Use of poziotinib for the treatment of cancers with NRG1 fusions

Abstract

Provided herein is a method for selecting cancer patients for treatment with poziotinib, and a method for treating the cancer patients thus selected.Cancer patients are selected for treatment when their cancer has NRG1 fusion.Then, the selected patients are treated with poziotinib alone or in combination with HER2 / HER3 targeting antibody.

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 967,265, filed January 29, 2020, the entire contents of which are incorporated herein by reference.

[0002] 1. Field The present invention relates generally to the fields of medicine and oncology. More particularly, the present invention relates to methods for selecting cancer patients for treatment with poziotinib, alone or in combination with a HER2 / HER3-targeting antibody, and methods of treating such selected cancer patients. [Background technology]

[0003] 2. Description of Related Technology NRG1 fusions occur in 0.3% of non-small cell lung cancers (NSCLCs) 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, leading to preferential heterodimerization with HER2 (Shin et al., 2018; Jung et al., 2015; Fernandez-Cuesta et al., 2014), 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 can be effective in inhibiting ErbB signaling caused by NRG1 fusions (Shin et al., 2018; Fernandez-Cuesta et al., 2014; Drilon et al., 2018). Previous reports have also shown that poziotinib can inhibit both EGFR (Robichaux et al., 2018) and HER2 (Robichaux et al., 2019) mutations. However, there are no approved targeted therapies for patients with NRG1 fusions. Summary of the Invention

[0004] overview In one aspect, there is provided a method of treating a patient having cancer, comprising: (a) determining or having determined whether the patient's cancer harbors an NRG1 fusion; (b) selecting or having selected the patient for treatment with poziotinib if the patient's cancer harbors an NRG1 fusion; and (c) administering or having administered to the selected patient a therapeutically effective amount of poziotinib. A method is provided herein, comprising: In some aspects, step (a) comprises: (i) obtaining or having obtained a biological sample from a patient; and (ii) performing or having performed an assay on the biological sample to determine that the patient's cancer has an NRG1 fusion.

[0005] In one aspect, provided herein is a method of treating a patient having cancer, the method comprising administering a therapeutically effective amount of poziotinib to the patient, wherein the cancer has an NRG1 fusion. In one aspect, provided herein is a composition comprising a therapeutically effective amount of poziotinib for use in treating a patient having cancer, wherein the patient's cancer has an NRG1 fusion.

[0006] In one aspect, there is provided a method of selecting a patient having cancer for treatment with poziotinib, comprising: (a) determining or having determined whether the patient's cancer harbors an NRG1 fusion; (b) selecting or having selected the patient for treatment with poziotinib if the patient's cancer harbors an NRG1 fusion. A method is provided herein, comprising: In some aspects, step (a) comprises: (i) obtaining or having obtained a biological sample from a 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 comprises: (c) administering or having administered to the selected patient a therapeutically effective amount of poziotinib. Further includes:

[0007] In one aspect, there is provided a method of treating a patient having cancer, comprising: (a) determining or having determined whether the patient's cancer harbors an NRG1 fusion; (b) selecting or having selected the patient for treatment with poziotinib and a HER2 / HER3-targeted antibody if the patient's cancer harbors an NRG1 fusion; and (c) administering or having administered to the selected patient a therapeutically effective amount of a combination of poziotinib and a HER2 / HER3 targeted antibody. A method is provided herein, comprising: In some aspects, step (a) comprises: (i) obtaining or having obtained a biological sample from a patient; and (ii) performing or having performed an assay on the biological sample to determine that the patient's cancer has an NRG1 fusion.

[0008] In one aspect, provided herein is a method of treating a patient having cancer, the method comprising administering to the patient a therapeutically effective amount of a combined poziotinib and a HER2 / HER3-targeting antibody, wherein the cancer has an NRG1 fusion. In one aspect, provided herein is a composition comprising a therapeutically effective amount of poziotinib and a HER2 / HER3-targeting antibody for use in treating a patient's cancer, wherein the patient's cancer has an NRG1 fusion.

[0009] In one embodiment, a method of selecting a patient having cancer for treatment with poziotinib and a HER2 / HER3 targeted antibody comprises: (a) determining or having determined whether the patient's cancer harbors an NRG1 fusion; (b) selecting or having selected the patient for treatment with poziotinib and a HER2 / HER3-targeted antibody if the patient's cancer harbors an NRG1 fusion. A method is provided herein, comprising: In some aspects, step (a) comprises: (i) obtaining or having obtained a biological sample from a 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 comprises: (c) administering or having administered to the selected patient a therapeutically effective amount of a combination of poziotinib and a HER2 / HER3 targeted antibody. Further includes:

[0010] In some aspects of any of the embodiments, 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.

[0011] In some aspects of any of the embodiments, the method further comprises administering to the patient a HER2 / HER3 targeting antibody. In some aspects, the HER2 / HER3 targeting antibody comprises trastuzumab, pertuzumab, or T-DM1.

[0012] In some aspects of any of the embodiments, the method further comprises administering to the patient an additional anti-cancer therapy, hi some aspects, the additional anti-cancer therapy is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, toxin therapy, immunotherapy, or cytokine therapy.

[0013] In some aspects of any of the embodiments, the cancer is breast cancer, lung cancer, colorectal cancer, neuroblastoma, pancreatic cancer, brain cancer, gastric 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, the cancer is breast cancer or lung cancer.

[0014] In some aspects of any of the embodiments, the patient has previously received at least one round of anti-cancer therapy. In some aspects of any of the embodiments, the method further comprises reporting the presence of an NRG1 fusion in the patient's cancer. In some aspects, the reporting comprises generating a paper or electronic report. In some aspects, the method further comprises submitting the report to the subject, a physician, a hospital, or an insurance company.

[0015] As used herein, "essentially free" in terms of specific components means that none of the specific components are intentionally incorporated into the composition, and / or only exist as contaminants or only exist in trace amounts.The total amount of the specific components resulting from any unintentional incorporation of the composition is therefore much less than 0.05%, preferably less than 0.01%.The most preferred composition is one in which the amount of the specific components cannot be detected by standard analytical methods.

[0016] As used herein, "a" or "an" may mean one or more. When used in the claims, the words "a" or "an," when used in conjunction with the word "comprising," may mean one or more than one.

[0017] The use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or unless the alternatives are mutually exclusive, although the present disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" may mean at least a second or more.

[0018] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, the variation that exists among study subjects, or a value within 10% of the stated value.

[0019] [The present invention 1001] 1. A method of treating a patient with cancer, comprising: administering a therapeutically effective amount of poziotinib to said patient. wherein the cancer has an NRG1 fusion. [The present invention 1002] 1. A method of treating a patient with cancer, comprising: (a) determining or having determined whether the patient's cancer harbors an NRG1 fusion; (b) selecting or having selected the patient for treatment with poziotinib if the patient's cancer harbors an NRG1 fusion; and (c) administering or having administered to said selected patient a therapeutically effective amount of poziotinib. A method comprising: [The present invention 1003] The method of claim 1001 or 1002, 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. [The present invention 1004] administering a HER2 / HER3 targeting antibody to said patient. The method of the present invention 1001 or 1002 further comprising: [The present invention 1005] 1005. The method of claim 1004, wherein said HER2 / HER3 targeting antibody comprises trastuzumab, pertuzumab, or T-DM1. [The present invention 1006] Step (a) (i) obtaining or having obtained a biological sample from said patient; and (ii) performing or having performed an assay on the biological sample to determine that the patient's cancer harbors an NRG1 fusion. Any of the methods 1001 to 1005 of the present invention, comprising: [The present invention 1007] administering to said patient an additional anti-cancer therapy. The method of any one of 1001 to 1006 of the present invention, further comprising: [The present invention 1008] 1007. The method of claim 1007, wherein said further anti-cancer therapy is surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, toxin therapy, immunotherapy, or cytokine therapy. [The present invention 1009] 9. The method of any one of claims 1001 to 1008, 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. [The present invention 1010] The method according to any one of claims 1001 to 1008, wherein said cancer is breast cancer or lung cancer. [The present invention 1011] The method of any of claims 1001 to 1010, wherein said patient has previously undergone at least one round of anti-cancer therapy. [The present invention 1012] reporting the presence of an NRG1 fusion in the patient's cancer. The method of any one of claims 1001 to 1011, further comprising: [The present invention 1013] The method of claim 1012, wherein the reporting step comprises generating a paper or electronic report. [The present invention 1014] submitting said report to said subject, physician, hospital, or insurance company. The method of the present invention 1012 or 1013 further comprising: [The present invention 1015] 1. A method of selecting a patient having cancer for treatment with poziotinib, comprising: (a) determining or having determined whether the patient's cancer harbors an NRG1 fusion; (b) selecting or having selected the patient for treatment with poziotinib if the patient's cancer harbors an NRG1 fusion. A method comprising: [The present invention 1016] Step (a) (i) obtaining or having obtained a biological sample from said patient; and (ii) performing or having performed an assay on the biological sample to determine that the patient's cancer harbors an NRG1 fusion. The method of the present invention 1015, comprising: [The present invention 1017] (c) administering or having administered to said selected patient a therapeutically effective amount of poziotinib. The method of claim 1015 or 1016, further comprising: [The present invention 1018] Any of the methods of claims 1015 to 1017, 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. [The present invention 1019] administering a HER2 / HER3 targeting antibody to said patient. The method of invention 1017 or 1018, further comprising: [The present invention 1020] 1019. The method of claim 1019, wherein said HER2 / HER3 targeting antibody comprises trastuzumab, pertuzumab, or T-DM1. [The present invention 1021] administering to said patient an additional anti-cancer therapy. Any of the methods of inventions 1017 to 1020, further comprising: [The present invention 1022] 1022. The method of claim 1021, wherein said further anti-cancer therapy is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, toxin therapy, immunotherapy, or cytokine therapy. [The present invention 1023] 10. The method of any one of claims 1015 to 1022, 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. [The present invention 1024] The method of any one of claims 1015 to 1023, wherein the cancer is breast cancer or lung cancer. [The present invention 1025] 1025. The method of any of claims 1015 to 1024, wherein said patient has previously undergone at least one round of anti-cancer therapy. [The present invention 1026] reporting the presence of an NRG1 fusion in the patient's cancer. Any of the methods of 1017 to 1025 of the present invention further comprising: [The present invention 1027] The method of claim 1026, wherein the reporting step comprises generating a paper or electronic report. [The present invention 1028] submitting said report to said subject, physician, hospital, or insurance company. The method of any one of claims 1026 to 1027, further comprising: [The present invention 1029] 1. A method of treating a patient with cancer, comprising: administering to said patient a combined therapeutically effective amount of poziotinib and a HER2 / HER3 targeted antibody. wherein the cancer has an NRG1 fusion. [The present invention 1030] 1. A method of treating a patient with cancer, comprising: (a) determining or having determined whether the patient's cancer harbors an NRG1 fusion; (b) selecting or having selected the patient for treatment with poziotinib and a HER2 / HER3 targeted antibody if the patient's cancer has an NRG1 fusion; and (c) administering or having administered to said selected patient a therapeutically effective amount of a combination of poziotinib and a HER2 / HER3 targeted antibody. A method comprising: [The present invention 1031] The method of any one of claims 1029 to 1030, 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. [The present invention 1032] 1032. The method of any of claims 1029 to 1031, wherein the HER2 / HER3 targeting antibody comprises trastuzumab, pertuzumab, or T-DM1. [The present invention 1033] Step (a) (i) obtaining or having obtained a biological sample from said patient; and (ii) performing or having performed an assay on the biological sample to determine that the patient's cancer harbors an NRG1 fusion. Any of the methods of claims 1029 to 1032, comprising: [The present invention 1034] administering to said patient an additional anti-cancer therapy. Any of the methods of claims 1029 to 1033, further comprising: [This invention 1035] 1035. The method of claim 1034, wherein said further anti-cancer therapy is surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, toxin therapy, immunotherapy, or cytokine therapy. [The present invention 1036] 1036. The method of any one of claims 1029 to 1035, 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. [This invention 1037] The method of any one of claims 1029 to 1036, wherein the cancer is breast cancer or lung cancer. [The present invention 1038] 8. The method of any of claims 1029 to 1037, wherein said patient has previously undergone at least one round of anti-cancer therapy. [This invention 1039] reporting the presence of an NRG1 fusion in the patient's cancer. Any of the methods of claims 1029 to 1038, further comprising: [The present invention 1040] The method of claim 1039, wherein the reporting step comprises generating a written or electronic report. [The present invention 1041] submitting said report to said subject, physician, hospital, or insurance company. The method of any one of claims 1039 to 1040, further comprising: [The present invention 1042] 1. A method of selecting a patient having cancer for treatment with poziotinib and a HER2 / HER3 targeted antibody, comprising: (a) determining or having determined whether the patient's cancer harbors an NRG1 fusion; (b) selecting or having selected the patient for treatment with poziotinib and a HER2 / HER3 targeted antibody if the patient's cancer has an NRG1 fusion. A method comprising: [This invention 1043] Step (a) (i) obtaining or having obtained a biological sample from said patient; and (ii) performing or having performed an assay on the biological sample to determine that the patient's cancer harbors an NRG1 fusion. The method of the present invention 1042, comprising: [This invention 1044] (c) administering or having administered to said selected patient a therapeutically effective amount of a combination of poziotinib and a HER2 / HER3 targeted antibody. The method of any one of claims 1042 to 1043, further comprising: [This invention 1045] Any of the methods of claims 1042 to 1044, 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. [The present invention 1046] 1046. The method of any of claims 1042 to 1045, wherein the HER2 / HER3 targeting antibody comprises trastuzumab, pertuzumab, or T-DM1. [This invention 1047] administering to said patient an additional anti-cancer therapy. Any of the methods of 1044 to 1046 of the present invention, further comprising: [This invention 1048] 1048. The method of claim 1047, wherein said further anti-cancer therapy is surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, toxin therapy, immunotherapy, or cytokine therapy. [This invention 1049] 9. The method of any one of claims 1042 to 1048, 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. [The present invention 1050] The method of any one of claims 1042 to 1049, wherein the cancer is breast cancer or lung cancer. [This invention 1051] 1050. The method of any of claims 1042 to 1050, wherein said patient has previously undergone at least one round of anti-cancer therapy. [This invention 1052] reporting the presence of an NRG1 fusion in the patient's cancer. Any of the methods of claims 1044 to 1051, further comprising: [This invention 1053] The method of claim 1052, wherein the reporting step comprises generating a written or electronic report. [This invention 1054] submitting said report to said subject, physician, hospital, or insurance company. The method of any one of claims 1052 to 1053, further comprising: Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0020] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Figure 1] Bar graph of IC50 values ​​for MDA175-VII (NRG1-DOC4 fusion) treated with poziotinib for 72 hours. [Figure 2] Figure 2A: Dose-response curves for the MDA175-VII (NRG1-DOC4 fusion) cell line treated with HER2 / HER3 antibodies for 72 hours, with and without low-dose poziotinib (0.1 nM). At 100 ng / mL on the x-axis, the lines represent, from top to bottom, T-DM1, trastuzumab, pertuzumab, trastuzumab + poziotinib 0.1 nM, T-DM1 + ​​poziotinib 0.1 nM, and pertuzumab + poziotinib 0.1 nM. Figure 2B: Bar graph of IC50 values ​​for the MDA175-VII (NRG1-DOC4 fusion) cell line treated with HER2 / HER3 antibodies for 72 hours, with and without low-dose poziotinib (0.1 nM). DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed Description Provided herein is a method for treating cancer patients with NRG1 fusion.In particular, this method comprises administering poziotinib (also known as HM781-36B) alone or in combination with HER2 / HER3 targeting antibody to cancer patients identified as having NRG1 fusion.Furthermore, this method comprises determining whether the patient's cancer has NRG1 fusion, thereby identifying and selecting cancer patients who may benefit from administering poziotinib alone or in combination with HER2 / HER3 targeting antibody.

[0022] I.NRG1 fusion An NRG1 fusion gene comprises 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 can be present in the fusion, or a portion thereof. The fusion can have coding sequences for at least exons 6, 7, and 8 of NRG1. Another way to define the NRG1 portion in an NRG1 fusion gene is that it contains the EGF-like domain of NRG1. The EGF-like domain is encoded at the 3' end of the gene and is necessary for binding to ErbB-3. An NRG1 fusion retains an in-frame coding region for the EGF-like domain. A portion of the NRG1 gene can be fused to a sequence from a different chromosomal location, such that the sequence is located 5' or 3' to the portion of the NRG1 gene.

[0023] Preferably, the 3' end of the NRG1 gene can be fused to 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 following genes: 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 Gene: 26011; Ensembl: ENSG00000149256; OMIM: 610084; and UniProtKB: Q6N022); CD74 (CD74 molecule; CD74 antigen (major histocompatibility complex invariant polypeptide, class II antigen-associated); CD74 molecule, major histocompatibility complex, class II invariant chain; HLA-DR antigen-associated invariant chain; class II antigen-gamma chain; la-associated invariant chain; MHC HLA-DR gamma chain; HLA-DR-gamma; DHLAG; P33; HLA class II histocompatibility antigen gamma chain; la antigen-associated invariant chain; la-gamma; HLADG; HGNC: 1697; Entrez 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; death receptor 5; TRAIL-R2; TRAILR2; KILLER; TRICK2; ZTNFR9; DR5; p53-regulated DNA damage-inducible 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 Gene:8795; Ensembl:ENSG00000120889; OMIM:603612; and UniProtKB:014763); CLU (clusterin; testosterone-suppressed prostate message 2; apolipoprotein J; complement-related protein SP-40,40; complement cytolysis inhibitor; complement lysis inhibitor; sulfated glycoprotein 2; Ku70-binding protein 1; NA1 / NA2; TRPM-2; APO-J; APOJ; KUB1; CLI; clusterin (complement lysis inhibitor, SP-40,40, sulfated glycoprotein 2, testosterone-suppressed prostate message 2, apolipoprotein J); senescence-associated gene 4 protein; senescence-associated protein 4; SGP-2; SP-40; TRPM2; AAG4; CLU1; CLU2; SGP2; HGNC:2095; Entrez Gene:1191; Ensembl:ENSG00000120885; OMIM:185430; and UniProtKB:P10909); VAMP2 (vesicle-associated membrane protein 2; synaptobrevin 2; SYB2; vesicle-associated membrane protein 2; synaptobrevin-2; HGNC:12643; Entrez Gene:6844; Ensembl:ENSG00000220205; OMIM:185881; and UniProtKB:P63027); SLC3A2 (solute carrier family 3 member 2; lymphocyte activation antigen 4F2 large subunit; solute carrier family 3 (activator of dibasic and neutral amino acid transport), member 2; monoclonal antibody 4F2, TRA1.10, TROP4, and T4 antigen defined by 3; solute transporter family 3 (amino acid transporter heavy chain), member 2; 4F2 cell surface antigen heavy chain; CD98 heavy chain; 4F2HC; MDU1; antigen defined by monoclonal antibody 4F2, heavy chain; antigen defined by monoclonal antibody 4F2; 4F2 heavy chain antigen; 4F2 heavy chain; CD98 antigen; CD98HC; 4T2HC; NACAE; CD98; 4F2; HGNC: 11026; Entrez Gene: 6520; Ensembl: ENSG00000168003; OMIM: 158070;and UniProtKB:P08195); RBPMS (RNA Binding Protein With Multiple Splicing; Heart and RRM Expressed Sequence; HERMES; RNA-Binding Protein With Multiple Splicing; RBP-MS; HGNC:19097; Entrez Gene:11030; Ensembl:ENSG00000157110; OMIM:601558; and 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 (also known as syndecan 4 (amphiglycan, ryudocan); syndecan proteoglycan 4; ryudocan core protein; amphiglycan; SYND4; ryudocan amphiglycan; syndecan-4; HGNC:10661; Entrez Gene:6385; Ensembl:ENSG00000124145; OMIM:600017; and UniProtKB:P31431); KIF13B; SLECA2; PDE7A; ATP1B1; CDK1; BMPRIB; MCPH1; and RAB2IL1.

[0024] Certain aspects of the present disclosure relate to determining whether a subject has an NRG1 fusion. Detection methods are known in the art, including PCR analysis, nucleic acid sequencing, fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH), and comparative genomic hybridization (CGH).

[0025] 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 scrapings from the lining of the oral cavity, but may also be extracted from other biological samples, including urine, tumors, or expectorated sputum. The sample itself is typically considered to include nucleated cells (e.g., blood or oral cells) or tissues removed from a subject, including tumor tissue. Methods and reagents for obtaining, processing, and analyzing samples are known in the art. In some embodiments, the sample is obtained with the assistance of a healthcare provider, for example, to draw blood or obtain a tumor biopsy. In some embodiments, the sample is obtained without the assistance of a healthcare provider, for example, when the sample is obtained non-invasively, such as a sample containing oral cells obtained using a cheek swab or brush, or a mouthwash sample.

[0026] In particular, the patient sample can be any tissue or fluid of the body that contains nucleic acid from the cancer of interest.In certain embodiments, the sample can be a blood sample containing circulating tumor cells or cell-free DNA.In other embodiments, the sample can be tissue such as tumor tissue.Tumor tissue can be fresh frozen or formalin-fixed, paraffin-embedded (FFPE).

[0027] In some cases, biological samples can be processed for DNA isolation. For example, DNA in cell or tissue samples can be separated from other components of the sample. Cells can be harvested from biological samples using standard techniques known in the art. For example, cells can be harvested by centrifuging a cell sample and resuspending the pelleted cells. The cells can be resuspended in a buffered solution such as phosphate-buffered saline (PBS). After centrifuging 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 DNA. All samples obtained from a subject, including those that undergo any type of further processing, are considered to be obtained from the subject. Genomic DNA can be extracted from biological samples using conventional methods, including, for example, phenol extraction. Alternatively, genomic DNA can be extracted using a kit such as the QIAamp® Tissue Kit (Qiagen, Chatsworth, Calif.) or the Wizard® Genomic DNA Purification Kit (Promega).

[0028] If desired, nucleic acid amplification can be achieved 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, as described herein, the DNA in the sample is examined to determine the identity of NRG1 fusion. NRG1 fusion can be detected by any method described herein, for example, by sequencing, or by hybridization of genes, RNA, or cDNA in genomic DNA to a nucleic acid probe, for example, a DNA probe (including cDNA and oligonucleotide probe) or an RNA probe. The nucleic acid probe can be designed to specifically or preferentially hybridize with a specific NRG1 fusion.

[0029] A set of probes typically refers to a set of primers, usually a primer pair, and / or a detectably labeled probe, used to detect a target genetic variation (e.g., an NRG1 fusion) used in the actionable treatment recommendations of the present disclosure. The primer pair is used in an amplification reaction to define an amplification product corresponding to the NRG1 fusion. The set of amplification products is detected with a matching set of probes. In an exemplary embodiment, the method uses a TaqMan PCR probe used to detect a set of target genetic variations, e.g., an NRG1 fusion. (商標) (Roche Molecular Systems, Pleasanton, Calif.) assays can be used. In one embodiment, the set of probes is a set of primers used to generate amplification products that are detected in a nucleic acid sequencing reaction, e.g., 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.

[0030] Analysis of nucleic acid markers can be performed using techniques known in the art, including, but not limited to, sequence analysis and electrophoretic analysis. Non-limiting 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 hybridization sequencing. Non-limiting examples of electrophoretic analysis include slab gel electrophoresis, such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis. Additionally, next-generation sequencing methods can be performed using commercially available kits and equipment from companies such as Life Technologies / Ion Torrent PGM or Proton, Illumina HiSEQ or MiSEQ, and Roche / 454 next-generation sequencing systems.

[0031] Other methods of nucleic acid analysis include direct manual sequencing (U.S. Pat. No. 5,288,644); automated fluorescent sequencing; single-strand conformation polymorphism analysis (SSCP); constant denaturing gel electrophoresis (CDGE); two-dimensional gel electrophoresis (2DGE or TDGE); conformational sensitive gel electrophoresis (CSGE); denaturing gradient gel electrophoresis (DGGE); denaturing 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; the 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, the entire contents of which are incorporated herein by reference.

[0032] In one example, a method for identifying an NRG1 fusion in a sample comprises contacting nucleic acid from the sample with a nucleic acid probe capable of specifically hybridizing to a nucleic acid encoding an NRG1 fusion, and detecting said hybridization. In certain embodiments, the probe is radioisotope-conjugated ( 3 H, 32 P, or 33 The probe is detectably labeled with, for example, phosphodiesterase (P), a fluorescent agent (rhodamine or fluorescein), or a chromogenic agent. In certain embodiments, the probe is an antisense oligomer, such as a PNA, morpholino-phosphoramidate, LNA, or 2'-alkoxyalkoxy. The probe can 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 comprising an oligonucleotide that specifically hybridizes to a particular NRG1 fusion. The kit can further comprise instructions for treating a patient with a tumor containing an NRG1 fusion with poziotinib, either alone or in combination with a HER2 / HER3-targeting antibody, based on the results of a hybridization test using the kit.

[0033] II.HER2 / HER3 targeting antibody As used herein, the term "HER2 / HER3 targeting antibody" includes any molecule that interferes with the function of HER2 and / or HER3. Thus, HER2 / HER3 targeting 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 targeting antibodies can prevent the formation of HER2 / HER2 dimers and / or HER2 / HER3 dimers (e.g., trastuzumab or pertuzumab). In some cases, HER2 / HER3 targeting antibodies can be antibody-drug conjugates (e.g., T-DM1 or U3-1402).

[0034] In certain embodiments, the HER2 / HER3 targeting antibody is selected from the group consisting of trastuzumab (Genentech and Roche), trastuzumab emtansine (T-DM1; Genentech and Roche), pertuzumab (Genentech), ertumaxomab (Fresenius), margetuximab (MacroGenics), MCLA-128 (zenoctuzumab; Merus), MM-111 (Merrimack), MM-121 (Merrimack), CT-P06 (Celltrion), GSK2849330 (GlaxoSmithKline), PF-05280014 (Pfizer), MM-302 (Merrimack), SB3 (Merck & Co), CMAB302 (Shanghai CP 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).

[0035] Trastuzumab (CAS 180288-69-1, HERCEPTIN®, huMAb4D5-8, rhuMAb HER2, Genentech) is a humanized IgG1 kappa monoclonal antibody that binds with high affinity and selectivity 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 human framework regions with the complementarity-determining regions of a murine antibody (4D5) that binds to HER2. Trastuzumab binds to the HER2 antigen, thereby inhibiting the growth of cancerous cells. Trastuzumab has been shown to inhibit the growth of HER2-overexpressing human tumor cells in both in vitro assays and in animals. Trastuzumab is a mediator of antibody-dependent cellular cytotoxicity, or ADCC.

[0036] Trastuzumab emtansine, also known as ado-trastuzumab emtansine and sold under the trade name KADCYLA®, is an antibody-drug conjugate consisting of the humanized monoclonal antibody trastuzumab covalently linked to the cytotoxic agent emtansine (DM1). Trastuzumab alone stops cancer cell growth by binding to the HER2 receptor, but trastuzumab emtansine undergoes receptor-mediated internalization into cells and is catabolized in lysosomes, where DM1-containing catabolites are released and subsequently bind to tubulin, causing mitotic arrest and cell death. Trastuzumab binding to HER2 prevents receptor homodimerization or heterodimerization (HER2 / HER3), ultimately inhibiting the activation of MAPK and PI3K / AKT cell signaling pathways. Because the monoclonal antibody targets HER2, which is overexpressed only in cancer cells, the conjugate specifically delivers the cytotoxic agent DM1 to tumor cells. The conjugate is abbreviated as T-DM1. T-DM1 can be administered at a dose of 2-3 mg / kg, e.g., 3.6 mg / kg. T-DM1 can be administered by intravenous infusion.

[0037] Pertuzumab (CAS Registry Number 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 of trastuzumab is located in 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 the ability of HER2 to cooperate with other HER family receptors, it blocks cell signaling, which can ultimately lead to the inhibition of cancer cell growth and cancer cell death.

[0038] Further exemplary HER2 / HER3 targeting antibodies include MM-121 / SAR256212, a fully human monoclonal antibody that targets the HER3 receptor and has been reported to be useful in the treatment of non-small cell lung cancer (NSCLC), breast cancer, and ovarian cancer. SAR256212 is an investigational fully human monoclonal antibody that targets the HER3 (ErbB3) receptor. Durigotuzumab (MEHD7945A, RG7597) is a humanized IgG1 monoclonal antibody that targets HER1 and HER3 and is described as useful for head and neck cancer. Margetuximab (MGAH22) is an Fc-optimized monoclonal antibody that targets HER2.

[0039] Antibodies according to the present disclosure can be defined primarily by their binding specificity. Those skilled in the art can determine whether a given antibody falls within the scope of the claims by assessing 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 truncation analysis, high-resolution electron microscopy using single-particle reconstruction, cryoEM, or tomography, crystallographic studies, and NMR analysis.

[0040] The present disclosure includes antibodies that can bind to the same epitope or a part of the epitope.In addition, the present disclosure also includes antibodies that compete with any of the specific exemplary antibodies described herein for binding to a target or a fragment thereof.Using conventional methods known in the art, 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.For example, to determine whether a test antibody binds to the same epitope as a reference antibody, the reference antibody is bound to the target under saturating conditions.Then, the ability of the test antibody to bind to the target molecule is evaluated.If the test antibody can bind to the target molecule after saturating binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope from the reference antibody.On the other hand, if the test antibody cannot bind to the target molecule after saturating binding with the reference antibody, the test antibody may bind to the same epitope as the epitope bound by the reference antibody.

[0041] Two antibodies bind to the same or overlapping epitopes if they each competitively inhibit (block) the binding of the other to the antigen. That is, when measured in a competitive binding assay, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, but preferably 75%, 90%, or even 99%. Alternatively, 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. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

[0042] Then, further conventional experiments (such as peptide mutation and binding analysis) can be carried out 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 blocking (or another phenomenon) is responsible for the observed lack of binding.This type of experiment can be carried out 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 show whether two antibodies that compete for binding recognize the same epitope.

[0043] In another aspect, antibodies can be defined by their variable sequences, including additional "framework" regions. Furthermore, antibody sequences can be varied from these sequences, optionally using methods described in more detail below. For example, nucleic acid sequences can be modified in the following ways: (a) the variable regions can be separated from the light and heavy chain constant domains; (b) the nucleic acid can be varied from those described above but without affecting the residues encoded thereby; (c) the nucleic acid can be varied from those described above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology; (d) the nucleic acid can be modified to a desired concentration, e.g., from about 0.02 M to about 0.15 M at a temperature of about 50° C. to about 70° C. (e) amino acids may vary from those described above by a given percentage of homology, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or (f) amino acids may vary from those described above by allowing for conservative substitutions (described below).

[0044] When comparing polynucleotide and polypeptide sequences, if the nucleotide or amino acid sequences of the two sequences are the same when aligned for maximum matching, as described below, the two sequences are said to be "identical." Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20 consecutive positions, usually 30 to about 75, 40 to about 50, in which a sequence can be compared with a reference sequence of the same number of consecutive positions after optimally aligning the two sequences.

[0045] Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.) using 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.

[0046] 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, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computer implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.

[0047] One specific example of an algorithm suitable for determining percent sequence identity and percent sequence similarity 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, for example, with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the present disclosure. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The rearranged nature of antibody sequences and the variable length of each gene necessitates multiple rounds of BLAST searches for a single antibody sequence. Furthermore, manual assembly of different genes is difficult and error-prone. The sequence analysis tool IgBLAST (available on the World Wide Web at ncbi.nlm.nih.gov / igblast / ) reveals matches to germline V, D, and J genes, details of rearrangement junctions, and delineation of Ig V domain framework regions and complementarity-determining regions. IgBLAST can analyze either nucleotide or protein sequences, can process sequences in batches, and allows simultaneous searches against germline gene databases and other sequence databases, minimizing the chance of missing potentially best-matching germline V genes.

[0048] In one example, the cumulative score can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Extension of word hits in each direction is stopped: until the cumulative alignment score is reduced by an amount X from its maximum achieved value; until the accumulation of one or more negative-scoring residue alignments causes the cumulative score to fall below zero; or until the end of either 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 as defaults a word length (W) of 11 and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignment, a word length (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0049] For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of word hits in each direction is terminated: until the cumulative alignment score is reduced by an amount X from its maximum achieved value; until the accumulation of one or more negative-scoring residue alignments causes the cumulative score to fall below zero; or until the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.

[0050] In one approach, "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) of up to 20 percent, typically 5-15 percent or 10-12 percent, compared to the reference sequence (which does not contain additions or deletions) due to 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 occur 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.

[0051] Yet another way to define an antibody is as a "derivative" of any of the described antibodies and antigen-binding fragments thereof. The term "derivative" refers to an antibody or antigen-binding fragment thereof that immunospecifically binds 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 can introduce naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. The term "derivative" encompasses, for example, variants with altered CH1, hinge, CH2, CH3, or CH4 regions to form, for example, antibodies with variant Fc regions that exhibit 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., altered 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, linkage to cellular ligands or other proteins, etc. In some embodiments, altered glycosylation modulates one or more of the following: antibody solubilization, facilitation of intracellular trafficking and secretion of the antibody, promotion of antibody assembly, conformational integrity, and antibody-mediated effector function. In certain embodiments, altered glycosylation enhances antibody-mediated effector function compared to an antibody lacking the glycosylation. Glycosylation modifications that lead to altered antibody-mediated effector function are well known in the art.

[0052] Derivative antibodies or antibody fragments can be generated with engineered sequences or glycosylation states to confer desired levels of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), or antibody-dependent complement deposition (ADCD) function activity as measured by bead- or cell-based assays or in vivo studies in animal models.

[0053] 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, metabolic synthesis of tunicamycin, etc. In one aspect, an antibody derivative will have similar or identical function as the parent antibody. In another aspect, an antibody derivative will exhibit altered activity compared to the parent antibody. For example, a derivative antibody (or fragment thereof) may bind to its epitope more tightly or be more resistant to proteolysis compared to the parent antibody.

[0054] III. Treatment Methods The present invention provides methods for treating cancer patients with poziotinib alone or in combination with a HER2 / HER3-targeting antibody. Such treatment 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 comprising the cancer may harbor NRG1 fusions, indicating that the patient is a candidate for treatment. In some aspects, the patient's cancer cells lack mutations at EGFR T790 and / or EGFR C797. In some aspects, the patient's cancer cells lack a mutation in HER2 T798 and / or HER2 C805.

[0055] In certain aspects, the presence of NRG1 fusion is determined by analyzing the genome sample from the subject.In some aspects, the genome sample is isolated from saliva, blood, urine or tumor tissue.In certain aspects, the presence of NRG1 fusion is determined by nucleic acid sequencing (for example, DNA sequencing of tumor tissue or plasma-derived circulating free DNA) or PCR analysis.

[0056] Certain embodiments relate to the administration of poziotinib (also known as HM781-36B, HM781-36, and 1-[4-[4-(3,4-dichloro-2-fluoroanilino)-7-methoxyquinazolin-6-yl]oxypiperidin-1-yl]prop-2-en-1-one) to a subject determined to have an NRG1 fusion. Poziotinib is a quinazoline-based pan-HER inhibitor that irreversibly blocks signaling through the HER family of tyrosine kinase receptors, including HER1, HER2, and HER4. Poziotinib or structurally similar compounds (e.g., U.S. Patent No. 8,188,102 and U.S. Patent Application Publication No. 20130071452; incorporated herein by reference) can be used in this method.

[0057] In some aspects, poziotinib is further defined as poziotinib hydrochloride. In certain aspects, poziotinib hydrochloride is formulated as a tablet. Poziotinib can be administered orally, such as in a tablet. Poziotinib can be administered at a dose of 4 to 25 mg, for example, at a dose of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mg. In certain aspects, poziotinib is administered at a dose of 6 mg, 8 mg, 12 mg, or 16 mg. Dosing can be twice daily, daily, every other day, every three days, or weekly. Dosing can be on a continuous schedule, such as a 28-day cycle.

[0058] In certain aspects, poziotinib and / or HER2 / HER3 targeting antibodies are administered intravenously, subcutaneously, intraosseously, orally, transdermally, sustained-release, controlled-release, delayed-release, as a suppository, or sublingually. In some aspects, administration of poziotinib and / or HER2 / HER3 targeting antibodies includes local, regional, or systemic administration. In certain aspects, poziotinib and / or HER2 / HER3 targeting antibodies are administered two or more times, for example, daily, every other day, or weekly.

[0059] In some aspects, poziotinib is administered before or after the HER2 / HER3 targeting antibody, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 1 month or more apart. In some aspects, poziotinib is administered simultaneously with the HER2 / HER3 targeting antibody.

[0060] As used herein, the term " subject " or " patient " refers to any individual that the subject method is carried out.Generally, patient is human, but as recognized by those skilled in the art, patient can also be animal.Therefore, other animals are included in the definition of patient, including mammals, for example, rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, livestock (including cows, horses, goats, sheep, pigs, etc.), and primates (including monkeys, chimpanzees, orangutans and gorillas).

[0061] "Treatment" and "treating" refer to the administration or application of a therapeutic agent to a subject or the performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit for a disease or health-related condition. For example, treatment can include administering chemotherapy, immunotherapy, radiation therapy, performing surgery, or any combination thereof.

[0062] The methods described herein are useful for inhibiting cell (e.g., tumor cell) survival or proliferation, treating proliferative diseases (e.g., cancer, psoriasis), and treating pathogenic infections. Generally, the terms "cancer" and "cancerous" refer to or describe a physiological state in a mammal that is typically characterized by unregulated 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 can occur in the lung, kidney, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, liver, nasopharynx, cervix, ovaries, pancreas, prostate, skin, stomach, testes, tongue, or uterus.

[0063] The cancer may be of the following histological types, among others, but not limited to: neoplasia, malignancy; carcinoma; non-small cell lung cancer; kidney cancer; renal cell carcinoma; renal clear cell carcinoma; lymphoma; blastoma; sarcoma; carcinoma, undifferentiated; meningioma; brain tumor; oropharyngeal cancer; nasopharyngeal carcinoma; biliary tract cancer; pheochromocytoma; pancreatic islet cell carcinoma; Li-Fraumeni tumor; thyroid cancer; parathyroid cancer; pituitary tumor; adrenal tumor; osteogenic sarcoma tumor; neuroendocrine tumor; breast cancer; lung cancer; head and neck cancer; prostate cancer; esophageal cancer; tracheal cancer; liver cancer; bladder cancer; gastric 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;Oral pharyngeal cancer;Nasopharyngeal cancer;Respiratory cancer;Genitourinary cancer;Squamous cell carcinoma;Lymphoepithelial carcinoma;Basal cell carcinoma;Hair stromal carcinoma;Transitional cell carcinoma;Papillary transitional cell carcinoma;Adenocarcinoma;Gastrointestinal cancer;Gastrinoma, malignant;Bile duct carcinoma;Hepatocellular carcinoma;Mixed hepatocellular and cholangiocarcinoma;Follicle adenocarcinoma;Adenoid cystic carcinoma;Adenomatous intrapolypoid adenocarcinoma;Adenocarcinoma, familial polyposis coli;Solid tumors;Carcinoid tumors, malignant;Bronchioloalveolar carcinoma;Papillary adenocarcinoma;Chromophobe Cancer;Eosinophilic carcinoma;Eosinophilic adenocarcinoma;Basophilic carcinoma;Clear cell adenocarcinoma;Granular cell carcinoma;Follicle adenocarcinoma;Papillary-follicular adenocarcinoma;Non-encapsulated sclerosing carcinoma;Adrenal cortical carcinoma;Endometrioid carcinoma;Cut adnexal carcinoma;Apocrine adenocarcinoma;Sebaceous gland carcinoma;Eurinary gland carcinoma;Mucous epidermoid 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 of the breast;Acinic cell carcinoma;Adenosquamous carcinoma;Adenocarcinoma with squamous metaplasia;Thymoma, malignant;Ovarian stromal tumor, malignant;Theca cell tumor, malignant ;Granulosa cell tumor, malignant;Androblastoma, malignant;Sertoli cell carcinoma;Leydig cell tumor, malignant;Lipocyte tumor, malignant;Paraganglioma, malignant;Extramammary paraganglioma, malignant;Pheochromocytoma;Hemangioangiosarcoma;Malignant melanoma;Amelanotic melanoma;Superficial spreading melanoma;Malignant melanoma in giant pigmented nevus;Lentigo maligna melanoma;Acral lentigo melanoma;Nodular melanoma;Epithelioid cell melanoma;Blue nevus, malignant;Sarcoma;Fibrosarcoma;Fibrous histiocytoma, malignant;Myxosarcoma;Liposarcoma;Leiomyosarcoma;Rhabdomyosarcoma;Embryonal rhabdomyosarcoma;Alveolar rhabdomyosarcoma;Stromatous sarcoma;Mixed tumor, malignant;Mixed Müllerian tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Mesenchymoma, malignant; Brenner tumor, malignant; Phyllodes tumor, malignant; Synovial sarcoma; Mesothelioma, malignant; Dysgerminoma; Embryonal carcinoma; Teratoma, malignant; Ovarian stromatosis, malignant; Chorioepithelioma; Mesonephroma, malignant; Angiosarcoma; Hemangioendothelioma, malignant; Kaposi's sarcoma; Hemangiopericytoma, malignant; Lymphangiosarcoma; Osteosarcoma; Parosteal osteosarcoma; Chondrosarcoma; Chondroblastic, malignant; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's Sarcoma; Odontogenic tumor, malignant; Ameloblastic odontosarcoma; Ameloblastoma, malignant; Ameloblastoma fibrosarcoma; Endocrine or neuroendocrine or hematopoietic carcinoma; Pinealoma, malignant; Chordoma; Cancer of central or peripheral nervous system tissue; Glioma, malignant; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrous astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal tumor; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Reticulum Membranoblastoma; Olfactory nerve tumor; Meningioma, malignant; Neurofibrosarcoma; Schwannoma, malignant; Granular cell tumor, malignant; B-cell lymphoma; Malignant lymphoma; Hodgkin's disease; Hodgkin's; Low-grade / follicular non-Hodgkin's lymphoma; Paragranuloma; Malignant lymphoma, small lymphoid; Malignant lymphoma, large cell, diffuse; Malignant lymphoma, follicular; Mycosis fungoides; Mantle cell lymphoma; Waldenstrom's macroglobulinemia; Other specified non-Hodgkin's lymphoma; Malignant Histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and hairy cell leukemia.

[0064] The term "therapeutic benefit" or "therapeutically effective," as used throughout this application, refers to anything that promotes or enhances the well-being of a subject in relation to the medical treatment of the condition. This includes, but is not limited to, reducing the frequency or severity of signs or symptoms of a disease. For example, cancer treatment can involve, for example, reducing the invasiveness of a tumor, reducing the rate of cancer growth, or preventing metastasis. Cancer treatment can also refer to extending the survival of a subject with cancer.

[0065] Similarly, a patient's effective response to treatment or a patient's "responsiveness" refers to the clinical or therapeutic benefit that is provided to a patient at risk of or suffering from a disease or disorder. Such benefit can include a cellular or biological response, a complete response, a partial response, a stable disease (no progression or recurrence), or a response with late recurrence. For example, an effective response can be a reduction in tumor size or progression-free survival in a patient diagnosed with cancer.

[0066] Regarding the treatment of neoplastic conditions, depending on the stage of the neoplastic condition, the treatment of the neoplastic condition involves one or a combination of the following therapies: surgery to remove neoplastic tissue, radiation therapy, and chemotherapy.Other therapeutic regimens can be combined with the administration of anti-cancer agents, such as therapeutic compositions and chemotherapeutic agents.For example, patients treated with such anti-cancer agents can also undergo radiation therapy and / or surgery.

[0067] For the treatment of a disease, the appropriate dosage of the therapeutic composition will depend on the type of disease being treated, as defined above, the severity and course of the disease, previous therapy, the patient's medical history and response to the agent, and the discretion of the physician. The agent may be suitably administered to the patient at one time or over a series of treatments.

[0068] Methods and compositions that include combination therapy enhance the therapeutic or protective effect and / or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy. Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect, such as killing cancer cells and / or inhibiting cell hyperproliferation. Tissues, tumors, or cells can be contacted with one or more compositions or pharmacological preparations containing one or more agents, or by contacting the tissues, tumors, and / or cells with two or more different compositions or preparations. It is also contemplated that such combination therapy can be used in conjunction with radiation therapy, surgery, or immunotherapy.

[0069] The combined administration can include simultaneous administration of two or more agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, the subject therapeutic composition and another therapeutic agent can be formulated together in the same dosage form and administered simultaneously. Alternatively, the subject therapeutic composition and another therapeutic agent can be administered simultaneously, in which case both agents are present in separate formulations. In another alternative, a therapeutic agent can be administered immediately followed by another therapeutic agent, or vice versa. In separate administration protocols, the subject therapeutic composition and another therapeutic agent can be administered several days apart, or several hours apart, or several days apart.

[0070] A first anticancer treatment can be administered before, during, or after a second anticancer treatment, or in various combinations. Administration can occur over a period ranging from simultaneous administration to minutes to days to weeks. In embodiments in which a first treatment is provided to a patient separately from a second treatment, there will generally be an effective period between the time of delivery, ensuring that the two compounds can still exert their beneficially combined effect on the patient. In such cases, it is contemplated that the first and second therapies can be provided to the patient within about 12 to 24 or 72 hours of each other, more particularly, within about 6 to 12 hours of each other. In some situations, it may be desirable to extend the period of treatment significantly, where days (2, 3, 4, 5, 6, or 7) to weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between each administration.

[0071] In certain embodiments, a course of treatment is contemplated to last from 1 to 90 days or more (such ranges inclusive). It is contemplated that one agent can be given on any day from day 1 to day 90 (such ranges inclusive), or any combination thereof, and another agent can be given on any day from day 1 to day 90 (such ranges inclusive), or any combination thereof. One or more doses of the agent can be administered to the patient within a single day (24-hour period). Furthermore, it is contemplated that after a course of treatment, there will be a period during which no anti-cancer treatment is administered. This period can last from 1 to 7 days, and / or 1 to 5 weeks, and / or 1 to 12 months or more (such ranges inclusive), depending on the patient's condition, e.g., the patient's prognosis, strength, health, etc. It is expected that the treatment cycle will be repeated as necessary.

[0072] Various combinations can be used. For the following examples, either (a) poziotinib is "A" and the HER2 / HER3 targeted antibody is "B"; or (b) poziotinib, either alone or in combination with a HER2 / HER3 targeted antibody, is "A" and another anti-cancer therapy is "B": TIFF0007777533000001.tif27128.

[0073] Administration of any compound or therapy of the present invention to a patient will follow standard protocols for the administration of such compounds, taking into account the toxicity, if any, of the agent. Thus, in some embodiments, there is a step of monitoring for toxicity that may result from the combination therapy.

[0074] 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. The term "chemotherapeutic agent" is used to include compounds or compositions administered in cancer therapy. These agents or drugs are categorized by 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 directly crosslink DNA, intercalate into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0075] Examples of chemotherapeutic agents include: alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines, such as altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptogens (especially cryptogens) ... tophicins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, e.g., chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquinone, fenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 11 and calicheamicin omega 11); dynemicins, such as dynemicin A; bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, authranycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo- L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, potfiromycin, piriformisin, anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamipiri and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenal agents such as mitotane and trilostane; folic acid supplements such as furoic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilon; etoglucide;Gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triazicone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vin Desine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Taxoids, e.g., paclitaxel and docetaxel, gemcitabine; 6-thioguanine; Mercaptopurine; Platinum coordination 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 inhibitors RFS 2000; difluoromethylornithine (DFMO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0076] 2. Radiation therapy Other widely used agents that cause DNA damage include gamma rays, X-rays, and / or what are commonly known as directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are also contemplated, such as microwaves, proton beam irradiation (U.S. Patent Nos. 5,760,395 and 4,870,287), and UV irradiation. All of these agents most likely affect a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50-200 roentgens for prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. The dose range for radioisotopes varies widely and depends on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

[0077] 3. Immunotherapy Those skilled in the art will understand that additional immunotherapy 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. The immune effector can be, for example, an antibody specific to some marker on the surface of tumor cells. The antibody alone can act as the therapeutic effector, or the antibody can recruit other cells to actually affect cell death. The antibody can also be conjugated to a drug or toxin (such as a chemotherapeutic agent, a radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and simply act as a targeting agent. Alternatively, the effector can be a lymphocyte bearing a surface molecule that interacts either directly or indirectly with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0078] In one aspect of immunotherapy, tumor cells must have some marker suitable for targeting, i.e., not present on the majority of other cells. Many tumor markers exist, any of which may be suitable for targeting in the present 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 to combine anti-cancer effects with immunostimulatory effects. There are also immunostimulatory molecules, 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.

[0079] Examples of immunotherapies currently under consideration or in use include immunoadjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patents 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 therapy, such as 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; U.S. Pat. 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 contemplated that one or more anti-cancer therapies can be used in conjunction with the antibody therapies described herein.

[0080] In some embodiments, immunotherapy can be adoptive immunotherapy, which involves transferring autoantigen-specific T cells generated ex vivo. T cells used for adoptive immunotherapy can be generated either by expanding antigen-specific T cells or by redirecting T cells through genetic engineering. The isolation and transfer of tumor-specific T cells has been shown to be successful in the treatment of melanoma. Genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) has successfully generated novel T cell specificities. CARs are synthetic receptors consisting of a targeting moiety linked 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 single-chain antibody (scFv), which contains the light chain variable fragment of a monoclonal antibody linked by a flexible linker. Binding moieties based on receptor or ligand domains have also been successfully used. The signaling domain of first-generation CARs is derived from the cytoplasmic region of CD3 zeta or the Fc receptor gamma chain. CARs have successfully redirected T cells against antigens expressed on the surface of tumor cells from a variety of malignancies, including lymphomas and solid tumors.

[0081] In one embodiment, the present application provides a combination therapy for cancer treatment, the combination therapy comprising an 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 to a tumor antigen.

[0082] Immunomodulators include immune checkpoint inhibitors, costimulatory molecule agonists, and immune inhibitory molecule antagonists. Immunomodulators can be drugs, such as small molecules, recombinant forms of ligands or receptors, or antibodies, such as human antibodies (e.g., International Patent Publication No. WO2015 / 016718; Pardoll, Nat Rev Cancer, 12(4): 252-264, 2012; both are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or their analogs can be used, particularly chimeric, humanized, or human forms of antibodies. As those skilled in the art will recognize, alternative and / or equivalent names may be used for certain antibodies referred to in this disclosure. Such alternative and / or equivalent names are interchangeable in this disclosure. For example, it is known that lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0083] Costimulatory 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 immunomodulatory agent is an anti-OX40 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human OX40 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present method can be generated using methods well known in the art. Alternatively, art-recognized anti-OX40 antibodies can be used. An exemplary anti-OX40 antibody is PF-04518600 (see, e.g., WO 2017 / 130076). ATOR-1015 is a bispecific antibody that targets CTLA4 and OX40 (see, e.g., WO 2017 / 182672, WO 2018 / 091740, WO 2018 / 202649, WO 2018 / 002339).

[0084] Another costimulatory 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), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human ICOS antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present method can be generated using methods well known in the art. Alternatively, art-recognized anti-ICOS antibodies can be used. Exemplary anti-ICOS antibodies include JTX-2011 (see, for example, WO 2016 / 154177, WO 2018 / 187191) and GSK3359609 (see, for example, WO 2016 / 059602).

[0085] Yet another costimulatory molecule that can be targeted by the methods provided herein is glucocorticoid-induced tumor necrosis factor receptor-related 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), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human GITR antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present method can be generated using methods well known in the art. Alternatively, art-recognized anti-GITR antibodies can be used. An exemplary anti-GITR antibody is TRX518 (see, for example, WO 2006 / 105021).

[0086] 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 attenuator (BTLA), CCL5, CD27, CD38, CD8A, CMKLR1, cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), CXCL9, CXCR5, HLA-DRB1, HLA-DQA1, HLA-E, killer cell immunoglobulin (KIR), lymphocyte activation genes These include CD19-3 (also known as CD223, LAG-3), Mer tyrosine kinase (MerTK), NKG7, programmed death 1 (PD-1), programmed death-ligand 1 (PD-L1, also known as CD274), PDCD1LG2, PSMB10, STAT1, T cell immunoreceptor with Ig and ITIM domains (TIGIT), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V domain Ig suppressor of T cell activation (VISTA, also known as C10orf54). 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.

[0087] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand-binding partner. In a specific aspect, the PD-1 ligand-binding partner is PD-L1 and / or PD-L2. In another embodiment, the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In a specific aspect, the PD-L1 binding partner is PD-1 and / or B7-1. In another embodiment, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In a specific aspect, the PD-L2 binding partner is PD-1. The antagonist can be an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 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 methods provided herein are known in the art and are described, for example, in U.S. Patent Application Publication Nos. 2014 / 0294898, 2014 / 022021, and 2011 / 0008369, all of which are incorporated herein by reference.

[0088] In some embodiments, the PD-1 binding 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 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion (e.g., an Fc region of an immunoglobulin sequence) of PD-L1 or PD-L2 fused to a constant region). In some embodiments, the PD-1 binding 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, KEYTRUDA, is an anti-PD-1 antibody described in WO2006 / 121168. (登録商標) Pembrolizumab, also known as PD-L2-Fc fusion soluble receptor (PD-L2-Fc), is described in WO2009 / 114335. Pembrolizumab, also known as PD-L2-Fc fusion soluble receptor (PD-L2-Fc), is described in WO2010 / 027827 and WO2011 / 066342. ...09 / 114335. Pembrolizumab, also known as PD-L2-Fc fusion soluble receptor (PD-L2-Fc), is described in WO2009 / 101611. Pembrolizumab, also known as PD-L2-Fc fusion soluble receptor (PD-L2

[0089] Another immune checkpoint protein that can be targeted by the methods 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 similar to the T cell costimulatory protein, CD28; 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 for their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for the B7 molecule.

[0090] 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), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, anti-CTLA-4 antibodies disclosed in U.S. Pat. No. 8,119,129; PCT Publication Nos. WO 01 / 14424, WO 98 / 42752, WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab); U.S. Pat. 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 methods disclosed herein. The teachings of each of the above publications are incorporated herein by reference.Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used.For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001 / 014424, WO2000 / 037504, and U.S. Patent No. 8,017,114; all are incorporated herein by reference.

[0091] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO 01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on CTLA-4 as the above-described antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-described antibodies (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. Pat. Nos. 5,844,905, 5,885,796, and International Patent Applications WO1995001994 and WO1998042752, which are incorporated herein by reference, and immunoadhesins such as those described in U.S. Pat. No. 8,329,867, which is incorporated herein by reference.

[0092] Another immune checkpoint protein that can be targeted in the methods provided herein is lymphocyte-activation 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 regulatory 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), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human LAG-3 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art-recognized anti-LAG-3 antibodies can be used. An exemplary anti-LAG-3 antibody is leratolimab (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.

[0093] Another immune checkpoint protein that can be targeted in the methods provided herein is V-domain Ig suppressor 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 white blood cells and inhibits the effector function of T cells. In some embodiments, the immune checkpoint inhibitor is an anti-VISTA antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human VISTA antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art-recognized anti-VISTA antibodies can be used. An exemplary anti-VISTA antibody is JNJ-61610588 (also known as onvatilimab) (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).

[0094] Another immune checkpoint protein that can be targeted by the methods provided herein is CD38. The complete protein sequence of human CD38 has 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), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human CD38 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art-recognized anti-CD38 antibodies can be used. An exemplary anti-CD38 antibody is daratumumab (see, e.g., U.S. Patent No. 7,829,673).

[0095] Another immune checkpoint protein that can be targeted by the methods provided herein is T cell immunoreceptor (TIGIT) with 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), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human TIGIT antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present method can be generated using methods well known in the art. Alternatively, art-recognized anti-TIGIT antibodies can be used. An exemplary anti-TIGIT antibody is MK-7684 (see, for example, WO 2017 / 030823, WO 2016 / 028656).

[0096] Other immunosuppressive 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 GenBank accession number NP_002155. In some embodiments, the immunomodulator is a small molecule IDO inhibitor. Exemplary small molecules include BMS-986205, epacadostat (INCB24360), and navoximod (GDC-0919).

[0097] 4.Surgery Approximately 60% of people with cancer will undergo some type of surgery, including preventive, diagnostic, or staging, curative, and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and this surgery can be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgical surgery (Mohs surgery).

[0098] When cancerous cells, tissues, or tumors are partially or completely removed, a cavity can be formed in the body.Treatment can be achieved by perfusion, direct injection, or local application of additional anti-cancer therapy to the area.Such treatment 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.These treatments can also vary in dosage.

[0099] 5. Other agents It is contemplated that other agents can be used in combination with certain aspects of the present invention to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, cell adhesion inhibitors, agents that sensitize hyperproliferative cells to apoptosis-inducing factors, or other biological agents. It is believed that increasing intercellular signaling by increasing the number of GAP junctions enhances the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present invention to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors are contemplated to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that sensitize hyperproliferative cells to apoptosis, such as the antibody c225, can be used in combination with certain aspects of the present invention to improve the efficacy of the treatment.

[0100] IV. Kit Various aspects of the present invention contemplate kits containing diagnostic, therapeutic, and / or delivery agents. In some embodiments, the present invention contemplates kits for detecting NRG1 fusions in tumor cells of a patient. In some embodiments, the present invention contemplates kits for preparing and / or administering therapies of the present invention. The kits can include reagents that can be used to administer the active or effective agents of the present invention. The kit reagents can include one or more anti-cancer components of the combination therapy, as well as reagents for preparing, formulating, and / or administering the components of the present invention or performing one or more steps of the methods of the present invention. In some embodiments, the kits can also include suitable container means, such as Eppendorf tubes, assay plates, syringes, bottles, or tubes that will not react with the components of the kit. The containers can be made of a sterilizable material, such as plastic or glass. The kits can further include an instruction sheet outlining the procedural steps of the method and following substantially the same procedures as described herein or known to those of skill in the art. [Example]

[0101] V. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. It will be understood by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and therefore can be considered to constitute preferred modes for its practice. However, those of skill in the art will, in light of the present disclosure, recognize that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result, without departing from the spirit and scope of the invention.

[0102] Example 1 The cell viability of the breast cancer cell line, MDA175-VII (NRG1-DOC4 fusion), was tested with poziotinib treatment with and without antibodies targeting HER2 and HER2 / HER3 dimerization and antibody-drug conjugates (ADCs). Cell viability was determined by Cell Titer Glo assay. Poziotinib demonstrated an average IC of 0.287 nM. 50 poziotinib potently inhibited NRG1 fusions in MDA175-VII cells at concentrations lower than previously reported TKIs (Figure 1).

[0103] Furthermore, treatment with the HER2-targeting antibodies trastuzumab, T-DM1, and pertuzumab resulted in IC of >10000 ng / mL, 634.5 ng / mL, and 53.7 ng / mL, respectively. 50 Furthermore, the combination of a HER2 antibody with low-dose poziotinib (0.1 nM) enhanced sensitivity to trastuzumab, pertuzumab, and T-DM1, resulting in an IC 50 The values ​​were reduced to 1.37 nM, 1.23 nM, and 1.32 nM, respectively (FIG. 2B).

[0104] Example 2 Generation of Ba / F3 cells. Ba / F3 cells stably co-expressing WT ErbB2 and WT ErbB3, or WT ErbB3 and WT ErbB4, were generated as previously described. Briefly, retroviral or lentiviral constructs were transfected into Phoenix 293T cells to generate viruses, which were then incubated with the Ba / F3 cell line overnight. The virus was removed, and the cells were cultured in puromycin for 10 days to select for Ba / F3 cell lines stably expressing the retroviral constructs. After selection, the cells were sorted using anti-HER2, anti-HER3, and anti-HER4 antibodies (Biolegend). The cell lines were then re-transduced with lentivirus containing the NRG fusion plasmids listed in Table 1A. The cells were then sorted for NRG1 expression by FACS. IL-3 was then removed from the stable cell lines. The resulting stable cells were used for downstream analyses, including drug screening.

[0105] Drug screening and IC50 determination. Drug screening was performed as previously described. Briefly, cells were plated in technical triplicates at 2000–3000 cells per well in 384-well plates (Greiner Bio-One). Seven different concentrations of TKI or DMSO vehicle were added to a final volume of 40 μL per well. After 72 h, 11 μL of Cell Titer Glo (Promega) was added to each well. Plates were incubated for a minimum of 10 min and bioluminescence was measured using a FLUOstar OPTIMA plate reader (BMG LABTECH). Raw bioluminescence values ​​were normalized to cells treated with the DMSO control and values ​​were plotted in GraphPad Prism. Nonlinear regression was used to fit the normalized data with a variable slope, and IC50 values ​​were determined by interpolation of the concentration at 50% inhibition in GraphPad Prism. 50 Drug screens are performed with technical triplicates on each plate and either duplicate or triplicate biological replicates.

[0106] Overexpression model. The overexpression model was generated by lentiviral transduction of the NRG1 fusions listed in Table 1A. Lentivirus was generated using the Lenti-X cells Lenti-X single shot kit (Takarabio). Lentivirus was generated as described by the manufacturer. The lentivirus was then added to the cell lines listed in Table 1B. 24 hours after viral transduction, the virus was removed, and the cells were placed in 2 μg / ml puromycin for selection. After 10 days of selection, protein and RNA were harvested from the cell lines, and expression of the NRG1 fusions was determined by Western blotting and RT-PCR, respectively. Stable cell lines expressing the NRG1 fusions were used for downstream analyses, including Western blotting and ELISA.

[0107] Inhibition of HER signaling in overexpressing cell lines was determined by Western blotting and ELISA. Parental and overexpressing (OE) cell lines were plated in 10 cm dishes and treated with increasing doses of poziotinib, ranging from 1 nM to 100 nM. Cells were incubated with inhibitors and / or antibodies for 4 hours, 1 day, and 3 days, and proteins were harvested using lysis buffer (Cell Signaling). Expression of NRG1 fusions, phosphorylated EGFR, HER2, HER3, and HER4, and total EGFR, HER2, HER3, and HER4 was determined by Western blotting, and blots were exposed using a BioRad Chemidoc imager. To quantify changes in protein expression, proteins from poziotinib-treated parental and OE-expressing cell lines were loaded into ELISA (Cell Signaling), and the ELISA was completed according to the manufacturer's instructions.

[0108] Table 1A. NRG1 fusion plasmids TIFF0007777533000002.tif16164

[0109] Table 1B. Human cell line models TIFF0007777533000003.tif21164

[0110] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present 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 changes can be made in the methods and method steps or sequence of steps described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related can be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications 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.

[0111] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF0007777533000004.tif128160

Claims

1. 1. A composition for treating a patient with cancer, comprising poziotinib and a HER2 / HER3 targeted antibody selected from trastuzumab, pertuzumab, or T-DM1, The composition, wherein the cancer is breast cancer and has an NRG1 fusion.

2. The composition of claim 1, 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.

3. 3. The composition of any one of claims 1 to 2 for use in combination with a further anti-cancer therapy.

4. 4. The composition of claim 3, wherein the additional anti-cancer therapy is surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, toxin therapy, immunotherapy, or cytokine therapy.

5. The composition of any one of claims 1 to 4, wherein the patient has previously undergone at least one round of anti-cancer therapy.

6. 1. A method of aiding in the selection of a patient with breast cancer for treatment with poziotinib and a HER2 / HER3 targeted antibody selected from trastuzumab, pertuzumab, or T-DM1, comprising: (a) determining whether the patient's breast cancer harbors an NRG1 fusion; (b) assisting in selecting the patient for treatment with poziotinib and a HER2 / HER3-targeted antibody selected from trastuzumab, pertuzumab, or T-DM1 if the patient's breast cancer has an NRG1 fusion. A method comprising:

7. The method of claim 6, 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.

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