reference). In some embodiments, known inhibitors of immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized or human forms of antibodies may be used.

[0276] In some embodiments, the checkpoint inhibitor is a PD-L1 axis binding antagonist. PD-1 (programmed death 1) is also referred to in the art as "programmed cell death 1," "PDCD1," "CD279," and "SLEB2." An exemplary human PD-1 is shown in UniProtKB / Swiss-Prot Accession No. Q15116. PD-L1 (programmed death ligand 1) is also referred to in the art as "programmed cell death 1 ligand 1,” "PDCD1 LG1," "CD274," "B7- H," and "PDL1." An exemplary human PD-L1 is shown in UniProtKB / Swiss-Prot Accession No.Q9NZQ7.1. PD-L2 (programmed death ligand 2) is also referred to in the art as "programmed cell death 1 ligand 2," "PDCD1 LG2," "CD273," "B7-DC," "Btdc," and "PDL2." An exemplary human PD-L2 is shown in UniProtKB / Swiss-Prot Accession No. Q9BQ51. In some instances, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1 and PD-L2.

[0277] In some instances, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific embodiment, the PD-1 ligand binding partners are PD-L1 and / or PD-L2. In another instance, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding ligands. In a specific embodiment, PD-L1 binding partners are PD-1 and / or B7-1. In another instance, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its ligand binding partners. In a specific embodiment, the PD-L2 binding ligand partner is PD-1. The antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the PD-1 binding antagonist is a small molecule, a nucleic acid, a polypeptide (e.g., antibody), a carbohydrate, a lipid, a metal, or a toxin.

[0278] In some instances, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), for example, as described 96MOFO-358156824Docket No: 197102019040 below. In some instances, the anti-PD-1 antibody is one or more of MDX-1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, MGA-012, JNJ-63723283, BI 754091, or BGB-108. In other instances, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). In some instances, the PD-1 binding antagonist is AMP-224. Other examples of anti-PD-1 antibodies include, but are not limited to, MEDI-0680 (AMP-514; AstraZeneca), PDR001 (CAS Registry No. 1859072-53-9; Novartis), REGN2810 (e.g., cemiplimab-rwlc), BGB-108 (BeiGene), BGB-A317 (BeiGene), BI 754091, JS-001 (Shanghai Junshi), STI- A1110 (Sorrento), INCSHR-1210 (Incyte), PF-06801591 (Pfizer), TSR-042 (also known as ANB011; Tesaro / AnaptysBio), AM0001 (ARMO Biosciences), ENUM 244C8 (Enumeral Biomedical Holdings), or ENUM 388D4 (Enumeral Biomedical Holdings). In some embodiments, the PD-1 axis binding antagonist comprises tislelizumab (BGB-A317), BGB- 108, STI-A1110, AM0001, BI 754091, sintilimab (IBI308), cetrelimab (JNJ-63723283), toripalimab (JS-001), camrelizumab (SHR-1210, INCSHR-1210, HR-301210), MEDI-0680 (AMP-514), MGA-012 (INCMGA 0012), nivolumab (BMS-936558, MDX1106, ONO- 4538), spartalizumab (PDR00l), pembrolizumab (MK-3475, SCH 900475), PF-06801591, cemiplimab (REGN-2810, REGEN2810), dostarlimab (TSR-042, ANB011), FITC-YT-16 (PD-1 binding peptide), APL-501 or CBT-501 or genolimzumab (GB-226), AB-122, AK105, AMG 404, BCD-100, F520, HLX10, HX008, JTX-4014, LZM009, Sym021, PSB205, AMP- 224 (fusion protein targeting PD-1), CX-188 (PD-1 probody), AGEN-2034, GLS-010, budigalimab (ABBV-181), AK-103, BAT-1306, CS-1003, AM-0001, TILT-123, BH-2922, BH-2941, BH-2950, ENUM-244C8, ENUM-388D4, HAB-21, H EISCOI 11-003, IKT-202, MCLA-134, MT-17000, PEGMP-7, PRS-332, RXI-762, STI-1110, VXM-10, XmAb-23104, AK-112, HLX-20, SSI-361, AT-16201, SNA-01, AB122, PD1-PIK, PF-06936308, RG-7769, CAB PD-1 Abs, AK-123, MEDI-3387, MEDI-5771, 4H1128Z-E27, REMD-288, SG-001, BY-24.3, CB-201, IBI-319, ONCR-177, Max-1, CS-4100, JBI-426, CCC-0701, or CCX- 4503, or derivatives thereof.

[0279] In some embodiments, the PD-L1 binding antagonist is a small molecule that inhibits PD-1. In some embodiments, the PD-L1 binding antagonist is a small molecule that inhibits PD-L1. In some embodiments, the PD-L1 binding antagonist is a small molecule that inhibits PD-L1 and VISTA or PD-L1 and TIM3. In some embodiments, the PD-L1 binding antagonist is CA-170 (also known as AUPM-170). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 97MOFO-358156824Docket No: 197102019040 antibody can bind to a human PD-L1, for example a human PD-L1 as shown in UniProtKB / Swiss-Prot Accession No.Q9NZQ7.1, or a variant thereof. In some embodiments, the PD-L1 binding antagonist is a small molecule, a nucleic acid, a polypeptide (e.g., antibody), a carbohydrate, a lipid, a metal, or a toxin.

[0280] In some instances, the PD-L1 binding antagonist is an anti-PD-L1 antibody, for example, as described below. In some instances, the anti-PD-L1 antibody is capable of inhibiting the binding between PD-L1 and PD-1, and / or between PD-L1 and B7-1. In some instances, the anti-PD-L1 antibody is a monoclonal antibody. In some instances, the anti-PD- L1 antibody is an antibody fragment selected from a Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment. In some instances, the anti-PD-L1 antibody is a humanized antibody. In some instances, the anti-PD-L1 antibody is a human antibody. In some instances, the anti-PD-L1 antibody is selected from YW243.55.S70, MPDL3280A (atezolizumab), MDX-1105, MEDI4736 (durvalumab), or MSB0010718C (avelumab). In some embodiments, the PD-L1 axis binding antagonist comprises atezolizumab, avelumab, durvalumab (imfinzi), BGB- A333, SHR-1316 (HTI-1088), CK-301, BMS-936559, envafolimab (KN035, ASC22), CS1001, MDX-1105 (BMS-936559), LY3300054, STI-A1014, FAZ053, CX-072, INCB086550, GNS-1480, CA-170, CK-301, M-7824, HTI-1088 (HTI-131, SHR-1316), MSB-2311, AK- 106, AVA-004, BBI-801, CA-327, CBA-0710, CBT-502, FPT-155, IKT- 201, IKT-703, 10-103, JS-003, KD-033, KY-1003, MCLA-145, MT-5050, SNA-02, BCD- 135, APL-502 (CBT-402 or TQB2450), IMC-001, KD-045, INBRX-105, KN-046, IMC- 2102, IMC-2101, KD-005, IMM-2502, 89Zr-CX-072, 89Zr-DFO-6E11, KY-1055, MEDI- 1109, MT-5594, SL-279252, DSP-106, Gensci-047, REMD-290, N-809, PRS-344, FS-222, GEN-1046, BH-29xx, or FS-118, or a derivative thereof.

[0281] In some embodiments, the checkpoint inhibitor is an antagonist of CTLA4. In some embodiments, the checkpoint inhibitor is a small molecule antagonist of CTLA4. In some embodiments, the checkpoint inhibitor is an anti-CTLA4 antibody. CTLA4 is part of the CD28-B7 immunoglobulin superfamily of immune checkpoint molecules that acts to negatively regulate T cell activation, particularly CD28-dependent T cell responses. CTLA4 competes for binding to common ligands with CD28, such as CD80 (B7-1) and CD86 (B7-2), and binds to these ligands with higher affinity than CD28. Blocking CTLA4 activity (e.g., using an anti-CTLA4 antibody) is thought to enhance CD28-mediated costimulation (leading to increased T cell activation / priming), affect T cell development, and / or deplete Tregs (such as intratumoral Tregs). In some embodiments, the CTLA4 antagonist is a small molecule, a nucleic acid, a polypeptide (e.g., antibody), a carbohydrate, a lipid, a metal, or a toxin. In 98MOFO-358156824Docket No: 197102019040 some embodiments, the CTLA-4 inhibitor comprises ipilimumab (IBI310, BMS-734016, MDX010, MDX-CTLA4, MEDI4736), tremelimumab (CP-675, CP-675,206), APL-509, AGEN1884, CS1002, AGEN1181, Abatacept (Orencia, BMS-188667, RG2077), BCD-145, ONC-392, ADU-1604, REGN4659, ADG116, KN044, KN046, or a derivative thereof.

[0282] In some embodiments, the anti-PD-1 antibody or antibody fragment is MDX-1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, MGA-012, JNJ-63723283, BI 754091, BGB-108, BGB-A317, JS-001, STI-A1110, INCSHR- 1210, PF-06801591, TSR-042, AM0001, ENUM 244C8, or ENUM 388D4. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 immunoadhesin. In some embodiments, the anti-PD-1 immunoadhesin is AMP-224. In some embodiments, the anti- PD-L1 antibody or antibody fragment is YW243.55.S70, MPDL3280A (atezolizumab), MDX-1105, MEDI4736 (durvalumab), MSB0010718C (avelumab), LY3300054, STI- A1014, KN035, FAZ053, or CX-072.

[0283] In some embodiments, the immune checkpoint inhibitor comprises a LAG-3 inhibitor (e.g., an antibody, an antibody conjugate, or an antigen-binding fragment thereof). In some embodiments, the LAG-3 inhibitor comprises a small molecule, a nucleic acid, a polypeptide (e.g., an antibody), a carbohydrate, a lipid, a metal, or a toxin. In some embodiments, the LAG-3 inhibitor comprises a small molecule. In some embodiments, the LAG-3 inhibitor comprises a LAG-3 binding agent. In some embodiments, the LAG-3 inhibitor comprises an antibody, an antibody conjugate, or an antigen-binding fragment thereof. In some embodiments, the LAG-3 inhibitor comprises eftilagimod alpha (IMP321, IMP-321, EDDP- 202, EOC-202), relatlimab (BMS-986016), GSK2831781 (IMP-731), LAG525 (IΜΡ701), TSR-033, EVIP321 (soluble LAG-3 protein), BI 754111, IMP761, REGN3767, MK-4280, MGD-013, XmAb22841, INCAGN-2385, ENUM-006, AVA-017, AM-0003, iOnctura anti- LAG-3 antibody, Arcus Biosciences LAG-3 antibody, Sym022, a derivative thereof, or an antibody that competes with any of the preceding.

[0284] In some embodiments, the anti-cancer therapy comprises an immunoregulatory molecule or a cytokine, e.g., alone or in combination with an ALK-targeted therapy. An immunoregulatory profile is required to trigger an efficient immune response and balance the immunity in a subject. Examples of suitable immunoregulatory cytokines include, but are not limited to, interferons (e.g., IFNα, IFNβ and IFNγ), interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 and IL-20), tumor necrosis factors (e.g., TNFα and TNFβ), erythropoietin (EPO), FLT-3 ligand, gIp10, TCA-3, MCP-1, MIF, MIP-1α, MIP-1β, Rantes, macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating 99MOFO-358156824Docket No: 197102019040 factor (G-CSF), or granulocyte-macrophage colony stimulating factor (GM-CSF), as well as functional fragments thereof. In some embodiments, any immunomodulatory chemokine that binds to a chemokine receptor, i.e., a CXC, CC, C, or CX3C chemokine receptor, can be used in the context of the present disclosure. Examples of chemokines include, but are not limited to, MIP-3α (Lax), MIP-3β, Hcc-1, MPIF-1, MPIF-2, MCP-2, MCP-3, MCP-4, MCP-5, Eotaxin, Tarc, Elc, I309, IL-8, GCP-2 Groα, Gro-β, Nap-2, Ena-78, Ip-10, MIG, I-Tac, SDF- 1, or BCA-1 (Blc), as well as functional fragments thereof. In some embodiments, the immunoregulatory molecule is included with any of the treatments provided herein.

[0285] In some embodiments, the immune checkpoint inhibitor is monovalent and / or monospecific. In some embodiments, the immune checkpoint inhibitor is multivalent and / or multispecific.

[0286] In some embodiments, the anti-cancer therapy comprises an anti-cancer agent that inhibits expression of a nucleic acid that comprises or encodes a mutant ALK nucleic acid molecule of the disclosure or a portion thereof, or a mutant ALK polypeptide of the disclosure, or a portion thereof. In some embodiments, the anti-cancer therapy comprises a nucleic acid molecule, such as a dsRNA, an siRNA, or an shRNA. As is known in the art, dsRNAs having a duplex structure are effective at inducing RNA interference (RNAi). In some embodiments, the anti-cancer therapy comprises a small interfering RNA molecule (siRNA). dsRNAs and siRNAs can be used to silence gene expression in mammalian cells (e.g., human cells). In some embodiments, a dsRNA of the disclosure comprises any of between about 5 and about 10 base pairs, between about 10 and about 12 base pairs, between about 12 and about 15 base pairs, between about 15 and about 20 base pairs, between about 20 and 23 base pairs, between about 23 and about 25 base pairs, between about 25 and about 27 base pairs, or between about 27 and about 30 base pairs. As is known in the art, siRNAs are small dsRNAs that optionally include overhangs. In some embodiments, the duplex region of an siRNA is between about 18 and 25 nucleotides, e.g., any of 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. siRNAs may also include short hairpin RNAs (shRNAs), e.g., with approximately 29-base-pair stems and 2-nucleotide 3’ overhangs. In some embodiments, a dsRNA, an siRNA, or an shRNA of the disclosure comprises a nucleotide sequence that is configured to hybridize to a nucleic acid that comprises or encodes a mutant ALK nucleic acid molecule of the disclosure or a portion thereof comprising a breakpoint. Methods for designing, optimizing, producing, and using dsRNAs, siRNAs, or shRNAs, are known in the art. 100MOFO-358156824Docket No: 197102019040

[0287] In some embodiments, the anti-cancer therapy comprises a chemotherapy, e.g., alone or in combination with an ALK-targeted therapy. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L- norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti- adrenals, such as mitotane and trilostane; folic acid replenishers such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; 101MOFO-358156824Docket No: 197102019040 elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-l l); topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, famesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0288] Some non-limiting examples of chemotherapeutic drugs which can be combined with anti-cancer therapies of the present disclosure are carboplatin (Paraplatin), cisplatin (Platinol, Platinol-AQ), cyclophosphamide (Cytoxan, Neosar), docetaxel (Taxotere), doxorubicin (Adriamycin), erlotinib (Tarceva), etoposide (VePesid), fluorouracil (5-FU), gemcitabine (Gemzar), imatinib mesylate (Gleevec), irinotecan (Camptosar), methotrexate (Folex, Mexate, Amethopterin), paclitaxel (Taxol, Abraxane), sorafinib (Nexavar), sunitinib (Sutent), topotecan (Hycamtin), vincristine (Oncovin, Vincasar PFS), and vinblastine (Velban).

[0289] In some embodiments, the anti-cancer therapy comprises a kinase inhibitor, e.g., alone or in combination with an ALK-targeted therapy. Examples of kinase inhibitors include those that target one or more receptor tyrosine kinases, e.g., BCR-ABL, B-Raf, EGFR, HER- 2 / ErbB2, IGF-IR, PDGFR-a, PDGFR- β, cKit, Flt-4, Flt3, FGFR1, FGFR2, FGFR3, FGFR4, CSF1R, c-Met, ROS1, RON, c-Ret, or ALK; one or more cytoplasmic tyrosine kinases, e.g., c-SRC, c-YES, Abl, or JAK-2; one or more serine / threonine kinases, e.g., ATM, Aurora A & B, CDKs, mTOR, PKCi, PLKs, b-Raf, c-Raf, S6K, or STK11 / LKB1; or one or more lipid kinases, e.g., PI3K or SKI. Small molecule kinase inhibitors include PHA-739358, nilotinib, dasatinib, PD166326, NSC 743411, lapatinib (GW-572016), canertinib (CI-1033), semaxinib (SU5416), vatalanib (PTK787 / ZK222584), sutent (SU11248), sorafenib (BAY 43-9006), or 102MOFO-358156824Docket No: 197102019040 leflunomide (SU101). Additional non-limiting examples of tyrosine kinase inhibitors include imatinib (Gleevec / Glivec) and gefitinib (Iressa).

[0290] In some embodiments, the anti-cancer therapy comprises an anti-angiogenic agent, e.g., alone or in combination with an ALK-targeted therapy. Angiogenesis inhibitors prevent the extensive growth of blood vessels (angiogenesis) that tumors require to survive. Non- limiting examples of angiogenesis-mediating molecules or angiogenesis inhibitors which may be used in the methods of the present disclosure include soluble VEGF (for example: VEGF isoforms, e.g., VEGF121 and VEGF165; VEGF receptors, e.g., VEGFR1, VEGFR2; and co- receptors, e.g., Neuropilin-1 and Neuropilin-2), NRP-1, angiopoietin 2, TSP-1 and TSP-2, angiostatin and related molecules, endostatin, vasostatin, calreticulin, platelet factor-4, TIMP and CDAI, Meth-1 and Meth-2, IFNα, IFN-β and IFN-γ, CXCL10, IL-4, IL-12 and IL-18, prothrombin (kringle domain-2), antithrombin III fragment, prolactin, VEGI, SPARC, osteopontin, maspin, canstatin, proliferin-related protein, restin and drugs such as bevacizumab, itraconazole, carboxyamidotriazole, TNP-470, CM101, IFN-a platelet factor-4, suramin, SU5416, thrombospondin, VEGFR antagonists, angiostatic steroids and heparin, cartilage-derived angiogenesis inhibitory factor, matrix metalloproteinase inhibitors, 2- methoxyestradiol, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, prolactina ν β3 inhibitors, linomide, or tasquinimod. In some embodiments, known therapeutic candidates that may be used according to the methods of the disclosure include naturally occurring angiogenic inhibitors, including without limitation, angiostatin, endostatin, or platelet factor- 4. In another embodiment, therapeutic candidates that may be used according to the methods of the disclosure include, without limitation, specific inhibitors of endothelial cell growth, such as TNP-470, thalidomide, and interleukin-12. Still other anti-angiogenic agents that may be used according to the methods of the disclosure include those that neutralize angiogenic molecules, including without limitation, antibodies to fibroblast growth factor, antibodies to vascular endothelial growth factor, antibodies to platelet derived growth factor, or antibodies or other types of inhibitors of the receptors of EGF, VEGF or PDGF. In some embodiments, anti-angiogenic agents that may be used according to the methods of the disclosure include, without limitation, suramin and its analogs, and tecogalan. In other embodiments, anti- angiogenic agents that may be used according to the methods of the disclosure include, without limitation, agents that neutralize receptors for angiogenic factors or agents that interfere with vascular basement membrane and extracellular matrix, including, without limitation, metalloprotease inhibitors and angiostatic steroids. Another group of anti- angiogenic compounds that may be used according to the methods of the disclosure includes, 103MOFO-358156824Docket No: 197102019040 without limitation, anti-adhesion molecules, such as antibodies to integrin alpha v beta 3. Still other anti-angiogenic compounds or compositions that may be used according to the methods of the disclosure include, without limitation, kinase inhibitors, thalidomide, itraconazole, carboxyamidotriazole, CM101, IFN-α, IL-12, SU5416, thrombospondin, cartilage-derived angiogenesis inhibitory factor, 2-methoxyestradiol, tetrathiomolybdate, thrombospondin, prolactin, and linomide. In one particular embodiment, the anti-angiogenic compound that may be used according to the methods of the disclosure is an antibody to VEGF, such as bevacizumab.

[0291] In some embodiments, the anti-cancer therapy comprises an anti-DNA repair therapy, e.g., alone or in combination with an ALK-targeted therapy. In some embodiments, the anti- DNA repair therapy is a PARP inhibitor (e.g., talazoparib, rucaparib, olaparib), a RAD51 inhibitor (e.g., RI-1), or an inhibitor of a DNA damage response kinase, e.g., CHCK1 (e.g., AZD7762), ATM (e.g., KU-55933, KU-60019, NU7026, or VE-821), and ATR (e.g., NU7026).

[0292] In some embodiments, the anti-cancer therapy comprises a radiosensitizer, e.g., alone or in combination with an ALK-targeted therapy. Exemplary radiosensitizers include hypoxia radiosensitizers such as misonidazole, metronidazole, and trans-sodium crocetinate, a compound that helps to increase the diffusion of oxygen into hypoxic tumor tissue. The radiosensitizer can also be a DNA damage response inhibitor interfering with base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), recombinational repair comprising homologous recombination (HR) and non-homologous end-joining (NHEJ), and direct repair mechanisms. Single strand break (SSB) repair mechanisms include BER, NER, or MMR pathways, while double stranded break (DSB) repair mechanisms consist of HR and NHEJ pathways. Radiation causes DNA breaks that, if not repaired, are lethal. SSBs are repaired through a combination of BER, NER and MMR mechanisms using the intact DNA strand as a template. The predominant pathway of SSB repair is BER, utilizing a family of related enzymes termed poly-(ADP-ribose) polymerases (PARP). Thus, the radiosensitizer can include DNA damage response inhibitors such as PARP inhibitors.

[0293] In some embodiments, the anti-cancer therapy comprises an anti-inflammatory agent, e.g., alone or in combination with an ALK-targeted therapy. In some embodiments, the anti- inflammatory agent is an agent that blocks, inhibits, or reduces inflammation or signaling from an inflammatory signaling pathway In some embodiments, the anti-inflammatory agent inhibits or reduces the activity of one or more of any of the following: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23; interferons (IFNs), e.g., 104MOFO-358156824Docket No: 197102019040 IFNα, IFNβ, IFNγ, IFN-γ inducing factor (IGIF); transforming growth factor-β (TGF-β); transforming growth factor-α (TGF-α); tumor necrosis factors, e.g., TNF-α, TNF-β, TNF-RI, TNF-RII; CD23; CD30; CD40L; EGF; G-CSF; GDNF; PDGF-BB; RANTES / CCL5; IKK; NF-κB; TLR2; TLR3; TLR4; TL5; TLR6; TLR7; TLR8; TLR8; TLR9; and / or any cognate receptors thereof. In some embodiments, the anti-inflammatory agent is an IL-1 or IL-1 receptor antagonist, such as anakinra, rilonacept, or canakinumab. In some embodiments, the anti-inflammatory agent is an IL-6 or IL-6 receptor antagonist, e.g., an anti-IL-6 antibody or an anti-IL-6 receptor antibody, such as tocilizumab, olokizumab, clazakizumab, sarilumab, sirukumab, siltuximab, or ALX-0061. In some embodiments, the anti-inflammatory agent is a TNF-α antagonist, e.g., an anti-TNFα antibody, such as infliximab, golimumab, adalimumab, certolizumab pegol, or etanercept. In some embodiments, the anti-inflammatory agent is a corticosteroid. Exemplary corticosteroids include, but are not limited to, cortisone (hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate), decadron (dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate), methylprednisolone (6-methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate), prednisolone, and prednisone, and bisphosphonates (e.g., pamidronate and zoledronic acid).

[0294] In some embodiments, the anti-cancer therapy comprises an anti-hormonal agent, e.g., alone or in combination with an ALK-targeted therapy. Anti-hormonal agents are agents that act to regulate or inhibit hormone action on tumors. Examples of anti-hormonal agents include anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, and anastrozole; anti-androgens such...

Claims

Docket No: 197102019040 CLAIMS What is claimed is:

1. A method of treating or delaying progression of cancer, comprising: (a) administering to an individual a treatment comprising a first ALK-targeted therapy; (b) after administration of the first ALK-targeted therapy, detecting a mutant ALK nucleic acid molecule or mutant ALK polypeptide in a sample obtained from an individual, wherein the mutant ALK nucleic acid molecule encodes an ALK polypeptide that comprises, or the mutant ALK polypeptide comprises, a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; and (c) after the detection of the mutant ALK nucleic acid molecule or mutant ALK polypeptide in the sample, administering to the individual a treatment comprising a second ALK-targeted therapy different from the first ALK-targeted therapy.

2. The method of claim 1, further comprising, after (b): ceasing administration of the first ALK-targeted therapy.

3. A method of treating or delaying progression of cancer, comprising: (a) detecting a mutant ALK nucleic acid molecule or mutant ALK polypeptide in a sample obtained from an individual, wherein the mutant ALK nucleic acid molecule encodes an ALK polypeptide that comprises, or the mutant ALK polypeptide comprises, a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; and (b) administering to the individual a treatment comprising an ALK-targeted therapy.

4. The method of claim 3, wherein the individual has received a prior treatment comprising a different ALK-targeted therapy, and optionally the method further comprises, prior to the administration: ceasing the prior treatment comprising the different ALK-targeted therapy. 148MOFO-358156824Docket No: 197102019040 5. A method of monitoring or screening for resistance of a cancer in an individual to a treatment comprising an ALK-targeted therapy, the method comprising detecting a mutant ALK nucleic acid molecule or mutant ALK polypeptide in a sample obtained from an individual, wherein the mutant ALK nucleic acid molecule encodes an ALK polypeptide that comprises, or the mutant ALK polypeptide comprises, a mutation selected from the group consisting of G1123A, T1151_L1152insT, I1171M, G1202del, I1268V, E1129V, and L1196Q, amino acid numbering based on SEQ ID NO:2; wherein presence of the mutant ALK nucleic acid molecule or polypeptide indicates resistance to a treatment comprising an ALK-targeted therapy; and wherein the individual has received a prior treatment comprising an ALK-targeted therapy.

6. The method of claim 5, wherein: (a) the prior treatment comprises crizotinib, and the ALK-targeted therapy comprises alectinib, brigatinib, ceritinib, ensartinib, or lorlatinib; or (b) the prior treatment comprises crizotinib, alectinib, brigatinib, ceritinib, or ensartinib, and the ALK-targeted therapy comprises lorlatinib.

7. The method of claim 1, wherein the cancer further comprises an ALK nucleic acid molecule that encodes an ALK polypeptide comprising, or comprises an ALK polypeptide comprising, a second ALK mutation associated with acquired resistance to an ALK-targeted therapy.

8. The method of claim 7, wherein: (a) the second ALK mutation associated with acquired resistance to an ALK-targeted therapy is selected from the group consisting of T1151M, T1151R, C1156Y, I1171N, I1171T, I1171S, I1171H, I1171V, F1174X, V1180L, R1192P, L1196M, L1198F, G1202X, G1202del, D1203N, S1206Y, E1210K, and G1269A, amino acid numbering based on SEQ ID NO:2; (b) the second ALK mutation associated with acquired resistance to an ALK-targeted therapy is selected from the group consisting of T1151M, L1152R, C1156Y, I1171T, F1174 (any missense), R1192P, L1196M, G1202R, G1269A, S1206Y, and E1210K, amino acid numbering based on SEQ ID NO:2, and optionally the second ALK mutation is associated with resistance to a first-generation ALK TKI, e.g., crizotinib; 149MOFO-358156824Docket No: 197102019040 (c) the second ALK mutation associated with acquired resistance to an ALK-targeted therapy is selected from the group consisting of T1151R, I1171N, I1171T, I1171S, I1171H, I1171V, F1174C, V1180L, G1202 (any missense), G1202R, G1202del, and D1203N, amino acid numbering based on SEQ ID NO:2, and optionally the second ALK mutation is associated with resistance to a second-generation ALK TKI, e.g., ceritinib, brigatinib, or alectinib; or (d) the second ALK mutation associated with acquired resistance to an ALK-targeted therapy comprises a set of multiple ALK mutations selected from the group consisting of C1156Y + L1198F, L1196M + D1203N, L1196M + G1202R, G1202R + G1269A, G1202R + F1174L, L1196M + F1174L, amino acid numbering based on SEQ ID NO:2, and optionally the second ALK mutation is associated with resistance to a third-generation ALK TKI, e.g., lorlatinib.

9. The method of claim 1, further comprising: detecting an ALK nucleic acid molecule that encodes an ALK polypeptide comprising, or comprises an ALK polypeptide comprising, a second ALK mutation associated with acquired resistance to an ALK-targeted therapy in a sample obtained from the individual.

10. The method of claim 9, wherein: (a) the second ALK mutation associated with acquired resistance to an ALK-targeted therapy is selected from the group consisting of T1151M, T1151R, C1156Y, I1171N, I1171T, I1171S, I1171H, I1171V, F1174X, V1180L, R1192P, L1196M, L1198F, G1202X, G1202del, D1203N, S1206Y, E1210K, and G1269A, amino acid numbering based on SEQ ID NO:2; (b) the second ALK mutation associated with acquired resistance to an ALK-targeted therapy is selected from the group consisting of T1151M, L1152R, C1156Y, I1171T, F1174 (any missense), R1192P, L1196M, G1202R, G1269A, S1206Y, and E1210K, amino acid numbering based on SEQ ID NO:2, and optionally the second ALK mutation is associated with resistance to a first-generation ALK TKI, e.g., crizotinib; (c) the second ALK mutation associated with acquired resistance to an ALK-targeted therapy is selected from the group consisting of T1151R, I1171N, I1171T, I1171S, I1171H, I1171V, F1174C, V1180L, G1202 (any missense), G1202R, G1202del, and D1203N, amino 150MOFO-358156824Docket No: 197102019040 acid numbering based on SEQ ID NO:2, and optionally the second ALK mutation is associated with resistance to a second-generation ALK TKI, e.g., ceritinib, brigatinib, or alectinib; or (d) the second ALK mutation associated with acquired resistance to an ALK-targeted therapy comprises a set of multiple ALK mutations selected from the group consisting of C1156Y + L1198F, L1196M + D1203N, L1196M + G1202R, G1202R + G1269A, G1202R + F1174L, L1196M + F1174L, amino acid numbering based on SEQ ID NO:2, and optionally the second ALK mutation is associated with resistance to a third-generation ALK TKI, e.g., lorlatinib.

11. The method of claim 1, wherein: (a) the ALK-targeted therapy is crizotinib, alectinib, brigatinib, ceritinib, ensartinib, lorlatinib, TPX-0131, NVL-655, entrectinib, repotrectinib, belizatinib, TQ-B3139, WX-0593, PLB-1003, SAF-189s, CT-707, gilteritinib, XMU-MP-5, AZD3463, CEP-37440, ASP3026, KRCA-0008, or TAE684; or (b) the ALK-targeted therapy comprises a tyrosine kinase inhibitor (TKI) or an ALK proteolysis-targeting chimeric (PROTAC) degrader.

12. The method of claim 1, further comprising: detecting a nucleic acid molecule that encodes, or a polypeptide comprising, a mutation in one or more genes selected from the group consisting of AXL, BRAF, MAP2K1, MET, NF2, PIK3CA, and NRAS in a sample obtained from the individual.

13. The method of claim 1, further comprising: detecting a gene fusion, gene rearrangement, gene amplification, or activating mutation in an ALK gene in a sample obtained from the individual.

14. The method of claim 1, wherein the cancer is lung cancer, anaplastic large cell lymphoma (ALCL), neuroblastoma, an inflammatory myofibroblastic tumor (IMT), spitzoid tumor, colorectal cancer, B-cell lymphoma, ovarian cancer, non-Hodgkin’s lymphoma (NHL), or thyroid cancer; optionally wherein the cancer is non-small cell lung cancer (NSCLC). 151MOFO-358156824Docket No: 197102019040 15. The method of claim 1, wherein the mutant ALK nucleic acid molecule is detected in the sample by one or more of: a nucleic acid hybridization assay, an amplification-based assay, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay, real-time PCR, a screening analysis, fluorescence in situ hybridization (FISH), spectral karyotyping, multicolor FISH (mFISH), comparative genomic hybridization, in situ hybridization, sequence-specific priming (SSP) PCR, high-performance liquid chromatography (HPLC), mass-spectrometric genotyping, or sequencing; optionally wherein the sequencing comprises a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique; and optionally wherein the massively parallel sequencing (MPS) technique comprises next-generation sequencing (NGS).

16. The method of claim 1, wherein detecting the mutant ALK nucleic acid molecule in the sample comprises: (a) providing a plurality of nucleic acid molecules obtained from the sample, wherein the plurality of nucleic acid molecules comprises nucleic acid molecules corresponding to the mutant ALK nucleic acid molecule; (b) optionally, ligating one or more adapters onto one or more nucleic acid molecules from the plurality of nucleic acid molecules; (c) optionally, amplifying the one or more ligated nucleic acid molecules from the plurality of nucleic acid molecules; (d) optionally, capturing amplified nucleic acid molecules from the amplified nucleic acid molecules; (e) sequencing, by a sequencer, the captured nucleic acid molecules to obtain a plurality of sequence reads that represent the captured nucleic acid molecules, wherein one or more of the plurality of sequence reads correspond to the mutant ALK nucleic acid molecule; (f) analyzing the plurality of sequence reads; and (g) based on the analysis, detecting the mutant ALK nucleic acid molecule in the sample.

17. The method of claim 1, wherein detecting the mutant ALK nucleic acid molecule in the sample comprises: 152MOFO-358156824Docket No: 197102019040 (a) providing a plurality of nucleic acid molecules obtained from the sample, wherein the plurality of nucleic acid molecules comprises nucleic acid molecules corresponding to the mutant ALK nucleic acid molecule; (b) preparing a nucleic acid sequencing library from the plurality of nucleic acid molecules in the sample; (c) amplifying said library; (d) selectively enriching for one or more nucleic acid molecules comprising nucleotide sequences corresponding to the mutant ALK nucleic acid molecule in said library to produce an enriched sample; (e) sequencing the enriched sample, thereby producing a plurality of sequence reads; (f) analyzing the plurality of sequence reads for the presence of the mutant ALK nucleic acid molecule; and (g) detecting, based on the analyzing step, the mutant ALK nucleic acid molecule in the sample from the individual.

18. The method of claim 1, wherein the sample comprises a tissue biopsy sample; optionally wherein the sample is from a tumor biopsy or tumor specimen; and optionally wherein the sample comprises cells and / or nucleic acids from the cancer.

19. The method of claim 1, wherein the sample comprises a liquid biopsy sample; optionally wherein the sample comprises: (a) circulating tumor cells (CTCs); (b) cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof; or (c) blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva; and optionally wherein the sample comprises cells and / or nucleic acids from the cancer.

20. The method of claim 1, further comprising obtaining the sample from the individual. 153MOFO-358156824

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