Use of EGFR / HER2 tyrosine kinase inhibitors and / or HER2 / HER3 antibodies for the treatment of cancers with NRG1 fusions

Abstract

The present invention provides a method for selecting cancer patients for treatment using a covalent EGFR / HER2 TKI, a HER2 / HER3 targeted antibody, or a combination of a covalent EGFR / HER2 TKI and a HER2 / HER3 targeted antibody, and a method for treating such selected cancer patients. [Solution] Cancer patients are selected for treatment if their cancer contains an NRG1 fusion. The selected patients are then treated with a covalent EGFR / HER2 TKI, a HER2 / HER3 targeted antibody, or a combination of a covalent EGFR / HER2 TKI and a HER2 / HER3 targeted antibody.

Description

[Technical Field]

[0001] Reference to related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 967,275, filed on 29 January 2020, the entire contents of which are incorporated herein by reference.

[0002] 1. Field The present invention relates in general to the fields of medicine and oncology. More specifically, the present invention relates to a method for selecting cancer patients for treatment using a covalent EGFR / HER2 tyrosine kinase inhibitor (TKI), an anti-HER2 antibody, or a combination of a covalent EGFR / HER2 TKI and an anti-HER2 antibody, and a method for treating cancer patients thus selected. [Background technology]

[0003] 2. Explanation of related technologies NRG1 fusions occur in 0.3% of non-small cell lung cancers (NSCLC) and have been observed in several other cancer types, including gallbladder (0.5%), breast (0.2%), ovarian (0.4%), and colorectal (0.1%) cancers (Jonna et al., 2019). Common NRG1 fusion partners are CD74 (29% of NRG1 fusions), ATP1B1 (10% of NRG1 fusions), and SDC4 (7% of NRG1 fusions) (Jonna et al., 2019). NRG1 binds to the HER3 receptor, causing preferential heterodimerization with HER2 (Shin et al., 2018; Jung et al., 2015; Fernandez-Cuesta et al., 2014), which is one of the most potent forms of ERBB family signaling (Holbro et al., 2003). Previous reports have shown that targeting the HER2 / HER3 signaling pathway with a single activator may be effective in inhibiting ErbB signaling mediated by NRG1 fusions (Shin et al., 2018; Fernandez-Cuesta et al., 2014; Drilon et al., 2018). Previous reports have also shown that TKI treatment induces HER2 stabilization on the cell surface, and therefore enhances antibody binding and antitumor activity (Scaltriti et al., 2009; Robichaux et al., 2019). However, there are no approved targeted therapies for patients with NRG1 fusions. [Overview of the project]

[0004] overview In one embodiment, a method for treating a patient with cancer, (a) the process of determining or having determined whether the patient's cancer has an NRG1 fusion; (b) If the patient's cancer has an NRG1 fusion, the patient was selected or had been selected for treatment with a covalent EGFR / HER2 tyrosine kinase inhibitor (TKI); and (c) The process of administering or having administered a therapeutically effective dose of a covalent EGFR / HER2 TKI to selected patients. Methods including the above are provided herein. In some situations, process (a) is, (i) the stage of obtaining or having obtained a biological sample from a patient; and (ii) The step of performing or having performed an assay on a biological sample to determine whether the patient's cancer has an NRG1 fusion.

[0005] In one embodiment, a method for treating a patient having cancer is provided herein, comprising the step of administering a therapeutically effective amount of a covalent EGFR / HER2 TKI to the patient, wherein the cancer has an NRG1 fusion. In one embodiment, a composition comprising a therapeutically effective amount of a covalent EGFR / HER2 TKI for use in the treatment of a patient's cancer is provided herein, wherein the patient's cancer has an NRG1 fusion.

[0006] In one embodiment, a method for selecting patients with cancer for treatment using a covalent EGFR / HER2 TKI, (a) the process of determining or having determined whether the patient's cancer has an NRG1 fusion; (b) The process of selecting or having selected a patient for treatment with a covalent EGFR / HER2 TKI when the patient's cancer has an NRG1 fusion. Methods including the above are provided herein. In some situations, process (a) is, (i) the stage of obtaining or having obtained a biological sample from a patient; and (ii) The step of performing or having performed an assay on a biological sample to determine whether the patient's cancer has an NRG1 fusion. In some situations, the method is (c) The process of administering or having administered a therapeutically effective dose of a covalent EGFR / HER2 TKI to selected patients. It also includes.

[0007] In one embodiment, a method for treating a patient with cancer, (a) the process of determining or having determined whether the patient's cancer has an NRG1 fusion; (b) The process of selecting or having selected a patient for treatment with a HER2 / HER3 targeted antibody if the patient's cancer has an NRG1 fusion; and (c) The process of administering or having administered a therapeutically effective dose of a HER2 / HER3-targeted antibody to selected patients. Methods including the above are provided herein. In some situations, process (a) is, (i) the stage of obtaining or having obtained a biological sample from a patient; and (ii) The step of performing or having performed an assay on a biological sample to determine whether the patient's cancer has an NRG1 fusion.

[0008] In one embodiment, a method for treating a patient having cancer is provided herein, comprising the step of administering to the patient a therapeutically effective amount of a HER2 / HER3 targeted antibody, wherein the cancer has an NRG1 fusion. In one embodiment, a composition for use in the treatment of a patient's cancer is provided herein, comprising a therapeutically effective amount of a HER2 / HER3 targeted antibody, wherein the patient's cancer has an NRG1 fusion.

[0009] In one embodiment, a method for selecting patients with cancer for treatment using a HER2 / HER3 targeted antibody, (a) the process of determining or having determined whether the patient's cancer has an NRG1 fusion; (b) The process of selecting or having selected a patient for treatment with a HER2 / HER3 targeted antibody when the patient's cancer has an NRG1 fusion. Methods including the above are provided herein. In some situations, process (a) is, (i) the stage of obtaining or having obtained a biological sample from a patient; and (ii) the stage of performing or having performed an assay on the biological sample to determine that the patient's cancer has an NRG1 fusion, including. In some aspects, the method (c) the step of administering or having administered a therapeutically effective amount of a HER2 / HER3 targeting antibody to a selected patient further includes.

[0010] In one aspect, a method of treating a patient having cancer, (a) the step of determining or having determined whether the patient's cancer has an NRG1 fusion; (b) the step of selecting or having selected a patient for treatment with a covalent EGFR / HER2 TKI and a HER2 / HER3 targeting antibody if the patient's cancer has an NRG1 fusion; and (c) the step of administering or having administered a combined therapeutically effective amount of a covalent EGFR / HER2 TKI and a HER2 / HER3 targeting antibody to the selected patient is provided herein.

[0011] In one aspect, a method of treating a patient having cancer, including the step of administering to the patient a combined therapeutically effective amount of a covalent EGFR / HER2 TKI and a HER2 / HER3 targeting antibody, wherein the cancer has an NRG1 fusion, is provided herein. In some aspects, step (a) (i) the stage of obtaining or having obtained a biological sample from a patient; and (ii) the stage of performing or having performed an assay on the biological sample to determine that the patient's cancer has an NRG1 fusion, including. In one aspect, there is provided herein a composition for use in treating a patient's cancer, the composition comprising a therapeutically effective amount of a covalent EGFR / HER2 TKI and a HER2 / HER3 targeting antibody, wherein the patient's cancer has an NRG1 fusion.

[0012] In one aspect, there is provided herein a method of selecting a patient having cancer for treatment with a covalent EGFR / HER2 TKI and a HER2 / HER3 targeting antibody, the method comprising: (a) determining or having determined whether the patient's cancer has an NRG1 fusion; (b) selecting or having selected the patient for treatment with a covalent EGFR / HER2 TKI and a HER2 / HER3 targeting antibody if the patient's cancer has an NRG1 fusion. In some aspects, step (a) comprises: (i) obtaining or having obtained a biological sample from the patient; and (ii) performing or having performed an assay on the biological sample to determine that the patient's cancer has an NRG1 fusion. In some aspects, the method further comprises: (c) administering or having administered to the selected patient a combined therapeutically effective amount of a covalent EGFR / HER2 TKI and a HER2 / HER3 targeting antibody.

[0013] In some aspects of any of the aspects, the NRG1 fusion is an NRG1-DOC4 fusion, an NRG1-VAMP2 fusion, an NRG1-CLU fusion, an NRG1-SLC3A2 fusion, an NRG1-CD74 fusion, an NRG1-ATP1B1 fusion, or an NRG1-SDC4 fusion.

[0014] In some aspects of any aspect, the covalent EGFR / HER2 TKI is afatinib, neratinib, dacomitinib, tarloxo-TKI, pirotinib, or ibrutinib.

[0015] In some aspects of any embodiment, the method further comprises the step of administering a HER2 / HER3-targeted antibody to a patient. In some aspects, the HER2 / HER3-targeted antibody includes trastuzumab, pertuzumab, or T-DM1.

[0016] In some aspects of any aspect of the method, the method further includes the step of administering further anti-cancer therapy to the patient. In some aspects, the further anti-cancer therapy is surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, toxin therapy, immunotherapy, or cytokine therapy.

[0017] In some aspects of any of the embodiments, cancer is breast cancer, lung cancer, colorectal cancer, neuroblastoma, pancreatic cancer, brain tumor, stomach cancer, skin cancer, testicular cancer, prostate cancer, ovarian cancer, liver cancer, esophageal cancer, cervical cancer, head and neck cancer, melanoma, or glioblastoma. In some aspects, cancer is breast cancer or lung cancer.

[0018] In some aspects of any aspect, the patient has previously received at least one round of chemotherapy. In some aspects of any aspect, the method further includes a step of reporting the presence of an NRG1 fusion in the patient's cancer. In some aspects, the reporting step includes a step of preparing a written or electronic report. In some aspects, the method further includes a step of submitting the report to the subject, physician, hospital, or insurance company.

[0019] As used herein, “essentially absent” in terms of a particular component means that none of the particular component is intentionally included in the composition, and / or is present only as an impurity or in trace amounts. The total amount of a particular component resulting from any unintentional impurity of the composition is therefore far less than 0.05%, preferably less than 0.01%. A composition in which the amount of a particular component cannot be detected by standard analytical methods is most preferred.

[0020] As used herein, “a” or “an” may mean one or more. As used in a claim, the word “a” or “an” may mean one or more when used with the word “comprising.”

[0021] The use of the term “or” in the claims is used to mean “and / or” unless it is explicitly indicated that it refers only to substitutes, or unless the substitutes are mutually exclusive, although this disclosure supports the definitions that refer only to substitutes and “and / or.” As used herein, “another” may mean at least a second or more.

[0022] Throughout this application, the term “about” is used to indicate that a value includes inherent variations in error of the device or method used to determine the value, variations present among the subjects of study, or values ​​within 10 percent of the stated value.

[0023] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while illustrating preferred embodiments of the present invention, are given for illustrative purposes only, as various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing]

[0024] The following drawings form part of this specification and are included to further illustrate certain aspects of the invention. The invention can be better understood by referring to one or more of these drawings in combination with the detailed description of the particular embodiments presented herein. [Figure 1] Figure 1A: Bar graph showing the mean ± SEM IC50 values ​​for MDA17-VII cell lines treated with the indicated inhibitor for 72 hours. Figure 1B: Representative dose-response curve for MDA175-VII (NRG1-DOC4 fusion) treated with the indicated inhibitor for 72 hours. Cell viability was determined by the Cell Titer Glo assay. [Figure 2] Bar graph showing the mutant / WT EGFR ratio between MDA175-VII cells and Ba / F3 cells expressing WT EGFR. [Figure 3] Figure 3A: Representative dose-response curves of MDA175-VII (NRG1-DOC4 fusion) treated with anti-HER2 therapy for 72 hours, with and without low-dose treatment with a HER2 inhibitor. Cell viability was determined by the Cell Titer Glo assay. Figure 3B: Bar graph of mean ± SEM IC50 values ​​for MDA17-VII cell lines treated with the indicated inhibitors for 72 hours. The bars for each group, from left to right, represent +DMSO, +0.5nM pirotinib, +0.5nM tarloxotinib, and +0.1nM neratinib. Combinations with the anti-HER2 antibody trastuzumab were not included in the bar graph because IC50 values ​​could not be calculated. [Modes for carrying out the invention]

[0025] Detailed explanation Methods for treating cancer patients having an NRG1 fusion are provided herein. In particular, the methods include administering a covalent EGFR / HER2 TKI, a HER2 / HER3 targeted antibody, or a combination of a covalent EGFR / HER2 TKI and a HER2 / HER3 targeted antibody to cancer patients identified as having an NRG1 fusion. Furthermore, the methods include identifying and selecting cancer patients who may benefit from administration of a covalent EGFR / HER2 TKI, a HER2 / HER3 targeted antibody, or a combination of a covalent EGFR / HER2 TKI and a HER2 / HER3 targeted antibody by determining whether the patient's cancer has an NRG1 fusion.

[0026] I.NRG1 fusion The NRG1 fusion gene contains at least a portion of the NRG1 gene fused to a sequence from a different chromosomal location. “At least a portion” indicates that the entire NRG1 gene may be present in the fusion or as a portion of it. The fusion may have the coding sequences for at least exons 6, 7, and 8 of NRG1. Another way to define the NRG1 portion in the NRG1 fusion gene is that it contains the EGF-like domain of NRG1. The EGF-like domain is encoded at the 3' end of this gene and is required for binding to ErbB-3. The NRG1 fusion retains the in-frame coding region for the EGF-like domain. The portion of the NRG1 gene may be fused to a sequence from a different chromosomal location, resulting in the sequence being located at 5' or 3' relative to the portion of the NRG1 gene.

[0027] Preferably, the 3' end of the NRG1 gene can fuse with a sequence from a different chromosomal location. In particular, the NRG1 fusion gene is a fusion of the 3' end of the NRG1 gene with the 5' sequence of one of the genes selected from the group consisting of: DOC4 (teneurin transmembrane protein 4 (TENM4); protein Odd Oz / Ten-M homolog 4; tenascin-M4; Ten-M4; Ten-4; ODZ4; TNM4; Odz, Odd Oz / Ten-M homolog 4 (Drosophila); Odz, Odd Oz^en-M homolog 4; teneurin-4; KIAA1302; Doc4; ETM5; HGNC:29945; Entrez Also known as Gene:26011;Ensembl:ENSG00000149256;OMIM:610084; and UniProtKB:Q6N022);CD74(CD74 molecule;CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-related);CD74 molecule, major histocompatibility complex, class II invariant chain;HLA-DR antigen-related invariant chain;gamma chain of class II antigen;1a-related invariant chain;MHC HLA-DR gamma chain;HLA-DR-gamma;DHLAG;P33;HLA class II histocompatibility antigen gamma chain;1a antigen-related invariant chain;1a-gamma;HLADG;HGNC:1697;Entrez Also known as Gene:972;Ensembl:ENSG00000019582;OMIM:142790, and UniProtKB:P04233);TNFRSF10B (TNF receptor superfamily member 10b; tumor necrosis factor receptor superfamily, member 10b; TNF-related apoptosis-inducing ligand receptor 2; cell death receptor 5; TRAIL-R2; TRAILR2; KILLER; TRICK2; ZTNFR9; DR5; P53 regulatory DNA damage-inducing cell death receptor (killer); tumor necrosis factor receptor superfamily member 10B; tumor necrosis factor receptor-like protein ZTNFR9; death domain-containing receptor for TRAIL / Apo-2L; apoptosis-inducing protein TRICK2A / 2B; apoptosis-inducing receptor TRAIL-R2; cytotoxic TRAIL receptor-2; Fas-like protein; TRAIL receptor 2; CD262 antigen; KILLER / DR5; TRICK2A;TRICK2B;TRICKB;CD262;HGNC:11905;Entrez Also known as Gene:8795;Ensembl:ENSG00000120889;OMIM:603612; and UniProtKB:014763);CLU(Clusterin;Testosterone-inhibiting prostate message 2;Apolipoprotein J;Complement-related proteins SP-40,40;Complement cell lysis inhibitor;Complement cell lysis inhibitor;Sulfated glycoprotein 2;Ku70-binding protein 1;NA1 / NA2;TRPM-2;APO-J;APOJ;KUB1;CLI;Clusterin (Complement cell lysis inhibitor, SP-40,40, Sulfated glycoprotein 2, Testosterone-inhibiting prostate message 2, Apolipoprotein J);Aging-related gene 4 protein;Aging-related protein 4;SGP-2;SP-40;TRPM2;AAG4;CLU1;CLU2;SGP2;HGNC:2095;Entrez Also known as Gene:1191;Ensembl:ENSG00000120885;OMIM:185430; and UniProtKB:P10909); VAMP2 (Vestile-binding membrane protein 2; Synaptobrevin 2; SYB2; Vesicle-binding membrane protein 2; Synaptobrevin-2;HGNC:12643;Entrez Gene:6844;Ensembl:ENSG00000220205;OMIM:185881; and UniProtKB:P63027); SLC3A2 (Solute transporter family 3 member 2; Lymphocyte-activating antigen 4F2 large subunit; Solute transporter family 3 (activator of dibasic and neutral amino acid transport), member 2; Monoclonal antibody 4F2, TRA1.10, TROP4, and T4 Antigen identified by 3; solute transporter family 3 (amino acid transporter heavy chain), member 2; 4F2 cell surface antigen heavy chain; CD98 heavy chain; 4F2HC; MDU1; monoclonal antibody 4F2, antigen defined by heavy chain; monoclonal antibody 4F2 defined antigen; 4F2 heavy chain antigen; 4F2 heavy chain; CD98 antigen; CD98HC; 4T2HC; NACAE; CD98; 4F2; HGNC:11026; Entrez Gene:6520; Ensembl:ENSG00000168003; OMIM:158070;Also known as UniProtKB:P08195); RBPMS (RNA Binding Protein With Multiple Splicing); cardiac and RRM expression sequence; HERMES; RNA-Binding Protein With Multiple Splicing; RBP-MS; HGNC:19097; Entrez Gene:11030; Ensembl:ENSG00000157110; OMIM:601558; and also known as UniProtKB:Q93062); WRN (Werner syndrome RecQ-like helicase; DNA helicase, RecQ-like type 3; RecQ protein-like 2; exonuclease WRN; RECQL2; RECQ3; Werner syndrome ATP-dependent helicase; Werner syndrome, RecQ helicase-like; Werner syndrome; EC 3.6.4.12; EC 3.1.-.-; EC 3.6.1;RECQL3;HGNC:12791;Entrez Gene:7486;Ensembl:ENSG00000165392;OMIM:604611 and UniProtKB:Q14191;SDC4 (Syndecane 4 (amphiglycan, lyudocan);Syndecane proteoglycan 4;Lyudocan core protein;Amphiglycan;SYND4;Lyudocan amphiglycan;Syndecane-4;HGNC:10661;Entrez Gene:6385;Ensembl:ENSG00000124145;OMIM:600017;and UniProtKB:P31431);KIF13B;SLECA2;PDE7A;ATP1B1;CDK1;BMPRIB;MCPH1;and RAB2IL1.

[0028] Certain aspects of this disclosure relate to determining whether a subject has an NRG1 fusion. Detection methods are known in the art, including PCR analysis, nucleic acid sequencing, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), and comparative genomic hybridization (CGH).

[0029] Samples suitable for use in the methods described herein include genetic material, such as genomic DNA (gDNA). Genomic DNA is typically extracted from biological samples such as blood or mucosal exfoliation of the oral cavity, but may also be extracted from other biological samples, including urine, tumors, or sputum. Samples are considered to contain, by themselves, nucleated cells (e.g., blood or oral cells) or tissues isolated from a subject, typically including tumor tissue. Methods and reagents for obtaining, processing, and analyzing samples are known in the art. In some embodiments, samples are obtained with the help of a healthcare provider, for example, by blood collection or obtaining a tumor biopsy. In some embodiments, samples are obtained without the help of a healthcare provider, for example, if the sample is obtained non-invasively, such as a sample containing oral cells obtained using a cheek swab or brush, or a mouthwash sample.

[0030] In particular, patient samples can be any tissue or fluid from the body containing nucleic acids from the cancer in question. In certain embodiments, the sample may be a blood sample containing circulating tumor cells or cell-free DNA. In other embodiments, the sample may be a tissue such as tumor tissue. Tumor tissue may be fresh-frozen or formalin-fixed and paraffin-embedded (FFPE).

[0031] In some cases, biological samples can be processed for DNA isolation. For example, DNA in a cell or tissue sample can be separated from other components of the sample. Cells can be collected from a biological sample using standard techniques known in the art. For example, cells can be collected by centrifugation of the cell sample and resuspending the pelleted cells. Cells can be resuspended in a buffering solution such as phosphate-buffered saline (PBS). After centrifugation of the cell suspension to obtain a cell pellet, the cells can be lysed to extract DNA, such as gDNA. The sample can be concentrated and / or purified to isolate the DNA. All samples obtained from a subject, including those subjected to any kind of further processing, are considered to be obtained from the subject. For example, genomic DNA can be extracted from a biological sample using conventional methods, including phenol extraction. Alternatively, genomic DNA can be extracted using kits such as the QIAamp® tissue kit (Qiagen, Chatsworth, Calif.) or the Wizard® genomic DNA purification kit (Promega).

[0032] Nucleic acid amplification can be achieved, if desired, using methods known in the art, such as PCR. For example, a sample (e.g., a sample containing genomic DNA) is obtained from a subject. Then, the DNA in the sample is examined to determine the identity of the NRG1 fusion, as described herein. The NRG1 fusion can be detected by any method described herein, for example, by sequencing, or by hybridization of genes, RNA, or cDNA in the genomic DNA to a nucleic acid probe, such as a DNA probe (including cDNA and oligonucleotide probes) or an RNA probe. Nucleic acid probes can be designed to hybridize specifically or preferentially with a particular NRG1 fusion.

[0033] A set of probes typically refers to a set of primers, usually a primer pair, and / or probes labeled to a detectable degree, used to detect a target genetic variant (e.g., NRG1 fusion) used in the viable therapeutic recommendations of this disclosure. The primer pair is used in an amplification reaction to define the amplification product corresponding to the NRG1 fusion. The set of amplification products is detected with the matching set of probes. In an exemplary embodiment, this method is used to detect a set of target genetic variants, e.g., NRG1 fusion, using TaqMan (商標) (Roche Molecular Systems, Pleasanton, Calif.) Assays can be used. In one embodiment, the probe set is a set of primers used to generate an amplified product to be detected in a nucleic acid sequencing reaction, such as a next-generation sequencing reaction. In these embodiments, for example, AmpliSEQ (商標) (Life Technologies / Ion Torrent, Carlsbad, Calif.) or TruSEQ (商標) (Illumina, San Diego, Calif.) Technology can be used.

[0034] The analysis of nucleic acid markers can be carried out using techniques known in the art, including, but not limited to, sequence analysis and electrophoretic analysis. Examples of sequence analysis include Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing, solid-phase sequencing, sequencing using mass spectrometry such as matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS), and sequencing by hybridization. Examples of electrophoretic analysis include slab gel electrophoresis such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturant concentration gradient gel electrophoresis. Furthermore, next-generation sequencing methods can be carried out using commercially available kits and instruments from companies, such as Life Technologies / Ion Torrent PGM or Proton, Illumina HiSEQ or MiSEQ, and Roche / 454 next-generation sequencing systems.

[0035] Other methods of nucleic acid analysis include direct manual sequencing (U.S. Patent No. 5,288,644); automated fluorescence sequencing; single-stranded higher-order polymorphism analysis (SSCP); gel electrophoresis using a constant concentration of denaturant without a gradient (CDGE); two-dimensional gel electrophoresis (2DGE or TDGE); conformational high-sensitivity gel electrophoresis (CSGE); denaturant concentration gradient gel electrophoresis (DGGE); denaturation high-performance liquid chromatography (DHPLC); infrared matrix-assisted laser desorption / ionization (IR-MALDI) mass spectrometry; mobility shift analysis; restriction enzyme analysis; quantitative real-time PCR; heteroduplex analysis; chemical mismatch cleavage (CMC); RNase protection assay; use of polypeptides that recognize nucleotide mismatches, such as the E. coli mutS protein; allele-specific PCR; and combinations of such methods. See, for example, U.S. Patent Application Publication No. 2004 / 0014095, which is incorporated in its entirety herein by reference.

[0036] For example, a method for identifying NRG1 fusions in a sample includes the steps of contacting a nucleic acid probe capable of specifically hybridizing to the nucleic acid encoding the NRG1 fusion with a nucleic acid derived from the sample, and detecting the hybridization. In certain embodiments, the probe is a radioactive isotope ( 3 H, 32 P, or 33 The probe is labeled to a detectable degree with a fluorescent agent (rhodamine or fluorescein), or a chromogenic agent, etc. In certain embodiments, the probe is an antisense oligomer, e.g., PNA, morpholino-phosphoramidate, LNA, or 2'-alkoxyalkoxy. The probe may be about 8 to about 100 nucleotides, or about 10 to about 75, or about 15 to about 50, or about 20 to about 30. In another aspect, the probe of the present disclosure is provided in a kit for identifying NRG1 fusions in a sample, the kit containing oligonucleotides that specifically hybridize to a particular NRG1 fusion. The kit may further include instructions for treating patients with tumors containing NRG1 fusions using a covalent EGFR / HER2 TKI, a HER2 / HER3 targeted antibody, or a combination of a covalent EGFR / HER2 TKI and a HER2 / HER3 targeted antibody, based on the results of a hybridization test using the kit.

[0037] II. Covalent EGFR / HER2 tyrosine kinase inhibitors Small molecule covalent inhibitors, also known as irreversible inhibitors, are a class of inhibitors that exert their biological function through irreversible covalent bonding to target protein residues. Compared to non-covalent inhibitors, covalent inhibitors have enhanced affinity for the target protein due to covalent bonding, which is the fundamental reason for the high biological activity of covalent inhibitors.

[0038] The covalent EGFR / HER2-TKIs used in this invention include, but are not limited to, afatinib (BIBW-2992), neratinib (HKI-272 or PB272), dacomitinib (PF-00299804; Pfizer), tarloxotinib (TH-4000; PR-610), pirotinib, ibrutinib, YH25448, rosiletinib (CO-1686), osimertinib (Tagrisso), olmutinib (Olita), and nacotinib (A SP8273), Nazartinib (EGF816), PF-06747775 (Pfizer), Abitinib (AC0010), EAI045, PF-06459988 (Pfizer), Tesebatinib (XL647; EXEL-7647; KD-019), TAS6417 (CL-081), AST2818, TAK-788, Transtinib, WZ-3146, WZ8040, CNX-2006, Brigatinib (Alunbrig; Ariad) Examples include CUDC-101, PD153035, peritinib, AEE788 (NVP-AEE788), AST-1306, AZ5104, rifilafenib (BGB-283), canertinib, and CL-387785 (EKI-785).

[0039] For example, afatinib is also known as BIBW-2992. Afatinib is marketed by Boehringer Ingelheim as GILOTRIF®. Afatinib covalently binds to the kinase domains of EGFR (ErbBl), HER2 (ErbB2), and HER4 (ErbB4), irreversibly inhibiting the autophosphorylation of tyrosine kinases and resulting in downregulation of ErbB signaling.

[0040] For example, "neratinib" is also known as HKI-272. Neratinib is marketed as NERLYNX® by Puma Biotechnology, Inc. Neratinib is a kinase inhibitor that irreversibly binds to the epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and HER4.

[0041] For example, "AST-1306" is also known as "allitinib" or "AST1306" and is manufactured by Shanghai Allist Pharmaceuticals, China. AST-1306 is a potent, selective, and irreversible ErbB2 and EGFR inhibitor. AST-1306 inhibits the enzymatic activity of wild-type epidermal growth factor receptor (EGFR) and ErbB2, as well as EGFR-resistant mutants, in both cell-free and cell-based systems.

[0042] III.HER2 / HER3 targeting antibody As used herein, "HER2 / HER3-targeted antibodies" include any molecule that interferes with the function of HER2 and / or HER3. Therefore, examples of HER2 / HER3-targeted antibodies include anti-HER2 antibodies (e.g., trastuzumab or pertuzumab), anti-HER3 antibodies, and anti-HER2 / HER3 bispecific antibodies (e.g., antibodies disclosed in WO2018 / 182422 or MCLA-128). HER2 / HER3-targeted antibodies can prevent the formation of HER2 / HER2 dimers and / or HER2 / HER3 dimers (e.g., trastuzumab or pertuzumab). In some cases, the HER2 / HER3-targeted antibody may also be an antibody-drug conjugate (e.g., T-DM1 or U3-1402).

[0043] In certain embodiments, HER2 / HER3-targeted antibodies include trastuzumab (Genentech and Roche), trastuzumab emtansine (T-DM1; Genentech and Roche), pertuzumab (Genentech), ertumaxomab (Fresenius), margetuximab (MacroGenics), MCLA-128 (xenoctuzumab; Merus), MM-111 (Merrimack), MM-121 (Merrimack), CT-P06 (Celltrion), GSK2849330 (GlaxoSmithKline), PF-05280014 (Pfizer), MM-302 (Merrimack), SB3 (Merck & Co), and CMAB302 (Shanghai CP). These include Guojian, RG7116 (lemretuzumab; Genentech / Roche), TrasGEX (Glycotope), ARX788 (Ambrx and Zhejiang Medicine), SYD985 (Synthon), FS102 (Bristol-Myers Squibb and f-star), BCD-022 (Biocad), ABP 980 (Amgen), DS-8201a (Daiichi Sankyo), HLX02 (Shanghai Henlius), SAR256212 (Sanofi Oncology), RG7597 (Genentech), U3-1402 (Daiichi Sankyo), or CANMAb (Biocon and Mylan).

[0044] Trastuzumab (CAS 180288-69-1, HERCEPTIN®, huMAb4D5-8, rhuMAb HER2, Genentech) is a humanized IgG1 kappa monoclonal antibody that selectively and with high affinity binds to the extracellular domain of the human epidermal growth factor receptor 2 protein, HER2 (ErbB2) (U.S. Patent Nos. 5,677,171; 5,821,337; 6,054,297; 6,165,464; 6,339,142; 6,407,213; 6,639,055; 6,719,971; 6,800,738; 7,074,404). Trastuzumab contains a human framework region with a complementarity-determining region of a mouse antibody (4D5) that binds to HER2. Trastuzumab binds to the HER2 antigen, thereby inhibiting the growth of cancer cells. Trastuzumab has been shown to inhibit the proliferation of HER2-overexpressing human tumor cells in both in vitro assays and in animals. Trastuzumab is a mediator of antibody-dependent cell-mediated cytotoxicity, or ADCC.

[0045] Trastuzumab emtansine, also known as ado-trastuzumab emtansine and marketed under the trade name KADCYLA®, is an antibody-drug conjugate consisting of trastuzumab, a humanized monoclonal antibody covalently bound to the cytotoxic substance emtansine (DM1). Trastuzumab alone stops cancer cell growth by binding to the HER2 receptor, but trastuzumab emtansine undergoes receptor-mediated internal translocation into cells, where it is catabolized in lysosomes, releasing DM1-containing catabolic products that subsequently bind to tubulin, causing mitotic arrest and cell death. Trastuzumab binding to HER2 prevents homodimerization or heterodimerization of the receptor (HER2 / HER3), ultimately inhibiting the activation of MAPK and PI3K / AKT cell signaling pathways. Since the monoclonal antibody targets HER2 and HER2 is overexpressed only in cancer cells, the conjugate specifically delivers the cytotoxic substance DM1 to tumor cells. The conjugate is abbreviated as T-DM1. T-DM1 can be administered at doses of 2-3 mg / kg, for example, 3.6 mg / kg. T-DM1 can be administered by intravenous infusion.

[0046] Pertuzumab (CAS Registry No. 380610-27-5, OMNITARG®, 2C4, Genentech) is a recombinant humanized monoclonal antibody that inhibits HER2 dimerization (U.S. Patent Nos. 6,054,297; 6,407,213; 6,800,738; 6,627,196; 6,949,245; 7,041,292). Pertuzumab contains a human IgG1(x) framework sequence. Pertuzumab and trastuzumab target different extracellular regions of the HER2 tyrosine kinase receptor. Pertuzumab binds to an epitope within subdomain 2 of HER2, while the epitope for trastuzumab is localized to subdomain 4. Pertuzumab blocks the ability of the HER2 receptor to cooperate with other HER receptor family members, namely HER1 / EGFR, HER3, and HER4 (U.S. Patent No. 6,949,245). In cancer cells, by interfering with HER2's ability to cooperate with other HER family receptors, it blocks cellular signaling, which can ultimately lead to inhibition of cancer cell growth and cancer cell death.

[0047] The antibodies described herein can be defined, firstly, by their binding specificity. Those skilled in the art can determine whether a given antibody falls within the scope of these claims by evaluating its binding specificity / affinity using techniques well known to those skilled in the art. Various techniques known to those skilled in the art can be used to determine whether an antibody interacts with a polypeptide or protein. Exemplary techniques include, for example, conventional cross-blocking assays. Cross-blocking can be measured using various binding assays such as ELISA, biolayer interferometry, or surface plasmon resonance. Other methods include alanine scanning mutation analysis, peptide blot analysis, peptide cleavage analysis, single-particle reconstruction, cryoEM, or high-resolution electron microscopy techniques using tomography, crystallographic studies, and NMR analysis.

[0048] This disclosure includes antibodies that can bind to the same epitope or a portion of said epitope. Furthermore, this disclosure also includes antibodies that compete with any of the specific exemplary antibodies described herein for binding to a target or fragment thereof. It can be easily determined whether an antibody binds to the same epitope as a reference antibody or competes with the reference antibody for binding using methods known and customary in the art. For example, to determine whether a test antibody binds to the same epitope as the reference, the reference antibody is bound to the target under saturated conditions. The ability of the test antibody to bind to the target molecule is then evaluated. If the test antibody can bind to the target molecule after saturated binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody cannot bind to the target molecule after saturated binding with the reference antibody, the test antibody may bind to the same epitope as the reference antibody.

[0049] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. That is, as measured by a competitive binding assay, one antibody in 1, 5, 10, 20, or 100-fold excess inhibits the binding of the other by at least 50%, but preferably 75%, 90%, or even 99%. Alternatively, the two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other. The two antibodies have overlapping epitopes if several amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other.

[0050] Next, further conventional experiments (e.g., peptide mutation and binding analysis) can be performed to determine whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody, or whether steric blockage (or another phenomenon) is involved in the observed lack of binding. These types of experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. Structural studies using EM or crystallography can also indicate whether two competing antibodies recognize the same epitope.

[0051] In another context, antibodies can be defined by their variable sequences, which may include further "framework" regions. Furthermore, antibody sequences may optionally be altered from these sequences using the methods described in more detail below. For example, nucleic acid sequences may be altered by (a) the variable region being separated from the constant domains of the light and heavy chains, (b) the nucleic acid being altered from the above but without affecting the residues encoded thereby, (c) the nucleic acid being altered by a given percentage of homology, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or (d) the nucleic acid being altered by (e.g., at a temperature of approximately 50°C to approximately 70°C and approximately 0.02M to approximately 0.15M (e) The amino acids may be altered from those described above by their ability to hybridize under high stringency conditions, such as those exemplified by low-salt and / or high-temperature conditions brought about by NaCl, (f) by a given percentage of homology, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or by enabling conservative substitutions (described below).

[0052] When comparing polynucleotide and polypeptide sequences, two sequences are said to be "identical" if, after being aligned to the greatest extent possible, their nucleotide or amino acid sequences are the same, as described below. Comparison between two sequences is typically performed by comparing the sequences across a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to at least about 20 consecutive positional segments, typically 30 to about 75 and 40 to about 50, over which the sequences can be compared to the same number of consecutive positional reference sequences after the two sequences have been optimally aligned.

[0053] Optimal alignment of sequences for comparison can be performed using the Megaalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.) with default parameters.This program incorporates several alignment schemes described in the following references: Dayhoff, MO (1978) A model of evolutionary change in proteins--Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogeny pp. 626-645; Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, DG and Sharp, PM (1989) CABIOS 5:151-153; Myers, EW and Muller W. (1988) CABIOS 4:11-17; Robinson, ED (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425;Sneath, PHA and Sokal, RR (1973) Numerical Taxonomy--the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, WJ and Lipman, DJ (1983) Proc. Natl. Acad., Sci. USA 80:726-730.

[0054] Alternatively, optimal alignment of sequences for comparison can be performed by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, the similarity search method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computer execution of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA from Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis), or by inspection.

[0055] One specific example of an algorithm suitable for determining sequence identity percentage and sequence similarity percentage is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used to determine the sequence identity percentage for the polynucleotides and polypeptides of this disclosure, for example, using the parameters described herein. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. Due to the rearranged nature of antibody sequences and the non-constant length of each gene, multiple rounds of BLAST searches are required for a single antibody sequence. Furthermore, manual assembly of different genes is difficult and prone to errors. The sequence analysis tool IgBLAST (World Wide Web, ncbi.nlm.nih.gov / igblast / ) reveals matches to germline V, D, and J genes, details of rearrangement junctions, and depictions of Ig V domain framework regions and complementarity-determining regions. IgBLAST can analyze nucleotide or protein sequences, process sequences in batches, and allows simultaneous searches against germline gene databases and other sequence databases to minimize the chance of missing potentially best-matching germline V genes.

[0056] In one example, the cumulative score can be calculated for a nucleotide sequence using parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). The extension of word hits in each direction stops when: the cumulative alignment score decreases by amount X from its maximum achieved value; the cumulative score becomes zero or less due to the accumulation of one or more negative-scoring residue alignments; or when the end of any sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses, by default, word lengths of 11 (W) and 10 (E), as well as BLOSUM62 score matrix alignment (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915), with M=5, N=-4, and a comparison of both strands.

[0057] For amino acid sequences, the cumulative score can be calculated using a score matrix. Word hit extension in each direction occurs when: the cumulative alignment score decreases by amount X from its maximum achieved value; the cumulative score becomes zero or less due to the accumulation of one or more negative-scoring residue alignments; or the extension stops when it reaches the end of any sequence. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.

[0058] One approach is to determine the "percentage of sequence identity" by comparing two optimally aligned sequences across a comparison window of at least 20 positions, where some of the polynucleotide or polypeptide sequences within the comparison window may contain no more than 20 percent, typically 5–15 percent or 10–12 percent, additions or deletions (i.e., gaps) compared to a reference sequence (which is free of additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues exist in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0059] Another way to define an antibody is as a “derivative” of any of the antibodies and their antigen-binding fragments described. The term “derivative” refers to an antibody or its antigen-binding fragment that binds immunospecifically to an antigen but contains one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications compared to the “parent” (or wild-type) molecule. Such amino acid substitutions or additions may introduce amino acid residues that are naturally present (i.e., encoded in DNA) or not naturally present. The term “derivative” also includes variants having altered CH1, hinge, CH2, CH3, or CH4 regions, for example, to form an antibody, etc., which has a variant Fc region exhibiting enhanced or impaired effector or binding properties. The term “derivative” further encompasses amino acids that may have undergone non-amino acid modifications, such as glycosylation (e.g., changes in the content of mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycolneuraminic acid, etc.), acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or linkage to cellular ligands or other proteins. In some embodiments, changes in glycosylation modulate one or more of the following: antibody solubilization, facilitation of intracellular transport and secretion of antibodies, enhancement of antibody assembly, conformational integrity, and antibody-mediated effector function. In certain embodiments, changes in glycosylation enhance antibody-mediated effector function compared to antibodies lacking glycosylation. Glycoscopy leading to changes in antibody-mediated effector function is well known in the art.

[0060] Derivative antibodies or antibody fragments can be generated using manipulated sequences or glycosylation states to confer preferred levels of activity in antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), or antibody-dependent complement deposition (ADCD) functions, as measured by bead-based or cell-based assays or in vivo studies in animal models.

[0061] Derivative antibodies or antibody fragments can be modified by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formulation, and metabolic synthesis of tunicamycin. In one embodiment, the antibody derivative will have a function similar to or identical to that of the parent antibody. In another embodiment, the antibody derivative will exhibit altered activity compared to the parent antibody. For example, the derivative antibody (or its fragment) may be able to bind more firmly to its epitope or be more resistant to proteolysis than the parent antibody.

[0062] IV. Treatment Methods The present invention provides a method for treating cancer patients using a covalent EGFR / HER2 TKI, a HER2 / HER3 targeted antibody, or a combination of a covalent EGFR / HER2 TKI and a HER2 / HER3 targeted antibody. Such treatments can also be combined with other therapeutic regimens, such as chemotherapy or immunotherapy. Certain aspects of the present invention can be used to select cancer patients for treatment based on the presence of NRG1 fusions in the patient's cancer cells. In various aspects, approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cells constituting the cancer may contain NRG1 fusions, indicating that the patient is a candidate for treatment. In some aspects, the patient's cancer cells lack mutations in EGFR T790 and / or EGFR C797. In some cases, the patient's cancer cells lack mutations in HER2 T798 and / or HER2 C805.

[0063] In certain scenarios, the presence of NRG1 fusions was determined by analyzing genomic samples derived from the subject. In some scenarios, genomic samples were isolated from saliva, blood, urine, or tumor tissue. In specific scenarios, the presence of NRG1 fusions was determined by nucleic acid sequencing (e.g., DNA sequencing of circulating free DNA from tumor tissue or plasma) or PCR analysis.

[0064] In certain situations, covalent EGFR / HER2 TKIs and / or HER2 / HER3 targeted antibodies are administered intravenously, subcutaneously, intraosseously, orally, percutaneously, by sustained release, controlled release, delayed release, as suppositories, or sublingually. In some situations, administration of covalent EGFR / HER2 TKIs and / or HER2 / HER3 targeted antibodies includes topical, regional, or systemic administration. In certain situations, covalent EGFR / HER2 TKIs and / or HER2 / HER3 targeted antibodies are administered two or more times, for example, daily, every other day, or weekly. These can be administered by different routes and on different schedules.

[0065] In some cases, covalent EGFR / HER2 TKIs are administered before or after HER2 / HER3-targeted antibodies, for example, with a delay of 1, 2, 3, 4, 5, 6, 7 days, 2 weeks, 3 weeks, 1 month, or more. In some cases, covalent EGFR / HER2 TKIs are administered concurrently with HER2 / HER3-targeted antibodies.

[0066] As used herein, the terms “subject” or “patient” refer to any individual on which the Method of Subject is performed. Generally, the patient is a human, but as will be recognized by those skilled in the art, the patient may also be an animal. Thus, other animals are included in the definition of patient, including mammals, e.g., rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, livestock, e.g., cattle, horses, goats, sheep, pigs, etc., as well as primates (including monkeys, chimpanzees, orangutans, and gorillas).

[0067] "Treatment" and "to treat" refer to the administration or application of a therapeutic agent to a subject or the implementation of a procedure or modality to a subject, with the aim of obtaining a therapeutic benefit from a disease or health-related condition. For example, treatment may include administered chemotherapy, immunotherapy, radiotherapy, surgery, or any combination thereof.

[0068] The methods described herein are useful for inhibiting the survival or proliferation of cells (e.g., tumor cells), treating proliferative disorders (e.g., cancer, psoriasis), and treating pathogenic infections. Generally, the terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell growth. More specifically, cancers treated in connection with the methods provided herein include, but are not limited to, solid tumors, metastatic cancers, or non-metastatic cancers. In certain embodiments, cancer may occur in the lungs, kidneys, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, liver, nasopharynx, neck, ovaries, pancreas, prostate, skin, stomach, testes, tongue, or uterus.

[0069] Cancer can be, but is not limited to, the following histological types: neoplasms, malignant; carcinoma; non-small cell lung cancer; kidney cancer; renal cell carcinoma; clear cell carcinoma; lymphoma; blastoma; sarcoma; carcinoma, undifferentiated; meningioma; brain tumor; oropharyngeal cancer; nasopharyngeal cancer; biliary tract cancer; pheochromocytoma; islet cell carcinoma; Li-Fraumeni tumor; thyroid cancer; parathyroid cancer; pituitary tumor; adrenal tumor; osteogenic sarcoma; neuroendocrine tumor; breast cancer; lung cancer; head and neck cancer; prostate cancer; esophageal cancer; tracheal cancer; liver cancer; bladder cancer; stomach cancer; pancreatic cancer; ovarian cancer; uterine cancer; cervical cancer; testicular cancer Colon cancer; rectal cancer; skin cancer; giant cell and spindle cell carcinoma; small cell carcinoma; small cell lung cancer; papillary carcinoma; oral cancer; oropharyngeal cancer; nasopharyngeal cancer; respiratory cancer; genitourinary cancer; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; hair matrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrointestinal cancer; gastrinoma, malignant; bile duct cancer; hepatocellular carcinoma; mixed type of hepatocellular carcinoma and bile duct cancer; cord-like adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma within adenomatous polyp; adenocarcinoma, familial adenomatous polyposis; solid tumors; carcinoid tumors, malignant; bronchioloalveolar carcinoma; papillary adenocarcinoma; chromophilic Eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenal cortical carcinoma; endometrioid carcinoma; cutaneous adnexal carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; ceruminous gland carcinoma; mucosal epidermal carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, breast; acinar cell carcinoma; adenosquamous cell carcinoma; adenocarcinoma associated with squamous metaplasia; thymoma, malignant; ovarian stromal carcinoma, malignant; theca cell carcinoma, malignant ; Granulosa cell tumor, malignant; Androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; Lipid cell tumor, malignant; Paraganglioma, malignant; Extramammary paraganglioma, malignant; Pheochromocytoma; Angioglobulosarcoma; Malignant melanoma; Melanin-deficient melanoma; Superficial spreading melanoma; Malignant melanoma within a giant pigmented nevus; Lentigo malignant melanoma; Acral mole melanoma; Nodular melanoma; Epithelioid cell melanoma; Blue nevus, malignant; Sarcoma; Fibrosarcoma; Fibrous histiocytoma, malignant; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Fetal rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Mixed tumor, malignant;Mixed Müllerian tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymal tumor, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; undifferentiated germ cell tumor; embryonic cancer; teratoma, malignant; ovarian goiter, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangioectocytoma, malignant; lymphangiosarcoma; osteosarcoma; paraosteal osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's tumor Sarcoma; odontogenic tumor, malignant; ameloblastoma; ameloblastoma, malignant; ameloblastoma-fibrosarcoma; endocrine or neuroendocrine cancer or hematopoietic cancer; pineal gland tumor, malignant; chordoma; central or peripheral nervous system tissue cancer; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectoderm tumor; cerebellar sarcoma; ganglioblastoma; neuroblastoma; reticular Membryoblastoma; Olfactory neuroma; Meningioma, malignant; Neurofibrosarcoma; Schwannoma, malignant; Granulocyte tumor, malignant; B-cell lymphoma; Malignant lymphoma; Hodgkin's disease; Hodgkin; Low-grade / follicular non-Hodgkin lymphoma; Paragranuloma; Malignant lymphoma, small lymphocytic; Malignant lymphoma, large cell type, diffuse; Malignant lymphoma, follicular; Mycosis fungoides; Mantle cell lymphoma; Waldenström macroglobulinemia; Other specific non-Hodgkin lymphomas; Malignant Histiocytic proliferative disorders; multiple myeloma; mast cell sarcoma; immunoproliferative bowel disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myelosarcoma; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and hairy cell leukemia.

[0070] As used throughout this application, the terms “therapeutic benefit” or “therapeutically effective” refer to any medical treatment of the condition that promotes or enhances the healthy state of the subject. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of the disease. For example, cancer treatment may involve, for instance, a reduction in the invasiveness of the tumor, a reduction in the rate of cancer growth, or a prevention of metastasis. Cancer treatment may also refer to extending the survival of a subject with cancer.

[0071] Similarly, a patient's effective response to treatment, or patient “responsiveness,” refers to the clinical or therapeutic benefit given to a patient who is at risk of or suffering from a disease or disability. Such benefits may include cellular or biological responses, complete responses, partial responses, stable disease (no progression or relapse), or responses with late relapse. For example, an effective response may be a reduction in tumor size or progression-free survival in a patient diagnosed with cancer.

[0072] Regarding the treatment of neoplasms, depending on the stage of the neoplasm, treatment involves one or a combination of the following therapies: surgery to remove neoplastic tissue, radiotherapy, and chemotherapy. Other treatment regimens can be combined with the administration of anticancer agents, such as therapeutic compositions and chemotherapeutic agents. For example, a patient treated with such anticancer agents may also undergo radiotherapy and / or surgery.

[0073] Regarding the treatment of a disease, the appropriate dosage of the therapeutic composition is considered to depend on the type of disease being treated as defined above, the severity and course of the disease, previous therapies, the patient's medical history and response to the drug, and the physician's discretion. The drug may be administered to the patient in a single dose or over a series of treatments.

[0074] Methods and compositions comprising combination therapies enhance therapeutic or protective effects and / or increase the therapeutic effect of another anti-cancer or anti-hypertrophy therapy. Therapeutic and prophylactic methods and compositions can be provided in combined amounts effective in achieving a desired effect, e.g., the death of cancer cells and / or the inhibition of cell hyperproliferation. Tissues, tumors, or cells can be brought into contact with one or more compositions or pharmacological preparations containing one or more agents, or by bringing tissues, tumors, and / or cells into contact with two or more different compositions or preparations. Such combination therapies are also intended to be used in conjunction with radiotherapy, surgery, or immunotherapy.

[0075] Combination administration can include simultaneous administration of two or more agents in the same dosage form, simultaneous administration of different dosage forms, and separate administration. That is, the therapeutic composition of the subject and another therapeutic agent can be formulated together in the same dosage form and administered simultaneously. Alternatively, the therapeutic composition of the subject and another therapeutic agent can be administered simultaneously, in which case both agents exist in separate formulations. In another alternative, a therapeutic agent may be administered, followed immediately by another therapeutic agent, or vice versa. In separate administration protocols, the therapeutic composition of the subject and another therapeutic agent may be administered separated by several hours, or separated by several days.

[0076] The first anticancer treatment can be administered before, between, or after the second anticancer treatment, or in various combinations. Administration can range from simultaneous to several minutes to several days to several weeks. In the manner in which the first treatment is delivered to the patient separately from the second treatment, it will generally be ensured that there is no gap in the effective period between each delivery, and as a result, the advantageous combined effect of the two compounds will still be exerted on the patient. In such cases, it is intended that the first and second therapies can be delivered to the patient within approximately 12-24 or 72 hours of each other, or more specifically, within approximately 6-12 hours of each other. In some situations, it may be desirable to significantly extend the treatment period, in which case several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) may pass between each administration.

[0077] In certain embodiments, one course of treatment is expected to last 1 to 90 days or more (including the days in between). One drug may be administered on any day between day 1 and day 90 (including the days in between), or in any combination thereof, and another drug may be administered on any day between day 1 and day 90 (including the days in between), or in any combination thereof. One or more doses of the drug may be administered to the patient within a single day (24-hour cycle). Furthermore, it is expected that there will be a period after one course of treatment during which no anti-cancer treatment is administered. This period may last 1 to 7 days and / or 1 to 5 weeks and / or 1 to 12 months or more (including the days in between), depending on the patient's condition, e.g., prognosis, physical strength, health status, etc. It is anticipated that the treatment cycle will be repeated as needed.

[0078] Various combinations can be used. For example, (a) the covalent EGFR / HER2 TKI is "A" and the HER2 / HER3 targeted antibody is "B"; or (b) the covalent EGFR / HER2 TKI is "A" and another anticancer therapy is "B," either alone or in combination with a HER2 / HER3 targeted antibody; or (c) the HER2 / HER3 targeted antibody is "A" and another anticancer therapy is "B," either alone or in combination with a covalent EGFR / HER2 TKI. TIFF2026065076000002.tif27128.

[0079] The administration of any compound or therapy of the present invention to a patient shall follow general protocols for the administration of such compounds, taking into consideration the toxicity of the agent, if any. Accordingly, in some embodiments, there is a step to monitor for toxicity that may result from the combination therapy.

[0080] 1.Chemotherapy A wide variety of chemotherapeutic agents can be used in accordance with the present invention. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agents" include compounds or compositions administered in cancer treatment. These agents or drugs are categorized according to their mode of activity within cells, for example, whether they affect the cell cycle and at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to induce chromosomal and mitotic abnormalities by directly crosslinking DNA, intercalating into DNA, or affecting nucleic acid synthesis.

[0081] Examples of chemotherapeutic agents include: alkylating agents, e.g., thiotepa and cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan, and picosulfan; aziridines, e.g., benzodopa, carbocon, meturedopa, and uredopa; ethyleneimines and methylamelamines, e.g., altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bratacin and bratacinone); camptothecin (including its synthetic analog topotecan); bryostatin; calistatin; CC-1065 (including its synthetic analogs adzeresin, calzelsin, and bizeresin); and crypt Toficin (especially cryptophycin 1 and cryptophycin 8); dorastatin; duocalmycin (including synthetic analogs, KW-2189 and CB1-TM1); eryuterobin; pancratistatin; sarcodictiin; spongistatin; nitrogen mustard, e.g., chlorambucil, chlornafadin, colophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, Nobuenviquin, fenesterine, prednimustine, trophosphamide, and uracil mustard; nitrosourea, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, e.g., engine antibiotics (e.g., calicheamicin, in particular calicheamicin gamma 1 and calicheamicin omega 1); dinemisins, e.g., dinemisin A; bisphosphonates, e.g., clodronate; esperamicin;Furthermore, neocardinostatin chromophore and related pigment proteins enegyoin antibiotic chromophores, acrasinomycin, actinomycin, authrarnycin, azaserin, bleomycin, kactinomycin, carabicin, carminomycin, cardinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinodo-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, e.g., mitomycin C, mycophenolic acid, nogalarnycin, olibomycin, peplomycin, potfiromycin, pi Thrombocytopenic acid, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and zolubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folate analogs, e.g., denopterin, pteropterin, and trimethrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamipri , and thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents, e.g., mitotane and trilostane; folic acid supplements, e.g., frolinic acid; acegraton; aldofamide glycoside; aminolevulinic acid; enyluracil; amsacrine; bestrabusil; bisanthren; edatraxate; defofamine; demecoltin; diaziquan; elformithine; eriptinium acetate; epotilone; etogluside;Gallium nitrate; hydroxyurea; lentinan; lonidainine; mytansinoids, e.g., mytansin and anthamitosin; mitogwazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triadicone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, loridine A, and anguidine); urethane; bin Decine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Taxoids, e.g., Paclitaxel and Docetaxel Gemcitabine; 6-Thiogunine; Mercaptopurine; Platinum-coordinated complexes, e.g., Cisplatin, Oxaliplatin, and Carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Xeloda; Ibandronate; Irinotecan (e.g., CPT-11); Topoisomerase inhibitor RFS 2000; difluoromethylornithine (DFMO); retinoids, e.g., retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl protein transferase inhibitor, trans platinum, and any pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0082] 2. Radiation therapy Other widely used factors that cause DNA damage include those commonly known as gamma rays, X-rays, and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damage factors are also considered, such as microwaves, proton beam irradiation (US Patents 5,760,395 and 4,870,287), and UV irradiation. All of these factors are most likely to affect a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dose range for X-rays ranges from a daily dose of 50–200 roentgens for long-term use (3–4 weeks) to a single-dose dose of 2,000–6,000 roentgens. The dose range for radioisotopes varies widely and depends on the half-life of the isotope, the intensity and type of radiation emitted, and uptake by neoplastic cells.

[0083] 3. Immunotherapy Those skilled in the art will understand that further immunotherapies can be used in combination with or in conjunction with the methods of the present invention. In cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (Rituxan®) is one such example. Immune effectors can be, for example, antibodies specific to some marker on the surface of tumor cells. Antibodies can act as effectors of therapy on their own, or antibodies can mobilize other cells to actually influence cell death. Antibodies can also be conjugated with drugs or toxins (chemotherapeutic agents, radionuclides, lysine A chain, cholera toxin, pertussis toxin, etc.) and can simply act as targeted substances. Alternatively, the effector can be a lymphocyte that possesses surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

[0084] In one aspect of immunotherapy, tumor cells must possess some marker suitable for targeting, i.e., markers not present in most other cells. Many tumor markers exist, and any of these may be suitable for targeting in this invention. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is combining anticancer effects with immunostimulatory effects. Immunostimulatory molecules also exist, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

[0085] Examples of immunotherapies currently under consideration or in use include immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (US Patents No. 5,801,005 and 5,739,169; Hui and Hashimoto, Infection Immun., 66(11):5329-5336, 1998; Christodoulides et al., Microbiology, 144(Pt 11):3027-3037, 1998); cytokine therapies, e.g., interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., Clinical Cancer Res., 4(10):2337-2347, 1998; Davidson et al., J. Immunother., 21(5):389-398, 1998;Hellstrand et al., Acta Oncologica, 37(4):347-353, 1998); gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., Proc. Natl. Acad. Sci. USA, 95(24):14411-14416, 1998;Austin-Ward and Villaseca, Revista Medica de Chile, 126(7):838-845, 1998; US Patent Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-p185 (Hanibuchi et al., Int. J. Cancer, 78(4):480-485, (1998; U.S. Patent No. 5,824,311). It is intended that one or more anticancer therapies may be used in conjunction with the antibody therapies described herein.

[0086] In some embodiments, immunotherapy may also be adoptive immunotherapy, which involves the transfer of ex vivo-generated autoantigen-specific T cells. T cells used for adoptive immunotherapy can be generated either by expanding antigen-specific T cells or by genetically engineered T cell redirection. Isolation and transfer of tumor-specific T cells have been shown to be successful in the treatment of melanoma. Genetic transmission of transgenic T cell receptors or chimeric antigen receptors (CARs) has successfully resulted in novel T cell specificity. CARs are synthetic receptors consisting of a target-directed moiety bound to one or more signaling domains in a single fusion molecule. Generally, the binding moiety of a CAR consists of the antigen-binding domain of a monoclonal antibody (scFv) containing a light chain variable fragment of a monoclonal antibody bound by a mobile linker. Binding moieties based on receptor or ligand domains have also been successfully utilized. The signaling domains of first-generation CARs are derived from the cytoplasmic region of the CD3 zeta or Fc receptor gamma chain. CAR successfully redirected T cells to antigens expressed on the surface of tumor cells derived from various malignant tumors, including lymphomas and solid tumors.

[0087] In one aspect, the present application provides a combination therapy for cancer treatment, the combination therapy comprising adoptive T cell therapy and a checkpoint inhibitor. In one aspect, the adoptive T cell therapy comprises autologous and / or allogeneic T cells. In another aspect, the autologous and / or allogeneic T cells are targeted against tumor antigens.

[0088] Immunomodulators include immune checkpoint inhibitors, agonists of costimulatory molecules, and antagonists of immunoinhibitory molecules. Immunomodulators may be drugs, e.g., small molecules, recombinant forms of ligands or receptors, or antibodies, e.g., human antibodies (e.g., International Patent Publication WO2015 / 016718; Pardoll, Nat Rev Cancer, 12(4): 252-264, 2012; both incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogues thereof may be used, and in particular, chimeric, humanized, or human-form antibodies may be used. As those skilled in the art will know, alternative and / or equivalent names may be used for certain antibodies referred to herein. Such alternative and / or equivalent names are interchangeable herein. For example, lambrolizumab is known to be also known by alternative and equivalent names, MK-3475 and pembrolizumab.

[0089] Co-stimulatory molecules are ligands that interact with receptors on the surface of immune cells, such as CD28, 4-1BB, OX40 (also known as CD134), ICOS, and GITR. As an example, the complete protein sequence of human OX40 has GenBank accession number NP_003318. In some embodiments, the immunomodulator is an anti-OX40 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), their antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. Anti-human OX40 antibodies (or VH and / or VL domains derived therefrom) suitable for use in this method can be produced using methods well known in the art. Alternatively, anti-OX40 antibodies recognized in the art can be used. An exemplary anti-OX40 antibody is PF-04518600 (see, for example, WO 2017 / 130076). ATOR-1015 is a bispecific antibody that targets CTLA4 and OX40 (see, for example, WO 2017 / 182672, WO 2018 / 091740, WO 2018 / 202649, and WO 2018 / 002339).

[0090] Another co-stimulatory molecule that can be targeted by the methods provided herein is ICOS, also known as CD278. The complete protein sequence of human ICOS has GenBank accession number NP_036224. In some embodiments, the immune checkpoint inhibitor is an anti-ICOS antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. An anti-human ICOS antibody (or its VH and / or VL domain) suitable for use in the present method can be produced using methods well known in the art. Alternatively, an anti-ICOS antibody recognized in the art can be used. Exemplary anti-ICOS antibodies include JTX-2011 (see, e.g., WO 2016 / 154177, WO 2018 / 187191) and GSK3359609 (see, e.g., WO 2016 / 059602).

[0091] Another co-stimulatory molecule that can be targeted by the methods provided herein is glucocorticoid-induced tumor necrosis factor receptor-associated protein (GITR), also known as TNFRSF18 and AITR. The complete protein sequence of human GITR has GenBank accession number NP_004186. In some embodiments, the immunomodulator is an anti-GITR antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. An anti-human GITR antibody (or its VH and / or VL domain) suitable for use in the present method can be produced using methods well known in the art. Alternatively, an anti-GITR antibody recognized in the art can be used. An exemplary anti-GITR antibody is TRX518 (see, for example, WO 2006 / 105021).

[0092] Immune checkpoint proteins that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuators (BTLA), CCL5, CD27, CD38, CD8A, CMKLR1, cytotoxic T lymphocyte-associated protein 4 (also known as CD152, CTLA-4), CXCL9, CXCR5, HLA-DRB1, HLA-DQA1, HLA-E, killer cell immunoglobulin (KIR), and lymphocyte-activating genes. Examples include LAG-3 (also known as CD223, LAG-3), Mer tyrosine kinase (MerTK), NKG7, programmed death 1 (PD-1), programmed death ligand 1 (also known as CD274, PD-L1), PDCD1LG2, PSMB10, STAT1, T-cell immune receptor with Ig and ITIM domains (TIGIT), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig inhibitor of T-cell activation (also known as C10orf54, VISTA). In particular, immune checkpoint inhibitors targeting the PD-1 axis and / or CTLA-4 have received widespread FDA approval across a variety of cancer types.

[0093] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand-binding partner. In specific embodiments, the PD-1 ligand-binding partner is PD-L1 and / or PD-L2. In other embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In specific embodiments, the PD-L1 binding partner is PD-1 and / or B7-1. In other embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In specific embodiments, the PD-L2 binding partner is PD-1. The antagonist may be an antibody, their antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patents 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the manner provided herein are known in the art, for example, described in U.S. Patent Application Publications 2014 / 0294898, 2014 / 022021, and 2011 / 0008369, all of which are incorporated herein by reference.

[0094] In some embodiments, the PD-1 conjugated antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 conjugated antagonist is an immunoadhesin (e.g., an immunoadhesin containing an extracellular or PD-1 binding portion (e.g., the Fc region of an immunoglobulin sequence) of PD-L1 or PD-L2 fused to a constant region). In some embodiments, the PD-1 conjugated antagonist is AMP-224. MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO (登録商標)Nivolumab, also known as MK-3475, Merck3475, lambrolizumab, and KEYTRUDA, is an anti-PD-1 antibody listed in WO2006 / 121168. (登録商標) Pembrolizumab, also known as SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0095] Another immune checkpoint protein that can be targeted in the manner provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has GenBank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA-4 is analogous to the T cell costimulatory protein, CD28, and both molecules bind to CD80 and CD68, also known as B7-1 and B7-2, respectively, on antigen-presenting cells. CTLA-4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA-4 is also found in regulatory T cells and may be important to their function. T cell activation via the T cell receptor and CD28 leads to increased expression of CTLA-4, which is an inhibitory receptor for the B7 molecule.

[0096] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. An anti-human CTLA-4 antibody (or its VH and / or VL domain) suitable for use in this method can be produced using methods well known in the art. Alternatively, an anti-CTLA-4 antibody recognized in the art can be used. For example, anti-CTLA-4 antibodies disclosed in U.S. Patent No. 8,119,129; PCT Publication Nos. WO 01 / 14424, WO 98 / 42752, WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly tisilimmab); U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA, 95(17): 10067-10071; Camacho et al. (2004) J Clin Oncology, 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res, 58:5301-5304 can be used in the manner disclosed herein. Each of the teachings in the aforementioned publications is incorporated herein by reference. Antibodies that compete with any of these antibodies recognized in the art for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application No. WO2001 / 014424, WO2000 / 037504, and U.S. Patent No. 8,017,114; all are incorporated herein by reference.

[0097] Exemplary anti-CTLA-4 antibodies are ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or its antigen-binding fragments and variants (see, e.g., WO 01 / 14424). In other embodiments, the antibody contains the CDR or VR of the heavy and light chains of ipilimumab. Thus, in one embodiment, the antibody contains the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, as well as the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to the same epitope on CTLA-4 as the antibody described above, and / or binds to said epitope. In yet another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the antibody described above (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab). Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors, such as those described in U.S. Patent No. 5,844,905, 5,885,796, and International Patent Applications WO1995001994 and WO1998042752, which are incorporated herein by reference, as well as immunoadhesins, such as those described in U.S. Patent No. 8,329,867, which are incorporated herein by reference.

[0098] Another immune checkpoint protein that can be targeted by the methods provided herein is lymphocyte-activating gene 3 (LAG-3), also known as CD223. The complete protein sequence of human LAG-3 has GenBank accession number NP-002277. LAG-3 is found on the surface of activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. LAG-3 acts as an "off" switch when bound to MHC class II on the surface of antigen-presenting cells. Inhibition of LAG-3 activates both effector T cells and inhibitor-regulated T cells. In some embodiments, the immune checkpoint inhibitor is an anti-LAG-3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), their antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. Anti-human LAG-3 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods herein can be produced using methods well known in the art. Alternatively, anti-LAG-3 antibodies recognized in the art can be used. Exemplary anti-LAG-3 antibodies include relatrimab (also known as BMS-986016) or its antigen-binding fragments and variants (see, e.g., WO 2015 / 116539). Other exemplary anti-LAG-3 antibodies include TSR-033 (see, e.g., WO 2018 / 201096), MK-4280, and REGN3767. MGD013 is an anti-LAG-3 / PD-1 bispecific antibody described in WO 2017 / 019846. FS118 is an anti-LAG-3 / PD-L1 bispecific antibody described in WO 2017 / 220569.

[0099] Another immune checkpoint protein that can be targeted by the methods provided herein is the V-domain Ig inhibitor of T cell activation (VISTA), also known as C10orf54. The complete protein sequence of human VISTA has GenBank accession number NP_071436. VISTA is found in leukocytes and inhibits the effector function of T cells. In some embodiments, the immune checkpoint inhibitor is an anti-VISTA3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), their antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. An anti-human VISTA antibody (or its VH and / or VL domains) suitable for use in the present method can be produced using methods well known in the art. Alternatively, an anti-VISTA antibody recognized in the art can be used. An exemplary anti-VISTA antibody is JNJ-61610588 (also known as ombachirimab) (see, e.g., WO 2015 / 097536, WO 2016 / 207717, WO 2017 / 137830, WO 2017 / 175058). VISTA can also be inhibited by the small molecule CA-170, which selectively targets both PD-L1 and VISTA (see, e.g., WO 2015 / 033299, WO 2015 / 033301).

[0100] Another immune checkpoint protein that can be targeted by the methods provided herein is CD38. The complete protein sequence of human CD38 has the GenBank accession number NP_001766. In some embodiments, the immune checkpoint inhibitor is an anti-CD38 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. An anti-human CD38 antibody (or its VH and / or VL domain) suitable for use in the present method can be produced using methods well known in the art. Alternatively, an anti-CD38 antibody recognized in the art can be used. An exemplary anti-CD38 antibody is daratumumab (see, for example, U.S. Patent No. 7,829,673).

[0101] Another immune checkpoint protein that can be targeted by the methods provided herein is the T cell immune receptor (TIGIT) having Ig and ITIM domains. The complete protein sequence of human TIGIT has GenBank accession number NP_776160. In some embodiments, the immune checkpoint inhibitor is an anti-TIGIT antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), their antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. An anti-human TIGIT antibody (or its VH and / or VL domain) suitable for use in the methods herein can be produced using methods well known in the art. Alternatively, an anti-TIGIT antibody recognized in the art can be used. An exemplary anti-TIGIT antibody is MK-7684 (see, for example, WO 2017 / 030823, WO 2016 / 028656).

[0102] Other immunoinhibitory molecules that can be targeted for immunomodulation include STAT3 and indoleamine 2,3-dioxygenase (IDO). For example, the complete protein sequence of human IDO has the GenBank accession number NP_002155. In some embodiments, immunomodulators are small molecule IDO inhibitors. Exemplary small molecules include BMS-986205, epacadostat (INCB24360), and napoximod (GDC-0919).

[0103] 4.Surgery Approximately 60% of people with cancer are expected to undergo some type of surgery, including prophylactic, diagnostic, or staging, curative, and palliative surgeries. Curative surgery includes excision, in which all or part of the cancerous tissue is physically removed, resected, and / or destroyed. This surgery can be used in conjunction with other therapies, such as the treatments of the present invention, chemotherapy, radiotherapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor excision refers to the physical removal of at least a portion of the tumor. In addition to tumor excision, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery).

[0104] When cancerous cells, tissue, or part or all of a tumor are removed, a cavity may form in the body. Treatment can be achieved by perfusion, direct injection, or local application of the area with further anticancer therapy. Such treatments can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. The dosage of these treatments may also vary.

[0105] 5. Other agents To enhance the therapeutic efficacy of the treatment, other agents may be used in combination with certain aspects of the present invention. These further agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cell proliferation inhibitors and differentiation agents, cell adhesion inhibitors, agents that enhance the sensitivity of hyperproliferating cells to apoptosis-inducing factors, or other biological agents. Increased intercellular signaling by increasing the number of GAP junctions is thought to enhance the anti-hyperproliferative effect on adjacent hyperproliferating cell populations. In other embodiments, cell proliferation inhibitors or differentiation agents may be used in combination with certain aspects of the present invention to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors are intended to enhance the efficacy of the present invention. Examples of cell adhesion inhibitors include adhesion plaque kinase (FAK) inhibitors and lovastatin. Further intended to enhance therapeutic efficacy, other agents that enhance the sensitivity of hyperproliferating cells to apoptosis, such as the antibody c225, may be used in combination with certain aspects of the present invention.

[0106] V. Kit In various aspects of the present invention, kits comprising diagnostic agents, therapeutic agents, and / or delivery agents are envisioned. In some embodiments, the present invention envisions a kit for detecting NRG1 fusions in a patient's tumor cells. In some embodiments, the present invention envisions a kit for preparing and / or administering the therapy of the present invention. The kit may include reagents that can be used for administering the active or effective agents of the present invention. The reagents of the kit may include one or more anticancer components of a combination therapy, as well as reagents for preparing, formulating, and / or administering the components of the present invention, or for carrying out one or more steps of the method of the present invention. In some embodiments, the kit may also include suitable container means, which are containers that are not expected to react with the components of the kit, e.g., Eppendorf tubes, assay plates, syringes, bottles, or tubes. The containers may be made from sterilizable materials, e.g., plastic or glass. The kit may further include an instruction sheet outlining the steps of the method and following substantially the same procedure as described herein, or which is known to those skilled in the art. [Examples]

[0107] VI. Examples The following embodiments are included to illustrate preferred embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques that the inventors have found to function well in carrying out the present invention and can therefore be considered to constitute a preferred form for its implementation. However, those skilled in the art will recognize that, in light of this disclosure, many modifications can be made in the particular embodiments disclosed without departing from the spirit and scope of the present invention, and similar or comparable results can still be obtained.

[0108] Example 1 The cell viability of the breast cancer cell line, MDA175-VII (NRG1-DOC4 fusion), was tested with a panel of EGFR and HER2 TKIs including lapatinib, afatinib, neratinib, dacomitinib, pyrotinib, taloraxotinib-TKI, ibrutinib, sapitinib, erlotinib, and osimertinib, alone and in combination with anti-HER2 therapies including trastuzumab, pertuzumab, and T-DM1. Cell viability was determined by the Cell Titer Glo assay. Covalent rather than non-covalent HER2 TKIs, and the HER2 / HER3-targeting antibody, pertuzumab, potently inhibited the cell viability of MDA175-VII cells (Table 1; Figures 1A-1B).

[0109] Furthermore, since inhibition of WT EGFR often leads to off-target adverse events in patients, IC 50 values were determined for Ba / F3 cells expressing WT EGFR (+10 ng / μL EGF) treated with EGFR and HER2 TKIs, and compared to the IC 50 values for cells with NRG1 fusions. Covalent HER2 TKIs were selective in the inhibition of MDA175-VII (NRG fusion) cells (Figure 2).

[0110] Finally, the addition of low-dose covalent HER2 TKI to anti-HER2 therapy decreased cell viability compared to anti-HER2 therapy alone (Table 2; Figures 3A-3B). These data indicate that the combination of HER2 TKI and HER2 antibody is more potent than single-agent inhibitors tested against NRG1 fusions.

[0111] (Table 1) Mean IC 50 values for MDA175-VII (NRG1-DOC4 fusion) cells treated with the HER2 inhibitors shown TIFF2026065076000003.tif62164

[0112] (Table 2) Mean IC2 for MDA175-VII (NRG1-DOC4 fusion) cells treated with anti-HER2 antibody, with or without low-dose HER2 TKI. 50 value TIFF2026065076000004.tif51164

[0113] Example 2 Generation of Ba / F3 cells. Ba / F3 cells stably co-expressing WT ErbB2 and WT ErbB3, or WT ErbB3 and WT ErbB4, are generated as previously described. Briefly, retroviral or lentiviral constructs are transfected into Phoenix 293T cells to generate viruses, which are incubated overnight with the Ba / F3 cell line. After virus removal, cells are cultured in puromycin for 10 days to select Ba / F3 cell lines that stably express the retroviral constructs. After selection, cells are sorted using anti-HER2, anti-HER3, and anti-HER4 antibodies (Biolegend). Then, lentiviruses containing the NRG fusion plasmid from Table 3A are transduced back into the cell line. Cells are then sorted for NRG1 expression by FACS. IL-3 is then removed from the stable cell line. The resulting stable cells are used for downstream analyses, including drug screening.

[0114] Drug screening and IC50 determination. Drug screening is performed as previously described. Briefly, cells are plated in a technical triplicate in a 384-well plate (Greiner Bio-One) with 2000-3000 cells per well. Seven different concentrations of covalent EGFR / HER2 TKI and / or HER2 / HER3 targeted antibody or DMSO vehicle are added to a final volume of 40 μL per well. After 72 hours, 11 μL of Cell Titer Glo (Promega) is added to each well. The plate is incubated for at least 10 minutes, and bioluminescence is measured using a FLUOstar OPTIMA plate reader (BMG LABTECH). The raw bioluminescence values ​​are normalized for cells treated with DMSO control, and the values ​​are plotted in GraphPad Prism. Nonlinear regression is used to fit the normalized data with a variable gradient, and IC50 is determined by interpolation of the concentration at 50% inhibition using GraphPad prism. 50 Determine the values. Drug screening is performed using either technical triplicates and biological repeats of 2 or 3 triplicates on each plate.

[0115] Overexpression model. The overexpression model is generated by lentiviral transduction of the NRG1 fusion shown in Table 3A. The lentivirus is generated using the Lenti-X cells Lenti-X single shot kit (Takarabio). The lentivirus is generated as described by the manufacturer. The lentivirus is then added to the cell line shown in Table 3B. After 24 hours of viral transduction, the virus is removed and the cells are placed in 2 μg / ml puromycin for selection. After 10 days of selection, proteins and RNA are collected from the cell line and the expression of the NRG1 fusion is determined by Western blotting and RT-PCR, respectively. Stable cell lines expressing the NRG1 fusion are used for downstream analyses, including Western blotting and ELISA.

[0116] Determination of HER signaling inhibition in overexpression cell lines by Western blotting and ELISA. Parental and overexpression (OE) cell lines were plated in 10 cm dishes and treated with escalating doses of covalent EGFR / HER2 TKI and / or HER2 / HER3 targeted antibody. Cells were incubated with inhibitor and / or antibody for 4 hours, 1 day, and 3 days, and proteins were collected using lysis buffer (Cell Signaling). Expression of NRG1 fusion, phosphorylated EGFR, HER2, HER3, and HER4, as well as total EGFR, HER2, HER3, and HER4, was determined by Western blotting, and the blots were exposed using a BioRad Chemidoc imager. To quantify changes in protein expression, proteins from parental and OE-expressing cell lines treated with covalent EGFR / HER2 TKI and / or HER2 / HER3 targeted antibody were loaded into ELISA (Cell Signaling), and the ELISA was completed according to the manufacturer's instructions.

[0117] (Table 3A) NRG1 fusion plasmid TIFF2026065076000005.tif16164

[0118] (Table 3B) Human cell line models TIFF2026065076000006.tif21164

[0119] All methods disclosed and asserted herein can be carried out and implemented without any experimentation beyond what is necessary in light of this disclosure. While the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the methods and steps or sets of steps described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically related active substances can be substituted for the active substances described herein, and that the same or similar results can be achieved at the same time. All such similar substitutions and modifications that are apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0120] References The following references are incorporated herein by reference to the extent that they provide exemplary procedures or other details that complement what is described herein. TIFF2026065076000007.tif136160

Claims

1. A method for treating patients with cancer, (a) the process of determining or having determined whether the patient's cancer has an NRG1 fusion; (b) the step of selecting or having selected a patient for treatment with a covalent EGFR / HER2 tyrosine kinase inhibitor (TKI) if the patient's cancer has an NRG1 fusion; and (c) The process of administering or having administered a therapeutically effective dose of a covalent EGFR / HER2 TKI to the selected patient. Methods that include...

2. A method for treating patients with cancer, The process of administering a therapeutically effective dose of a covalent EGFR / HER2 TKI to the patient. A method comprising the cancer having an NRG1 fusion.

3. The method according to claim 1 or 2, wherein the NRG1 fusion is an NRG1-DOC4 fusion, an NRG1-VAMP2 fusion, an NRG1-CLU fusion, an NRG1-SLC3A2 fusion, an NRG1-CD74 fusion, an NRG1-ATP1B1 fusion, or an NRG1-SDC4 fusion.

4. The method according to any one of claims 1 to 3, wherein the covalent EGFR / HER2 TKI is afatinib, neratinib, dacomitinib, tarlox-TKI, pirotinib, or ibrutinib.

5. The process of administering a HER2 / HER3-targeted antibody to the patient. The method according to any one of claims 1 to 4, further comprising:

6. The method according to claim 5, wherein the HER2 / HER3-targeting antibody comprises trastuzumab, pertuzumab, or T-DM1.

7. Step (a) is, (i) the stage of obtaining or having obtained a biological sample from the patient; and (ii) The stage in which an assay is performed or was being performed on a biological sample to determine whether the patient's cancer has an NRG1 fusion. A method according to any one of claims 1 to 6, including the method described in any one of claims 1 to 6.

8. The process of administering further anti-cancer therapy to the aforementioned patient. The method according to any one of claims 1 to 7, further comprising:

9. The method according to claim 8, wherein the further anti-cancer therapy is surgery, chemotherapy, radiotherapy, cryotherapy, hormone therapy, toxin therapy, immunotherapy, or cytokine therapy.

10. The method according to any one of claims 1 to 9, wherein the cancer is breast cancer, lung cancer, colorectal cancer, neuroblastoma, pancreatic cancer, brain tumor, stomach cancer, skin cancer, testicular cancer, prostate cancer, ovarian cancer, liver cancer, esophageal cancer, cervical cancer, head and neck cancer, melanoma, or glioblastoma.

11. The method according to any one of claims 1 to 10, wherein the cancer is breast cancer or lung cancer.

12. The method according to any one of claims 1 to 11, wherein the patient has previously received at least one round of anticancer therapy.

13. A step to report the presence of an NRG1 fusion in the cancer of the aforementioned patient. The method according to any one of claims 1 to 12, further comprising:

14. The method according to claim 13, wherein the reporting step includes the step of preparing a written or electronic report.

15. The process of submitting the aforementioned report to the aforementioned subject, physician, hospital, or insurance company. The method according to claim 13 or 14, further comprising:

16. A method for selecting patients with cancer for treatment with a covalent EGFR / HER2 TKI, (a) the process of determining or having determined whether the patient's cancer has an NRG1 fusion; (b) The process of selecting or having selected a patient for treatment with a covalent EGFR / HER2 TKI if the patient's cancer has an NRG1 fusion. Methods that include...

17. Step (a) is, (i) the stage of obtaining or having obtained a biological sample from the patient; and (ii) The stage in which an assay is performed or was being performed on a biological sample to determine whether the patient's cancer has an NRG1 fusion. The method according to claim 15, including the method described in claim 15.

18. (c) The process of administering or having administered a therapeutically effective dose of a covalent EGFR / HER2 TKI to the selected patient. The method according to claim 16 or 17, further comprising:

19. The method according to any one of claims 16 to 18, wherein the NRG1 fusion is an NRG1-DOC4 fusion, an NRG1-VAMP2 fusion, an NRG1-CLU fusion, an NRG1-SLC3A2 fusion, an NRG1-CD74 fusion, an NRG1-ATP1B1 fusion, or an NRG1-SDC4 fusion.

20. The method according to any one of claims 16 to 19, wherein the covalent EGFR / HER2 TKI is afatinib, neratinib, dacomitinib, tarloxo-TKI, pirotinib, or ibrutinib.

21. The process of administering a HER2 / HER3-targeted antibody to the patient. The method according to any one of claims 18 to 20, further comprising:

22. The method according to claim 21, wherein the HER2 / HER3-targeting antibody comprises trastuzumab, pertuzumab, or T-DM1.

23. The process of administering further anti-cancer therapy to the aforementioned patient. The method according to any one of claims 18 to 22, further comprising:

24. The method according to claim 23, wherein the further anti-cancer therapy is surgery, chemotherapy, radiotherapy, cryotherapy, hormone therapy, toxin therapy, immunotherapy, or cytokine therapy.

25. The method according to any one of claims 16 to 24, wherein the cancer is breast cancer, lung cancer, colorectal cancer, neuroblastoma, pancreatic cancer, brain tumor, stomach cancer, skin cancer, testicular cancer, prostate cancer, ovarian cancer, liver cancer, esophageal cancer, cervical cancer, head and neck cancer, melanoma, or glioblastoma.

26. The method according to any one of claims 16 to 25, wherein the cancer is breast cancer or lung cancer.

27. The method according to any one of claims 16 to 26, wherein the patient has previously received at least one round of anticancer therapy.

28. A step to report the presence of an NRG1 fusion in the cancer of the aforementioned patient. The method according to any one of claims 16 to 27, further comprising:

29. The method according to claim 28, wherein the reporting step includes the step of preparing a written or electronic report.

30. The process of submitting the aforementioned report to the aforementioned subject, physician, hospital, or insurance company. The method according to claim 28 or 29, further comprising:

31. A method for treating patients with cancer, (a) the process of determining or having determined whether the patient's cancer has an NRG1 fusion; (b) the step of selecting or having selected a patient for treatment with a HER2 / HER3 targeted antibody if the patient's cancer has an NRG1 fusion; and (c) The process of administering or having administered a therapeutically effective dose of a HER2 / HER3-targeted antibody to the selected patient. Methods that include...

32. A method for treating patients with cancer, The process of administering a therapeutically effective amount of HER2 / HER3-targeted antibody to the patient. A method comprising the cancer having an NRG1 fusion.

33. The method according to claim 31 or 32, wherein the NRG1 fusion is an NRG1-DOC4 fusion, an NRG1-VAMP2 fusion, an NRG1-CLU fusion, an NRG1-SLC3A2 fusion, an NRG1-CD74 fusion, an NRG1-ATP1B1 fusion, or an NRG1-SDC4 fusion.

34. The method according to any one of claims 31 to 33, wherein the HER2 / HER3-targeting antibody comprises trastuzumab, pertuzumab, or T-DM1.

35. The process of administering a covalent EGFR / HER2 TKI to the patient. The method according to any one of claims 31 to 34, further comprising:

36. The method according to any claim 35, wherein the covalent EGFR / HER2 TKI is afatinib, neratinib, dacomitinib, tarloxo-TKI, pirotinib, or ibrutinib.

37. Step (a) is, (i) the stage of obtaining or having obtained a biological sample from the patient; and (ii) The stage in which an assay is performed or was being performed on a biological sample to determine whether the patient's cancer has an NRG1 fusion. The method according to any one of claims 31 to 36, including the method described in any one of claims 31 to 36.

38. The process of administering further anti-cancer therapy to the aforementioned patient. The method according to any one of claims 31 to 37, further comprising:

39. The method according to claim 38, wherein the further anti-cancer therapy is surgery, chemotherapy, radiotherapy, cryotherapy, hormone therapy, toxin therapy, immunotherapy, or cytokine therapy.

40. The method according to any one of claims 31 to 39, wherein the cancer is breast cancer, lung cancer, colorectal cancer, neuroblastoma, pancreatic cancer, brain tumor, stomach cancer, skin cancer, testicular cancer, prostate cancer, ovarian cancer, liver cancer, esophageal cancer, cervical cancer, head and neck cancer, melanoma, or glioblastoma.

41. The method according to any one of claims 31 to 40, wherein the cancer is breast cancer or lung cancer.

42. The method according to any one of claims 31 to 41, wherein the patient has previously received at least one round of anticancer therapy.

43. A step to report the presence of an NRG1 fusion in the cancer of the aforementioned patient. The method according to any one of claims 31 to 42, further comprising:

44. The method according to claim 43, wherein the reporting step includes the step of preparing a written or electronic report.

45. The process of submitting the aforementioned report to the aforementioned subject, physician, hospital, or insurance company. The method according to claim 43 or 44, further comprising:

46. A method for selecting patients with cancer for treatment using HER2 / HER3 targeted antibodies, (a) the process of determining or having determined whether the patient's cancer has an NRG1 fusion; (b) The process of selecting or having selected a patient for treatment with a HER2 / HER3 targeted antibody if the patient's cancer has an NRG1 fusion. Methods that include...

47. Step (a) is, (i) the stage of obtaining or having obtained a biological sample from the patient; and (ii) The stage in which an assay is performed or was being performed on a biological sample to determine whether the patient's cancer has an NRG1 fusion. The method according to claim 46, including the method described in claim 46.

48. (c) The process of administering or having administered a therapeutically effective amount of HER2 / HER3-targeted antibody to the selected patient. The method according to claim 46 or 47, further comprising:

49. The method according to any one of claims 46 to 48, wherein the HER2 / HER3-targeting antibody comprises trastuzumab, pertuzumab, or T-DM1.

50. The method according to any one of claims 46 to 49, wherein the NRG1 fusion is an NRG1-DOC4 fusion, an NRG1-VAMP2 fusion, an NRG1-CLU fusion, an NRG1-SLC3A2 fusion, an NRG1-CD74 fusion, an NRG1-ATP1B1 fusion, or an NRG1-SDC4 fusion.

51. The process of administering a covalent EGFR / HER2 TKI to the patient. The method according to any one of claims 48 to 50, further comprising:

52. The method according to claim 51, wherein the covalent EGFR / HER2 TKI is afatinib, neratinib, dacomitinib, tarloxo-TKI, pirotinib, or ibrutinib.

53. The process of administering further anti-cancer therapy to the aforementioned patient. The method according to any one of claims 48 to 52, further comprising:

54. The method according to claim 53, wherein the further anti-cancer therapy is surgery, chemotherapy, radiotherapy, cryotherapy, hormone therapy, toxin therapy, immunotherapy, or cytokine therapy.

55. The method according to any one of claims 46 to 54, wherein the cancer is breast cancer, lung cancer, colorectal cancer, neuroblastoma, pancreatic cancer, brain tumor, stomach cancer, skin cancer, testicular cancer, prostate cancer, ovarian cancer, liver cancer, esophageal cancer, cervical cancer, head and neck cancer, melanoma, or glioblastoma.

56. The method according to any one of claims 46 to 55, wherein the cancer is breast cancer or lung cancer.

57. The method according to any one of claims 46 to 56, wherein the patient has previously received at least one round of anticancer therapy.

58. A step to report the presence of an NRG1 fusion in the cancer of the aforementioned patient. The method according to any one of claims 46 to 57, further comprising:

59. The method according to claim 58, wherein the reporting step includes the step of preparing a written or electronic report.

60. The process of submitting the aforementioned report to the aforementioned subject, physician, hospital, or insurance company. The method according to claim 58 or 59, further comprising:

61. A method for treating patients with cancer, (a) the process of determining or having determined whether the patient's cancer has an NRG1 fusion; (b) the step of selecting or having selected a patient for treatment with a covalent EGFR / HER2 TKI and a HER2 / HER3 targeted antibody if the patient's cancer has an NRG1 fusion; and (c) The process of administering or having administered a combined therapeutically effective dose of a covalent EGFR / HER2 TKI and a HER2 / HER3 targeted antibody to the selected patient. Methods that include...

62. A method for treating patients with cancer, The process of administering a combined therapeutically effective dose of a covalent EGFR / HER2 TKI and a HER2 / HER3 targeted antibody to the patient. A method comprising the cancer having an NRG1 fusion.

63. The method according to claim 61 or 62, wherein the NRG1 fusion is an NRG1-DOC4 fusion, an NRG1-VAMP2 fusion, an NRG1-CLU fusion, an NRG1-SLC3A2 fusion, an NRG1-CD74 fusion, an NRG1-ATP1B1 fusion, or an NRG1-SDC4 fusion.

64. The method according to any one of claims 61 to 63, wherein the covalent EGFR / HER2 TKI is afatinib, neratinib, dacomitinib, tarloxo-TKI, pirotinib, or ibrutinib.

65. The method according to any one of claims 61 to 64, wherein the HER2 / HER3-targeting antibody comprises trastuzumab, pertuzumab, or T-DM1.

66. Step (a) is, (i) the stage of obtaining or having obtained a biological sample from the patient; and (ii) The stage in which an assay is performed or was being performed on a biological sample to determine whether the patient's cancer has an NRG1 fusion. The method according to any one of claims 61 to 65, including the method described in any one of claims 61 to 65.

67. The process of administering further anti-cancer therapy to the aforementioned patient. The method according to any one of claims 61 to 66, further comprising:

68. The method according to claim 67, wherein the further anti-cancer therapy is surgery, chemotherapy, radiotherapy, cryotherapy, hormone therapy, toxin therapy, immunotherapy, or cytokine therapy.

69. The method according to any one of claims 61 to 68, wherein the cancer is breast cancer, lung cancer, colorectal cancer, neuroblastoma, pancreatic cancer, brain tumor, stomach cancer, skin cancer, testicular cancer, prostate cancer, ovarian cancer, liver cancer, esophageal cancer, cervical cancer, head and neck cancer, melanoma, or glioblastoma.

70. The method according to any one of claims 61 to 69, wherein the cancer is breast cancer or lung cancer.

71. The method according to any one of claims 61 to 70, wherein the patient has previously received at least one round of anticancer therapy.

72. A step to report the presence of an NRG1 fusion in the cancer of the aforementioned patient. The method according to any one of claims 61 to 71, further comprising:

73. The method according to claim 72, wherein the reporting step includes the step of preparing a written or electronic report.

74. The process of submitting the aforementioned report to the aforementioned subject, physician, hospital, or insurance company. The method according to claim 72 or 73, further comprising:

75. A method for selecting patients with cancer for treatment using covalent EGFR / HER2 TKIs and HER2 / HER3 targeted antibodies, (a) the process of determining or having determined whether the patient's cancer has an NRG1 fusion; (b) The process of selecting or having selected a patient for treatment with a covalent EGFR / HER2 TKI and a HER2 / HER3 targeted antibody, if the patient's cancer has an NRG1 fusion. Methods that include...

76. Step (a) is, (i) the stage of obtaining or having obtained a biological sample from the patient; and (ii) The stage in which an assay is performed or was being performed on a biological sample to determine whether the patient's cancer has an NRG1 fusion. The method according to claim 75, including the method described in claim 75.

77. (c) The process of administering or having administered a combined therapeutically effective amount of a covalent EGFR / HER2 TKI and a HER2 / HER3 targeted antibody to the selected patient. The method according to claim 75 or 76, further comprising:

78. The method according to any one of claims 75 to 77, wherein the NRG1 fusion is an NRG1-DOC4 fusion, an NRG1-VAMP2 fusion, an NRG1-CLU fusion, an NRG1-SLC3A2 fusion, an NRG1-CD74 fusion, an NRG1-ATP1B1 fusion, or an NRG1-SDC4 fusion.

79. The method according to claim 77 or 78, wherein the covalent EGFR / HER2 TKI is afatinib, neratinib, dacomitinib, tarloxo-TKI, pirotinib, or ibrutinib.

80. The method according to any one of claims 77 to 79, wherein the HER2 / HER3-targeting antibody comprises trastuzumab, pertuzumab, or T-DM1.

81. The process of administering further anti-cancer therapy to the aforementioned patient. The method according to any one of claims 77 to 80, further comprising:

82. The method according to claim 81, wherein the further anti-cancer therapy is surgery, chemotherapy, radiotherapy, cryotherapy, hormone therapy, toxin therapy, immunotherapy, or cytokine therapy.

83. The method according to any one of claims 75 to 82, wherein the cancer is breast cancer, lung cancer, colorectal cancer, neuroblastoma, pancreatic cancer, brain tumor, stomach cancer, skin cancer, testicular cancer, prostate cancer, ovarian cancer, liver cancer, esophageal cancer, cervical cancer, head and neck cancer, melanoma, or glioblastoma.

84. The method according to any one of claims 75 to 83, wherein the cancer is breast cancer or lung cancer.

85. The method according to any one of claims 75 to 84, wherein the patient has previously received at least one round of anticancer therapy.

86. A step to report the presence of an NRG1 fusion in the cancer of the aforementioned patient. The method according to any one of claims 75 to 85, further comprising:

87. The method according to claim 86, wherein the reporting step includes the step of preparing a written or electronic report.

88. The process of submitting the aforementioned report to the aforementioned subject, physician, hospital, or insurance company. The method according to claim 86 or 87, further comprising:

Images