Methods and compositions for efg / egfr pathway inhibition in conjunction with anaplastic lymphoma kinase inhibitors

Abstract

A method of treating a patient suffering from a cancer driven by a dysregulated human epidermal growth factor receptor (HER1 / human EGFR) comprising administering to a patient in need of such treatment a flexible proactive regimen to combine an anaplastic lymphoma kinase inhibitor (ALK inhibitor) with an anti-EGF antibody (mAb) for inhibiting the pathway activated by EGF-EGFR binding. The anti-EGF antibody can be produced by proactive immunization or provided passively by administration of an anti-EGF antibody. The method comprises administering the ALK inhibitor according to a continuous regimen based on an average daily dose ranging from 10 to 250 mg, and co-administering the mAb, either proactively or passively, according to a dosing regimen that achieves a therapeutically effective amount repeated three times a week, twice or once, once every two weeks, once every three weeks, or at least once a month.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 715,351, filed August 7, 2018; U.S. Provisional Patent Application No. 62 / 727,056, filed September 5, 2018; U.S. Provisional Patent Application No. 62 / 748,772, filed October 22, 2018; U.S. Provisional Patent Application No. 62 / 760,529, filed November 13, 2018; and U.S. Provisional Patent Application No. 62 / 822,290, filed March 22, 2019, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] Some embodiments of the present invention are directed to methods for the treatment and prevention of diseases (such as cancer), particularly for individuals who have developed drug resistance or are unresponsive to treatment with anaplastic lymphoma kinase inhibitors (ALK inhibitors). Background Technology

[0004] Non-small-cell lung cancer (NSCLC) is a leading cause of cancer-related deaths worldwide. Despite recent advances in treatment and diagnosis, the five-year survival rate remains around 16%. Poor prognosis is primarily due to advanced disease stage, disease refractory nature, and the extent of metastasis at diagnosis. While significant progress has been made in elucidating the genetic abnormalities that lead to malignant tumor cells, currently available chemotherapy therapies are still insufficient, and the prognosis for most patients diagnosed with cancer remains precarious.

[0005] Most chemotherapeutic agents act on specific molecular targets, and researchers believe these specific targets are involved in the development of malignant phenotypes. However, a complex network of signaling pathways regulates cell proliferation, and abnormalities in multiple genes within these pathways promote the formation of most malignant tumors. While the efficacy of standard cytotoxic chemotherapy for lung cancer has been optimized, the latest treatments for NSCLC are based on classifying NSCLC into different molecular subgroups according to their unique oncogene drivers. In treatment, the molecular drivers of NSCLC are attacked directly by targeted agents that target specific oncogenes.

[0006] Most previous chemotherapy drugs for cancer were non-selective in their activity. While their exact mechanisms of action are varied and complex, they generally exert their effects by damaging dividing cells, a phenomenon more common in malignant tumors compared to most normal tissues. Targeted agents aim to achieve their selective action by modulating the activity of proteins essential for tumorigenesis and cancer maintenance, particularly enzymes that drive the uncontrolled growth, angiogenesis, invasiveness, and metastasis characteristic of malignant tumors. Increased differential activity typically reduces side effects that distress cancer patients, particularly nausea, vomiting, and cell death in the bone marrow and gastrointestinal tract, and improves effectiveness against tumor cells.

[0007] A number of promising targets for therapeutic intervention in cancer treatment include members of the HER-kinase axis. These are frequently upregulated in solid epithelial tumors such as prostate, lung, and breast tumors, and also in glioblastoma. Epidermal growth factor receptor (EGFR) is a member of the HER-kinase axis and has become a target of choice for developing several different cancer therapies. Because EGFR pathway activation requires reversible phosphorylation of tyrosine residues, these therapies include EGFR tyrosine kinase inhibitors (EGFR-TKIs). In other words, EGFR-TKIs block cell surface receptors, which are responsible for triggering and / or maintaining cell signaling pathways that lead to tumor cell growth and differentiation. In particular, researchers believe that these inhibitors interfere with the EGFR kinase domain.

[0008] Recently, researchers discovered gene rearrangements in the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase in a subset of non-small cell lung cancer (NSCLC) patients. Subsequent studies demonstrated that crizotinib (a small-molecule ATP-competitive ALK inhibitor) was more effective than chemotherapy in ALK-positive NSCLC patients. Crizotinib, along with two other ATP-competitive ALK inhibitors (ceretinib and alectinib), has been approved for first-line treatment of these patients. Currently, immunohistochemistry and in situ hybridization are used to diagnose ALK rearrangements. The clinical success of these three ALK inhibitors has paved the way for the development of next-generation ALK inhibitors with greater potency and selectivity. Unfortunately, however, patients inevitably develop resistance to ALK inhibitors, leading to tumor recurrence, often manifesting as brain metastases.

[0009] Anaplastic lymphoma kinase (ALK) rearrangements define a unique molecular subtype of non-small cell lung cancer (NSCLC). Recently, the treatment prospects for ALK-positive advanced NSCLC patients have been transformed with the continuous development of the potency and selectivity of ALK inhibitors. Crizotinib is the first ALK inhibitor to enter clinical development. In a randomized phase III trial, crizotinib significantly improved objective response rate (ORR) and progression-free survival (PFS) compared to cytotoxic chemotherapy, establishing crizotinib as a standard of care for ALK-positive advanced NSCLC patients.

[0010] Most patients respond to crizotinib, but all eventually relapse after treatment, typically within one to two years. Analysis of post-progression biopsy specimens has proven extremely valuable, contributing to a better understanding of the molecular mechanisms of crizotinib resistance. These mechanisms are generally categorized into those involving target gene alterations (such as ALK resistance mutations and ALK gene amplification) or off-target resistance mechanisms (such as upregulation of bypass signaling pathways), such as EGFR, KIT, IGF-1R, SRC, and MEK / ERK. Typically, target resistance mechanisms are found in approximately one-third of patients treated with crizotinib.

[0011] Recently, several second-generation ALK inhibitors have demonstrated significant activity in ALK-positive NSCLC. Two of these inhibitors, ceritinib and alectinib, have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of crizotinib-refractory ALK rearranged NSCLC. A third inhibitor, brigatinib, received Breakthrough Therapy Designation from the FDA and was recently approved. In preclinical study models, these second-generation ALK inhibitors overcame several ALK mutations associated with crizotinib resistance. Furthermore, in phase I-II studies, these drugs showed high objective response rates (ORR) (48-71%) in patients resistant to crizotinib. Importantly, second-generation ALK inhibitors are also active in patients without ALK resistance mutations or fusion gene amplifications, suggesting that many cancers develop resistance to crizotinib due to insufficient ALK inhibition. However, despite the efficacy of second-generation ALK inhibitors, patients almost always relapse.

[0012] Although several new approaches aim to overcome the various resistance mechanisms that arise in ALK-positive NSCLC, including knowledge-based alternation and sequential use of different ALK inhibitors and combination therapies targeting alternative ALK+ signaling pathways, new approaches are still needed to address the resistance problem. Summary of the Invention

[0013] One object of the present invention is a method for treating patients with cancer driven by ALK mutations and ROS1 rearrangements, comprising administering a flexible and proactive regimen to patients requiring such treatment to combine an anaplastic lymphoma kinase inhibitor (ALK inhibitor) with active EGF pathway immunization (EGF PTI) to inhibit the pathway activated by EGF-EGFR, wherein in this method, the ALK inhibitor is administered according to a continuous regimen based on an average daily dose in the range of about 10 to 250 mg, and the EGF-PTI is co-administered according to the dosing regimen to achieve a therapeutically effective dose repeated three times, twice, or once a week, once every two weeks, once every three weeks, or at least once a month.

[0014] Another object of the present invention is a method for treating a patient with non-small cell lung cancer (NSCLC) driven by ALK mutations and ROS1 rearrangements, wherein the patient has a tumor expressing a mutated form of EGFR, comprising administering a flexible and active regimen to the patient requiring such treatment to combine an ALK inhibitor with active immunization targeting EGF, wherein in the method, the ALK inhibitor is administered according to a continuous regimen based on an average daily dose in the range of about 10 to 250 mg and active immunization, and co-administered with an EGF-PTI according to a therapeutically effective dose repeated three times a week, twice or once a week, once every two weeks, once every three weeks, or at least once a month, wherein the method results in the prevention of the acquisition of resistance to ALK inhibitor treatment.

[0015] Another object of the present invention is a pharmaceutical kit comprising: a first compartment containing an effective amount of an anti-EGF targeting antibody; and a second compartment containing an effective amount of an ALK inhibitor.

[0016] Another object of the present invention is a pharmaceutical kit comprising: a first compartment containing an effective amount of a vaccine that generates an immune response to EGF; and a second compartment containing an effective amount of an ALK inhibitor.

[0017] Another object of the present invention is a pharmaceutical kit comprising: a first compartment containing an effective amount of a vaccine that generates an immune response to EGFR; and a second compartment containing an effective amount of an ALK inhibitor.

[0018] Another object of the present invention is an ALK inhibitor for treating patients with cancer driven by ALK mutations and ROS1 rearrangements by co-administration with a vaccine that generates an immune response to EGF, wherein the ALK inhibitor is administered according to a continuous regimen based on an average daily dose in the range of about 10 to 250 mg, and the vaccine that generates an immune response to EGF is co-administered to the patient in need of such treatment according to a therapeutically effective dose repeated three times a week, twice or once a week, once every two weeks, once every three weeks, or at least once a month.

[0019] Another object of the present invention is the use of an ALK inhibitor in the preparation of a pharmaceutical kit for treating patients with cancers driven by ALK mutations and ROS1 rearrangements, the pharmaceutical kit comprising: a first compartment containing an effective amount of a vaccine that generates an immune response to EGF; and a second compartment containing an effective amount of an ALK inhibitor administered according to a continuous regimen based on an average daily dose in the range of about 10 to 250 mg, wherein the vaccine is administered to patients requiring such treatment prior to initiation of ALK inhibitor treatment according to a dosage regimen, i.e., from an average weekly dose to a therapeutically effective dose repeated three, two, or one week, once every two weeks, once every three weeks, or at least once a month. Attached Figure Description

[0020] The invention will be further described in the following detailed description through non-limiting embodiments and with reference to the accompanying drawings, wherein similar reference numerals denote similar portions throughout the several accompanying views, wherein:

[0021] A more comprehensive understanding of the invention can be obtained by referring to the specific description and the following figures, wherein:

[0022] Figure 1 The cell lines shown represent approximately 75% of NSCLC patients with ALK rearrangements. H3122 carries an EML4-ALK v1 fusion, and H2228 carries an EML4-ALK v3 fusion.

[0023] Figure 2A -B shows the effects of brigatinib and alectinib on cell viability in the H3122 cell line with and without EGF.

[0024] Figure 3 The study showed the effect of crizotinib on cell viability in the H3122 cell line with and without EGF after 72 hours of incubation.

[0025] Figure 4The effect of BVN22E antibody on cell viability after 72 hours of incubation in the H3122 cell line was shown. C-Ab is the control antibody.

[0026] Figure 5 The study demonstrated the effect of the combination of BVN22E antibody and alectinib on cell viability after 72 hours of incubation in H3122 cells.

[0027] Figure 6 The combined use of BVN22E antibody and crizotinib after 72 hours of incubation in H3122 cells was shown to have an effect on cell viability.

[0028] Figure 7A -B shows the effect of BVN22E antibody in combination with brigantinib and alectinib on cell viability in H3122 and H2228 cells;

[0029] Figure 8 The effects on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473) and ERK1 / 2 (THR 202 / TYR 204) in H3122 cells in response to 2 hours of incubation with BVN22E antibody were shown.

[0030] Figure 9 The effects on pEGFR (TYR1068), pSTAT3 (TYR 705), pAKT (SER 473), and ERK1 / 2 (THR 202 / TYR 204) in H3122 cells in response to 24 hours of incubation with BVN22E antibody were shown.

[0031] Figure 10 The effects of crizotinib on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473) and ERK1 / 2 (THR 202 / TYR204) in H3122 cells in response to 2 hours of incubation with crizotinib were shown.

[0032] Figure 11 shows the effects of brigatinib and alectinib on cell viability in the H2228 cell line with and without EGF.

[0033] Figure 12 The effect of crizotinib on cell viability after 72 hours of incubation in the H2228 cell line with and without EGF was shown.

[0034] Figure 13 The effect of BVN22E antibody on cell viability after 72 hours of incubation in the H2228 cell line was shown. C-Ab is the control antibody.

[0035] Figure 14 The study demonstrated the effect of the combination of BVN22E antibody and alectinib on cell viability after 72 hours of incubation in H2228 cells.

[0036] Figure 15 The combined use of BVN22E antibody and crizotinib on cell viability was demonstrated after 72 hours of incubation in H2228 cells.

[0037] Figure 16A -B shows the effect of the BVN22E antibody, used in combination with brigantinib and alectinib, on cell viability in H2228 cells;

[0038] Figure 17 The effects on pEGFR (TYR1068), pSTAT3 (TYR 705), pAKT (SER 473), and ERK1 / 2 (THR 202 / TYR 204) in H2228 cells in response to 2 hours of incubation with BVN22E antibody were shown.

[0039] Figure 18 The effects on pEGFR (TYR1068), pSTAT3 (TYR 705), pAKT (SER 473), and ERK1 / 2 (THR 202 / TYR 204) in H2228 cells in response to 24 hours of incubation with BVN22E antibody were shown.

[0040] Figure 19 The effects of crizotinib on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473) and ERK1 / 2 (THR 202 / TYR 204) in H2228 cells in response to 2 hours of incubation with crizotinib were shown.

[0041] Figure 20 The effects of brigatinib on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473) and ERK1 / 2 (THR 202 / TYR 204) in H2228 cells in response to brigatinib incubation for 2 hours were shown.

[0042] Figure 21 The effects on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473), and ERK1 / 2 (THR 202 / TYR 204) in H2228 cells in response to incubation for 2 hours with the BVN22E antibody and brigatinib were shown.

[0043] Figure 22 The effects of brigatinib on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473) and ERK1 / 2 (THR 202 / TYR 204) in H3122 cells in response to brigatinib incubation for 2 hours were shown.

[0044] Figure 23 The effects of incubation with high concentrations of brigatinib on pEGFR (TYR1068), pSTAT3 (TYR 705), pAKT (SER 473), and ERK1 / 2 (THR 202 / TYR 204) in H3122 cells for 2 hours were shown.

[0045] Figure 24 The effects on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473) and ERK1 / 2 (THR 202 / TYR 204) in H3122 cells in response to incubation for 2 hours with the BVN22E antibody and brigatinib were shown.

[0046] Figure 25 The results show the time it takes for H2228 cells to develop resistance to crizotinib and alectinib under different growth conditions, with and without BVN22E antibody, as shown by the appearance of resistance colonies (top column) and the delay in cell death (bottom column).

[0047] Figure 26 The study showed that H3122 cells exhibited resistance to crizotinib 0.1 μM in both the presence and absence of BVN22E antibody.

[0048] Figure 27 The study showed that H3122 cells exhibited resistance to crizotinib 0.2 μM in both the presence and absence of BVN22E antibody.

[0049] Figure 28 The BRAF-mutated cell line used in this invention is shown. The HT29 strain is a cell line derived from colon adenocarcinoma and contains the V600E BRAF mutation;

[0050] Figure 29 This demonstrates the effect of EGF on the viability of HT29 cells;

[0051] Figure 30 The effect of BVN22E antibody on cell viability in HT29 cells was shown in the presence and absence of EGF.

[0052] Figure 31This demonstrates the effect of the BVN22E antibody, used in combination with trametinib, on cell viability in the HT29 cell line;

[0053] Figure 32 The effects of trametinib on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473) and pERK1 / 2 (THR 202 / TYR 204) in HT29 cells in response to 2 hours of incubation with trametinib were shown.

[0054] Figure 33 The effects on pEGFR (TYR 1068) and pERK1 / 2 (THR 202 / TYR 204) in HT29 cells were shown in response to incubation with BVN22E antibody and trametinib for 2 hours.

[0055] Figure 34 A schematic diagram is shown illustrating how flow cytometry, using fluorescent dye staining, can determine the cell cycle and apoptotic status of individual cells. This method has been applied to KRAS mutant (A549 and DLD1) and ALK transposable (H2228 and H3122) cell lines.

[0056] Figure 35 The combined use of BVN22E antibody and trametinib showed the effect on the relative number of cells in the S and G2M phases of cell circulation in DLD1 cells;

[0057] Figure 36 The combined use of BVN22E antibody and trametinib showed the effect on the relative number of cells in the S and G2M phases of cell circulation in A549 cells;

[0058] Figure 37 The effects of BVN22E antibody in combination with brigatinib on the relative number of cells in the S and G2M phases of cell circulation in H2228 cells were demonstrated.

[0059] Figure 38 The effects of BVN22E antibody in combination with brigatinib on the relative number of cells in the S and G2M phases of cell circulation in H3122 cells were demonstrated.

[0060] Figure 39 The KRAS mutations in the cell lines used in this invention are shown. A549 cells contain the G12S KRAS mutation, H23 cells contain the G12C KRAS mutation, DLD1 cells contain the G13D KRAS mutation, and LS174T cells contain the G12C KRAS mutation;

[0061] Figure 40This study demonstrated the effect of BVN22E antibody on cell viability in DLD1 cells.

[0062] Figure 41 The study demonstrated the effect of the combination of BVN22E antibody and trametinib on cell viability after 72 hours of incubation in DLD1 cells.

[0063] Figure 42 The effects of BVN22E antibody on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473) and pERK1 / 2 (THR 202 / TYR 204) in response to 2 hours of incubation in DLD1 cells were demonstrated.

[0064] Figure 43 The effects of BVN22E antibody on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473) and pERK1 / 2 (THR 202 / TYR 204) in response to 24 hours of incubation in DLD1 cells were demonstrated.

[0065] Figure 44 The study demonstrated the effect of trametinib on cell viability in DLD1 cells with and without EGF.

[0066] Figure 45 The effects of trametinib on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473) and pERK1 / 2 (THR 202 / TYR 204) in response to 2 hours of incubation in DLD1 cells were demonstrated.

[0067] Figure 46 This study demonstrated the effect of the combination of BVN22E antibody and trametinib on cell viability in DLD1 cells.

[0068] Figure 47 The effects of BVN22E antibody in combination with trametinib on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473) and pERK1 / 2 (THR 202 / TYR 204) were demonstrated in DLD1 cells in the presence and absence of EGF.

[0069] Figure 48 This demonstrates the effect of BVN22E antibody in DLD1 cells on the development of trametinib resistance;

[0070] Figure 49 This demonstrates the effect of BVN22E antibody on cell viability in the A549 cell line;

[0071] Figure 50 The effects on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER473) and pERK1 / 2 (THR 202 / TYR 204) in A549 cells in response to 2 hours of incubation with BVN22E antibody were shown.

[0072] Figure 51 The effects on pEGFR (TYR1068), pSTAT3 (TYR 705), pAKT (SER 473) and pERK1 / 2 (THR 202 / TYR 204) in A549 cells in response to 24 hours of incubation with BVN22E antibody were shown.

[0073] Figure 52 The effect of trametinib on cell viability in the A549 cell line was shown in the presence and absence of EGF.

[0074] Figure 53 The effects of trametinib on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473) and pERK1 / 2 (THR 202 / TYR 204) in A549 cells in response to 2 hours of incubation were shown.

[0075] Figure 54 This study demonstrates the effect of the BVN22E antibody, used in combination with trametinib, on cell viability in A549 cells.

[0076] Figure 55 The effects on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473), and pERK1 / 2 (THR 202 / TYR 204) in response to 2 hours of incubation with BVN22E antibody in combination with trametinib, and in A549 cells with and without EGF, were demonstrated.

[0077] Figure 56 This demonstrates the effect of BVN22E antibody on cell viability in H23 cells;

[0078] Figure 57 The effects of BVN22E antibody incubation on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473) and pERK1 / 2 (THR 202 / TYR 204) in H23 cells were shown.

[0079] Figure 58The effects of BVN22E antibody incubation on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473) and pERK1 / 2 (THR 202 / TYR 204) in H23 cells were shown.

[0080] Figure 59 The effect of trametinib on cell viability in H23 cells with and without EGF was demonstrated.

[0081] Figure 60 The effects of trametinib on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473) and pERK1 / 2 (THR 202 / TYR 204) in H23 cells in response to 2 hours of incubation were shown.

[0082] Figure 61 This study demonstrates the effect of the BVN22E antibody, used in combination with trametinib, on cell viability in H23 cells.

[0083] Figure 62 The effects on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473), and pERK1 / 2 (THR 202 / TYR 204) in response to 2 hours of incubation with BVN22E antibody in combination with trametinib, and in H23 cells with and without EGF, were demonstrated.

[0084] Figure 63 This demonstrates the effect of BVN22E antibody on cell viability in the LS174T cell line;

[0085] Figure 64 The effects of BVN22E antibody incubation on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473) and pERK1 / 2 (THR 202 / TYR 204) in LS174T cells were shown.

[0086] Figure 65 The effects of BVN22E antibody incubation on pEGFR (TYR1068), pSTAT3 (TYR 705), pAKT (SER 473) and pERK1 / 2 (THR 202 / TYR 204) in LS174T cells were shown.

[0087] Figure 66The effect of trametinib on cell viability in the LS174T cell line was shown in the presence and absence of EGF.

[0088] Figure 67 The effects of trametinib on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473) and pERK1 / 2 (THR 202 / TYR 204) in LS174T cells in response to 2 hours of incubation were demonstrated.

[0089] Figure 68 The effects on pEGFR (TYR1068), pSTAT3 (TYR 705), pAKT (SER 473), and pERK1 / 2 (THR 202 / TYR 204) in response to 2 hours of incubation with BVN22E antibody in combination with trametinib, and in LS174T cells with and without EGF, were demonstrated.

[0090] Figure 69 The study demonstrated the effects of trametinib alone and in combination with BVN22E antibody on the viability of LS174T cells.

[0091] Figure 70 This study demonstrated the effect of human patient anti-EGF serum on pEGFR (TYR1068) in SW900 cells in the presence of EGF.

[0092] Figure 71 The effects of two human patient anti-EGF sera on pEGFR (TYR 1068), pAKT (SER 473), and pERK1 / 2 (THR 202 / TYR 204) were shown in SW900 cells in the presence of EGF.

[0093] Figure 72 The effects of three human patient anti-EGF sera on pEGFR (TYR 1068), pAKT (SER 473), and pERK1 / 2 (THR 202 / TYR 204) were shown in SW900 cells in the presence of EGF.

[0094] Figure 73A -B shows Figures 69-71 The quantitative summary of Western blots from patients' anti-EGF serum is shown; and

[0095] Figure 74A -D shows the output from Figures 69-71 The Western blot quantification of anti-EGF serum from patients in SW900 cells is shown. Detailed Implementation

[0096] In 2007, the EML4-ALK (echinoderm microtubule-associated protein 4) fusion oncogene was first discovered in a patient with non-small cell lung cancer (NSLC). Following an inversion of the short arm of chromosome 2, the N-terminus of the EML4 gene promoter was juxtaposed with the kinase domain of the anaplastic lymphoma kinase (ALK) gene, forming a fusion gene. After the formation of the EML4-ALK fusion gene, the EML4 fusion partner mediates ligand-independent activation and constitutive kinase activity of ALK, contributing to cancer cell proliferation and survival in 3%-7% of NSLC cases.

[0097] ALK inhibitors target ALK mutations that contribute to tumor growth, such as EML4-ALK translocation. Crizotinib, the first ALK inhibitor approved by the FDA in August 2011, is currently the standard of care for patients with metastatic ALK-positive NSCLC. Unfortunately, most NSCLC patients who initially respond to crizotinib develop resistance within a year of starting treatment, and their condition regresses to a more advanced stage. Crizotinib resistance appears to arise from NSCLC cells acquiring additional ALK mutations. The next-generation ALK inhibitors soon to be approved by the FDA, namely ceritinib (Zykadia) and alectinib (Alecensa), inhibit most clinically observed ALK mutations that drive resistance to crizotinib; these two therapies are currently approved for the treatment of patients with metastatic ALK-positive NSCLC who have either progressed on crizotinib or initially failed to improve with it.

[0098] Ceritinib, alectinib, brigatinib, and lorlatinib are all next-generation inhibitors that have been approved by the FDA for ALK-positive NSCLC. ALK inhibitors include ceritinib (LDK378), alectinib (RO5424802 / CH5424802), brigatinib (AP26113), ASP3026, bezatinib (TSR-011), lorlatinib (PF-06463922), entrectinib (RXDX-101), ensartinib (X-396), and CEP-37440.

[0099] Crizotinib is a highly potent c-Met and ALK inhibitor with IC50 values ​​of 11 nM and 24 nM, respectively, in cellular assays. Ceritinib (LDK378) is a highly potent and specific ALK inhibitor with an IC50 of 0.2 nM. Alectinib (CH5424802; RO5424802; AF802) is a highly potent and selective oral ALK inhibitor with an IC50 of 1.9 nM. Brigatinib is a highly potent and selective ALK inhibitor with an IC50 of 0.6 nM. ASP3026 is a novel selective ALK (anaplastic lymphoma kinase) inhibitor with an IC50 of 3.5 nM. Bezatinib is a highly potent oral dual inhibitor of ALK and TRKA, TRKB and TRKC, with an IC50 of 0.7 nM against wild-type recombinant ALK kinase. Lorlatinib (PF-06463922) is a highly potent dual ALK / ROS1 inhibitor with Kis values ​​of 0.02 nM, 0.07 nM, and 0.7 nM for ROS1, wild-type ALK, and ALK-L1196M, respectively. Entricinib is a highly potent oral inhibitor of Trk, ROS1, and ALK; it inhibits TrkA, TrkB, TrkC, ROS1, and ALK with IC50 values ​​of 1, 3, 5, 12, and 7 nM, respectively. Ensartinib (X-396) is a highly potent dual ALK / MET inhibitor with IC50 values ​​of <0.4 nM and 0.74 nM, respectively. CEP-37440 is a novel, highly potent, and selective dual FAK / ALK inhibitor with IC50 values ​​of 2.3 nM (FAK) and 120 nM (ALK cell IC50 in 75% human plasma). IC50 values: 2.3 nM (FAK); 120 nM (IC50 of ALK cells in 75% human plasma) (WO 2013134353, A1 20130912).

[0100] Crizotinib

[0101] Two subsequent randomized phase III studies compared crizotinib with standard chemotherapy, leading to its full approval and, until recently, its designation as the "gold standard" for first-line treatment of ALK-positive NSLC. Consistent with the phase II data, crizotinib showed impressive results compared to chemotherapy, demonstrating improvements in orrr (65% vs. 20%) and pfs [hazard ratio (hr): 0.49; 7.7 months vs. 3 months]. Crizotinib was then compared to platinum-based pemetrexed chemotherapy in previously untreated ALK-positive patients. Again, compared to chemotherapy, crizotinib was associated with improvements in orrr (74% vs. 45%), mpfs (hr: 0.45; 10.9 months vs. 7 months), and, importantly, quality of life. No difference was observed in overall survival (Rothenstein et al., Current Oncology, 2018).

[0102] Ceritinib

[0103] Ceritinib, the first next-generation inhibitor, has shown promising efficacy in treating ALK-positive NSLC resistant to crizotinib. Compared to crizotinib, ceritinib is 20 times more potent in inhibiting ALK. Biopsy studies of patients who have developed crizotinib resistance have demonstrated that ceritinib is a highly effective inhibitor of some identified resistance mutations.

[0104] Alectinib

[0105] Alectinib is another highly selective ALK inhibitor, active against ALK mutations resistant to crizotinib. A unique feature of alectinib is that it is not a substrate of P-glycoprotein, which is considered a mechanism of CNS resistance in patients taking crizotinib. Alectinib has now been established as the new standard of care for first-line treatment in patients with advanced ALK-positive NSCLC. While most responses to first-line alectinib are durable, virtually all patients develop resistance, leading to clinical relapse. Two other second-generation ALK inhibitors—ceritinib and brigatinib—have only been tested in settings where patients have not received crizotinib treatment and / or are resistant to crizotinib.

[0106] The third-generation ALK / ROS1 inhibitor lorlatinib has completed Phase I and II trials and has been tested in a large number of settings, including patients who have not received ALK TKI therapy, patients resistant to crizotinib, or patients resistant to one or more second-generation ALK-TKIs. The trials demonstrated that lorlatinib has antitumor activity in all settings, including patients who have failed treatment with second-generation ALK inhibitors (such as alectinib). Lorlatinib has high CNS penetration, demonstrating potent intracranial activity even in patients who have failed brain-penetrating TKIs (such as alectinib). Based on the efficacy and safety of the Phase I / II studies, lorlatinib received Breakthrough Therapy designation from the FDA last year and is expected to receive accelerated approval this year for ALK-positive patients previously treated with one or more ALK inhibitors. Therefore, the paradigm of sequential therapy with crizotinib and subsequent second-generation ALK inhibitors is being replaced by a new paradigm: alectinib and subsequently lorlatinib.

[0107] Embodiments of the technology described in this application are based, at least in part, on the finding that physiological concentrations of anti-EGF antibodies inhibit EGFR phosphorylation, and that the effects of Akt and ERK1 / 2 are at least as significant as those of ALK inhibitors on these signaling molecules. Further research has shown that combination therapy with anti-EGF antibodies and ALK inhibitors exhibits additional effects of inhibiting pEGFR, pAkt, pERK1 / 2, and pSTAT-3. In some embodiments, such antibodies or antigen-binding fragments thereof may be used in NSLC treatments. Within the scope of the invention, it is contemplated that anti-EGF antibodies can be actively generated in vivo by administering a vaccine that produces an immune response to EGF. Further within the scope of the invention, it is envisioned that passive monoclonal anti-EFG antibodies can be administered.

[0108] Of the 25 patients with non-small cell lung cancer (NSCLC—the most common type of lung cancer), one had ALK-positive (anaplastic lymphoma kinase positive, or ALK+) lung cancer. Young, non-smoking patients—usually 55 years of age or younger—were most likely to be diagnosed with ALK+.

[0109] Anaplastic lymphoma kinase inhibitors (ALK inhibitors) exert their inhibitory effect by interfering with (or inhibiting) the ALK signaling pathway. For a signal to be transmitted from one kinase (key) to another (lock), the two kinases need to temporarily bind together. Kinase inhibitors inhibit one kinase by temporarily blocking it, preventing the other kinase from receiving the first. Therefore, a certain concentration of kinase inhibitor must be achieved to prevent signal transmission sufficiently frequently (potentially exceeding 90%) to prevent the kinase from regulating the expression of certain genes.

[0110] Most anti-ALK tumor drugs currently under development aim to interfere with (or inhibit) the ALK signaling pathway (by blocking ALK signal transmission, or by blocking signal transmission from another kinase in the pathway). For example, one ALK inhibitor aims to inhibit AKT kinase, which is downstream of ALK. Therefore, such inhibitors are being tested to determine their effectiveness in inhibiting ALK tumors and other types of tumors.

[0111] As with other kinase inhibitors, resistance is a concern. However, data on resistance mechanisms to next-generation ALK inhibitors are limited; almost all data pertain to patients who received crizotinib before taking these drugs. Similar to other TKIs, target alterations, activation of alternative signaling pathways, and tumor phenotypic changes have been observed in tumors resistant to next-generation ALK inhibitors. Studies have shown that ALK target alterations are more common in tumors following next-generation ALK inhibitor treatment compared to tumors in patients treated with crizotinib. The most common mutation observed is G1202R, a mutation in the ALK solvent-exposed region that leads to steric hindrance effects from most ALK inhibitors. It remains unclear whether the same resistance mechanisms or the same proportions will be observed in tumors from patients treated with next-generation ALK inhibitors as first-generation ALK inhibitors.

[0112] Because cancer eventually develops resistance to the first-generation ALK inhibitor crizotinib within 1-2 years, ALK-positive NSCLC exhibits variable progression-free survival (PFS). Similar to EGFR, the TK domain of ALK also harbors resistance mutations, most commonly L1196M. This mutation leads to steric hindrance at the binding site, reducing responsiveness to crizotinib. However, unlike EGFR-TKI resistance, ALK-TKI resistance involves multiple kinase domain mutations (G1269A, G1202R, S1206Y, F1174C / L, D1203N) and mutations far from the binding site (threonine insertions at 1151, C1156Y, and L1152R). In vitro studies have shown that different resistance mutations induce varying degrees of resistance to structurally different TKIs, highlighting the need to identify secondary resistance mutations in acquired resistance using repeat cancer samples and more detailed sequencing methods rather than FISH analysis [69, 70]. Furthermore, FISH analysis revealed that other ALK-TKI resistance cases only showed amplification of the fusion product; some cases showed one of the identified resistance mutations, while others only showed amplification.

[0113] Crizotinib is an oral MET / ALK inhibitor used as a first-line treatment for advanced NSCLC with ALK rearrangements. In addition, clinical trials are evaluating newer second-generation ALK inhibitors with higher potency and selectivity for ALK. Similar to crizotinib, although these drugs are structurally different from tyrosine kinases, they are ATP-competitive inhibitors of ALK tyrosine kinases. Ceritinib belongs to the second-generation inhibitors and has shown activity in ALK-positive lung cancer patients, including individuals with acquired resistance to crizotinib. Ceritinib is FDA-approved for use in patients with advanced ALK-rearranged NSCLC who have previously received crizotinib treatment. However, responses to ALK inhibitors are often transient, with resistance typically developing within one year.

[0114] Since the introduction of crizotinib as treatment for ALK-driven NSCLC, EML4-ALK gene amplification or secondary mutations have been found in approximately one-third of tumors that have developed acquired resistance to crizotinib. In vitro studies have shown that secondary mutations can drive resistance to crizotinib, but not all mutations lead to resistance to structurally different second-generation ALK inhibitors. Furthermore, activation of EGFR, KIT, and IGF-1R was individually identified in a group of crizotinib-resistant tumors. Although secondary mutations in EML4-ALK have been found in a subset of tumors with acquired ceritinib resistance, resistance to second-generation ALK inhibitors is less pronounced due to the recent clinical adoption of these drugs.

[0115] EGFR mutations are associated with impaired responses to immune checkpoint inhibitors, making the development of novel immunotherapies for EGFR-mutant NSCLC particularly important. Epidermal growth factor (EGF) immunization has shown efficacy in phase III trials, including unselected NSCLC patients; however, researchers do not fully understand the mechanisms involved in the action of anti-EGF antibodies (anti-EGF VacAbs) generated by the vaccine, or their activity in EGFR-mutant tumor cells.

[0116] EGF vaccination represents a novel strategy, unlike programmed death-1 blockade, that aims not to reverse tumor-induced immunosuppression by activating T cells. Instead, it aims to stimulate B cells to produce neutralizing antibodies that dissociate circulating EGF, thereby preventing its binding to EGFR. EGF vaccination, also known as EGF-pathway targeted immunization, is well-tolerated with few serious adverse events and has shown promising results in two trials involving unselected advanced NSCLC patients (11, 12, and 21). However, beyond its ability to block ligand binding and EGFR phosphorylation, little is known about the molecular and cellular mechanisms involved in the action of anti-EGF antibodies, or their differential activity in tumors with EGFR mutations or other genetic alterations (22).

[0117] This invention demonstrates that anti-EGF VacAb cultured in rabbits inhibits the effects of EGF on cell proliferation, cell cycle, and signal transduction pathways in EGFR-mutant NSCLC cell lines, particularly those from untreated patients. The EGF concentration used in the experiment (10 ng / mL) was close to that reported in human studies, with a median of approximately 1 ng / mL, and showed significant inter-individual variability (11, 23). Notably, the researchers also found that in the absence of exogenous EGF, anti-EGF VacAb not only persistently reduced pErk1 / 2 levels in PC9 cells but also in PC9-GR4 cells, where growth factors did not exhibit any significant effect. One possible explanation for this observation is the presence of a receptor / ligand feedback loop in the cell lines used. Serum from patients immunized with the anti-EGF vaccine also effectively blocked EGF activation of pErk1 / 2. The control serum from unimmunized patients had little effect on Erk1 / 2 but exhibited strong activation of Akt, indicating that healthy individuals' blood contains growth factors that can specifically trigger pAkt in EGFR mutant cells. In contrast, analysis showed that the serum from the four immunized patients had lower activity in inducing Akt phosphorylation. Significant differences were observed in the efficacy of serum from vaccinated individuals in blocking Erk1 / 2 phosphorylation and in activating Akt to a lesser extent. As a novel therapeutic approach that has only completed Phase III trials, the sample size obtained from patients vaccinated with EGF is limited and unlikely to be correlated with clinical outcomes.

[0118] Previous studies have found that EGF significantly reduces the antiproliferative effects of TKIs (such as gefitinib, erlotinib, afatinib, and osimertinib) in several EGFR-mutant NSCLC cells, which are both sensitive to and resistant to EGFR TKIs. This finding is related to the results of Western blot experiments, in which the pErk1 / 2 level in cells was significantly increased in the presence of EGF after EGFR TKI treatment.

[0119] Patients with EGFR mutations and high EGF levels may have a poorer prognosis for EGFR-TKIs. Elevated serum levels of both EGFR ligands, transforming growth factor α and bimodalin, have been reported in unselected NSCLC patients and are associated with adverse responses to EGFR-TKIs. Regarding EGF, in the only study published to date, serum EGF was associated with shorter progression-free survival in 11 EGFR-mutant and 21 EGFR wild-type NSCLC patients treated with erlotinib. Studies have found that in EGFR-mutant cell lines tested, gefitinib, erlotinib, afatinib, and osimertinib combined with anti-EGF VacAbs had a stronger anti-proliferative effect than EGFR TKIs alone, a finding associated with a sustained decrease in pErk1 / 2 (Figure 78, comparing flow-through TKI+EGF versus TKI+Ab+EGF). Combined use also showed significant advantages in blocking the anti-apoptotic and G2 / M stimulatory effects of EGF.

[0120] In EGFR mutant cell line models, the anti-EGFR monoclonal antibody cetuximab was also tested. Similar to anti-EGF VacAb, cetuximab blocked ligand binding in vitro, demonstrating its ability to prevent ligand-induced EGFR, Erk1 / 2, and Akt phosphorylation in PC9 and H1975 cells. However, in other EGFR mutant cell lines, such as H3255 or DFCILU-011.29, its effect on downstream EGFR signaling was relatively small. Regarding receptor downregulation, there appeared to be a significant difference between the two antibodies. After incubation for 1 to 2 hours in EGFR mutant cells (such as PC9, H1975, or H3255), cetuximab significantly reduced total EGFR levels, while anti-EGF VacAb did not cause significant receptor downregulation after 24 hours of incubation. Finally, although there are reports that cetuximab amplifies the induction of apoptosis and tumor regression in EGFR wild-type, head and neck cancer cell lines and subcutaneous tumors, it failed to enhance the effect of gefitinib in PC9 xenografts.

[0121] Conversely, anti-EGF VacAb enhanced the antiproliferative activity of EGFR TKIs in PC9 and other EGFR mutant cell lines. This enhancement was statistically significant in all cases, with the sole exception of PC9-GR4 cells. The fact that anti-EGF VacAb targets the ligand rather than the receptor and does not induce EGFR downregulation may explain the difference in action between cetuximab and anti-EGF antibodies.

[0122] Previous studies have shown that the addition of anti-EGF VacAb significantly delayed the emergence of gefitinib and afatinib-resistant clones in the PC9 cell line. The addition of anti-EGF VacAb also consistently inhibited TKI-induced STAT3 activation. Conversely, studies have shown elevated pSTAT3 levels in head and neck human tumors that have progressed to cetuximab, suggesting that STAT3 activation is involved in resistance to this drug.

[0123] In summary, in EGFR-mutant NSCLC cell lines, anti-EGF VacAbs inhibited the effects of EGF while significantly enhancing the antitumor activity of EGFR-TKIs. They also blocked STAT3 activation, reduced AXL expression, and delayed the acquisition of resistance. Therefore, it is believed that ALK inhibitors may also benefit from anti-EGF VacAbs.

[0124] Some researchers believe that ALK plays a crucial role in the development and function of the nervous system, acting as a fundamental mechanism for controlling cell proliferation, survival, and differentiation in response to extracellular stimuli. The most prevalent ALK gene abnormality in human cancers is chromosomal rearrangement, leading to fusion genes. ALK fusion occurs when the 3′ half of ALK (originating from chromosome 2, which retains its kinase catalytic domain) fuses with the 5′ half of a different gene that provides its promoter. Several distinct 5′ partners have been identified. Wild-type ALK is typically activated by the binding of a ligand to its extracellular domain, leading to dimerization and autophosphorylation of the kinase domain. Structural studies have shown that fusion with multiple 5′ partners can help bypass this requirement, enhancing ALK's oncogenic potential, as demonstrated by NPM1-ALK and EML4-ALK in non-small cell lung cancer (NSCLC). Increased copy number and the presence of activating point mutations leading to kinase activation are also associated with ALK's oncogenic activity. These gene alterations are present in a variety of malignant tumors, including but not limited to lung cancer, neuroblastoma, rhabdomyosarcoma, renal cell carcinoma, inflammatory myofibroblastoma (IMT), and inflammatory breast cancer.

[0125] For convenience, this application includes certain terms, embodiments, and appended claims used in this application and specification. Unless otherwise stated or implied by the context, the following terms and phrases include the meanings provided below. Unless otherwise expressly stated or from the context, the following terms and phrases do not exclude the meanings already acquired in the art to which they pertain. Since the scope of the invention is limited only by the claims, the definitions are provided to help describe particular embodiments and are not intended to limit the claimed invention. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0126] The terms “decline,” “reduction,” “reduced,” “less,” “inhibition,” and “suppression” are generally used in this application to indicate a statistically significant decrease relative to a reference value. However, for the avoidance of doubt, “decline,” “reduction,” or “suppression” generally means a decrease of at least 10% compared to a reference level, and may include, for example, a decrease of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, up to and including, for example, a decrease of 10% to 99% compared to a reference level with no given entity or parameter, or compared to no given treatment.

[0127] In this application, “increased,” “enhanced,” or “activated” generally refers to an increase in a statistically significant amount. For the avoidance of doubt, “increased,” “enhanced,” or “activated” means an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including an increase of 100%, or any increase between 10 and 100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or an increase of 2 to 10 times or more compared to a reference level.

[0128] In the case of nucleic acids or peptides, the terms "isolated" or "partially purified" as used in this application refer to nucleic acids or peptides isolated from at least one other component (e.g., nucleic acids or peptides), present together with nucleic acids or peptides found in their natural sources, and / or coexisting with nucleic acids or peptides when expressed in cells or secreted in the case of secretory peptides. Chemically synthesized nucleic acids or peptides, or nucleic acids or peptides synthesized using in vitro transcription / translation, may be considered "isolated" nucleic acids or peptides. "Purified" or "substantially purified" refers to isolated nucleic acids or peptides that constitute at least 95% (by weight) of the subject's nucleic acids or peptides, for example, including at least 96%, at least 97%, at least 98%, at least 99%, or more.

[0129] The terms "protein" and "peptide" are used interchangeably in this application to refer to a series of amino acid residues linked to another series of amino acid residues via peptide bonds between the α-amino and carboxyl groups of adjacent residues. The interchangeable terms "protein" and "peptide" in this application refer to a polymer of protein amino acids, including modified amino acids (such as phosphorylated, glycosylated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. "Protein" and "peptide" are generally used to refer to relatively large polypeptides, while "peptide" is generally used to refer to smaller polypeptides; however, there is overlap in the use of these terms in the art. When referring to gene products and fragments thereof, "protein" and "peptide" are used interchangeably in this application.

[0130] Therefore, typical polypeptides or proteins include gene products, naturally occurring proteins, homologues, orthologs, paralogs, fragments and other equivalents, variants, fragments and the like.

[0131] The term "antibody" as used in this application includes any immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, etc., through at least one antigen recognition site within the variable region of the immunoglobulin molecule. The term is used in the broadest sense and includes fully polyclonal antibodies, fully monoclonal antibodies, antibody fragments (such as Fab, Fab′, F(ab′).sub.2, and Fv fragments), single-chain Fv (scFv) mutants, multispecific antibodies (e.g., bispecific antibodies generated from at least two whole antibodies), fusion proteins containing antibody portions, and any other modified immunoglobulin molecule containing an antigen recognition site (provided the antibody exhibits the desired biological activity). Based on the identity of the heavy chain constant regions (referred to as α, δ, ε, γ, and μ, respectively), antibodies may be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Different types of immunoglobulins have different well-known subunit structures and three-dimensional structures. Antibodies can be naked antibodies or bound to other molecules such as cytotoxins, toxins, and radioisotopes. Antibodies can be administered through active production in the body or passive application of monoclonal antibodies.

[0132] In this application, the terms "polynucleotide" or "nucleic acid" used interchangeably refer to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides, or bases and / or their analogues, or any substrate that can be incorporated into the polymer by DNA or RNA polymerases or through a synthetic reaction. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogues.

[0133] Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins with similar structural features. While antibodies have specific binding specificity to a particular antigen, immunoglobulins include both antibodies and other antibody-like molecules that typically lack antigen specificity. For example, the latter type of polypeptide is produced in lower amounts by the lymphatic system but in higher amounts by myeloma.

[0134] The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full-length monoclonal antibodies or all-monoclonal antibodies), polyclonal antibodies, monovalent, multivalent, and multispecific antibodies (e.g., bispecific antibodies, provided they exhibit the desired antibody activity), and may also include certain antibody fragments (described in more detail in this application). Antibodies can be chimeric antibodies, human antibodies, humanized antibodies, and / or affinity-matured antibodies.

[0135] Antibodies (immunoglobulins) can be classified into different classes based on the amino acid sequence of their heavy chain constant regions. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG-1, IgG-2, IgA-1, and IgA-2. The heavy chain constant regions corresponding to different types of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different types of immunoglobulins are well known and are generally described, for example, in Abbas et al., *Cellular and Molecular Immunology*, 4th edition (2000). Antibodies can be part of a larger fusion molecule, formed through covalent or non-covalent physical association between the antibody and one or more other proteins or peptides.

[0136] In this application, the terms "full-length antibody," "whole antibody," and "intact antibody" are used interchangeably to refer to an antibody with a generally intact morphology, rather than an antibody fragment as defined below. These terms specifically refer to antibodies having a heavy chain containing an Fc region.

[0137] An "antibody fragment" comprises only a portion of a whole antibody, wherein, when present in the whole antibody, the portion preferably retains at least one function, preferably most or all of the functions, typically associated with that portion. In one embodiment, the antibody fragment includes the antigen-binding site of the whole antibody, thus retaining antigen-binding capacity. In another embodiment, the antibody fragment (e.g., an antibody fragment including an Fc region) retains at least one biological function typically associated with the Fc region, such as FcRn binding, antibody half-life regulation, ADCC function, and complement binding, when present in the whole antibody. In one embodiment, the antibody fragment is a monovalent antibody whose in vivo half-life is substantially similar to that of the whole antibody. For example, such an antibody fragment may include an antibody on an antigen-binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.

[0138] As used in this application, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies; that is, individual antibodies comprising this group of antibodies comprise substantially the same amino acid sequence, except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific antibodies against a single antigen. Furthermore, unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen.

[0139] The monoclonal antibodies in this application specifically include “chimeric” antibodies, wherein a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remaining portion of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of those antibodies, provided they exhibit the desired biological activity (US Patent No. 4,816,567; Morrison et al., Proceedings of the National Academy of Sciences 81: 6851-6855 (1984)).

[0140] "Human antibody" is an antibody having an amino acid sequence corresponding to that of human-produced antibodies and / or having an amino acid sequence prepared using any technique disclosed in this application for preparing human antibodies. This definition of human antibody specifically excludes humanized antibodies that include non-human antigen-binding residues.

[0141] The range provided in this application can be understood as an abbreviation for all values ​​within the range. For example, the range 1 to 50 can be understood as including any number, combination of numbers, or subranges derived from groups of the following numbers: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, and all intermediate decimal values ​​between the above integers, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With regard to subranges, special consideration should be given to “nested subranges” that extend from either end of the range. For example, nested subranges of the exemplary range 1 to 50 may include 1 to 10, 1 to 20, 1 to 30 and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20 and 50 to 10 in another direction.

[0142] As used in this application, the term "subject" refers to any organism capable of bacterial infection. Such organisms include, but are not limited to, humans, dogs, cats, horses, cattle, sheep, goats, mice, rats, guinea pigs, monkeys, birds, reptiles, etc.

[0143] As used in this application, “tumor” refers to all proliferative cell growth and proliferation (whether malignant or benign), as well as all precancerous and cancerous cells and tissues. “Cancer,” “carcinoma,” “cell proliferation disorder,” “proliferative disorder,” and “tumor” are not mutually exclusive as used in this application.

[0144] "Cancer" and "carcinoma" refer to or describe a physiological state in mammals characterized by uncontrolled cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, germ cell tumor, sarcoma, and leukemia. More specific examples of this type of cancer include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, and various types of head and neck cancers.

[0145] As used in this application, "treatment" refers to a clinical intervention that attempts to alter the natural processes of an individual or cells receiving treatment, and can be used for preventative medication or during clinicopathological processes. Ideal therapeutic effects include preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing or reducing inflammation and / or tissue / organ damage, slowing disease progression, improving or alleviating disease states, and achieving disease remission or improved prognosis. In some embodiments, the antibodies of this invention are used to delay the development of a disease or condition.

[0146] "BVN22E nucleic acid molecule" refers to a polynucleotide encoding the BVN22E polypeptide. An exemplary BVN22E nucleic acid molecule (sequence ID: 1) is reproduced below:

[0147]

[0148] "BVN22E polypeptide" refers to a polypeptide or fragment thereof having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the following amino acid sequences (Sequence ID: 2) (excluding the following amino acid variations: T2S, E3D, N4S, D5E, E11D, A12G, V38I, F44Y, R48K, D51E, and A52L):

[0149]

[0150] "Pharmaceutical excipients" refers to excipients commonly used in pharmaceutical technology, especially those in the "Handbook of Pharmaceutical Excipients" (Raymond C. Rowe, Paul J. Sheskey and Paul J. Weller, 4th edition, 2003), the full text of which is incorporated into this document.

[0151] The "therapeutic effective amount" of the substance / molecule of this invention may vary depending on factors such as an individual's disease state, age, sex, weight, and the ability of the substance / molecule to elicit the desired response in the individual. Therapeutic effective amount also refers to the amount at which any toxic or harmful effects of the substance / molecule are offset by the beneficial effects of treatment. "Prophylactic effective amount" refers to the effective amount at the required dose and time to achieve the desired preventative effect. Because the prophylactic dose is used in subjects before or in the early stages of disease, the prophylactic effective amount is usually, but not necessarily, less than the therapeutic effective amount.

[0152] Chemotherapy agents are chemical compounds that can be used to treat cancer. 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; vinylamines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetic acid derivatives (especially bulbatacin and bulbatacinone); delta-9-tetrahydrocannabinol (dronabinol, ... ); β-lapachone; laparol; colchicine; betulinic acid; camptothecin (including the synthetic analogue topotecan) CPT-11 (irinotecan) Acetylcamptothecin, scopolectin, and 9-aminocamptothecin; bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllinic acid acid); teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, and mechlorethamine oxide. Hydrochloride, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, uracil mustard; chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omega1I (see, for example, Agnew ChemIntl. Ed. Engl.)Journal, 33: 183-186 (1994); dynemicin, including dynemicin A; espmeramicin; and neocarzinostatin chromophore and related chromoprotein enediyne antibacterial chromophores. Chromophores), aclacinomysins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin C, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including). Morpholino-doxorubicin, cyanomorpholino-doxorubicin, pyrrolino-doxorubicin, and doxorubicin hydrochloride liposome injection ( And deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid (acid), nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate, gemcitabine ( ), tegafur ( Capecitabine Epothilone and 5-fluorouracil (5-FU), folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, etc. Examples include ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and fluxuridine; and male hormones such as calusterone and dromostanolone. Propionate, epitiostanol, mepitiostane, testolactone; anti-adrenergic drugs, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate. acetate); etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansine compounds, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine;Pentostatin; Phenamet; Pirarubicin; Podophyllin; 2-Ethylhydrazide; Procarbazine; Polysaccharide complex (JHS Natural Products, Eugene, Oregon, USA); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 22,2′,2″-trichlorotriethylamine; trichothecene toxins (especially T-2 toxin, verracurin A, baculosporin A, and anguidine); urethan; vindesine ); dacarbazine; mannomustine; mitobronitol; mitolactalol; piperobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxanes, such as paclitaxel ( Albumin-coated paclitaxel lipid nanoparticle formulation (ABRAXANE) TM ) and taxanes ( ); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine ( Platinum; etoposide (VP-16); isophosphoramide; mitoxantrone; vincristine ( ); oxaliplatin; leucovovin; vinorelbine Nogantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; pharmaceutically acceptable salts, acids, or derivatives of any of the above substances; and combinations of two or more of the above substances, such as CHOP, an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, oxaliplatin. TM () is an abbreviation for a combination therapy regimen with 5-FU and leucovorin.

[0153] "Patient response" may be assessed using any endpoint that indicates patient benefit, including but not limited to: (1) inhibiting disease progression to some extent, including slowing and stopping it completely; (2) reducing the frequency of disease attacks and / or symptom occurrences; (3) reducing lesion size; (4) inhibiting (i.e. reducing, slowing or stopping) disease cell infiltration into adjacent peripheral organs and / or tissues; (5) inhibiting (i.e. reducing, slowing or stopping) disease spread; (6) reducing cell proliferation, invasion or metastasis (which may but not necessarily lead to lesion regression or ablation); (7) alleviating one or more symptoms associated with the disease to some extent; (8) prolonging the time to disease-free status after treatment; and / or (9) reducing mortality at a given time point after treatment.

[0154] A “tissue or cell sample” refers to a collection of similar cells obtained from the tissue of a subject or patient. The source of a tissue or cell sample can be a fresh, frozen, and / or preserved solid tissue sample or biopsy or aspirate; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage of the subject’s pregnancy or development. Tissue samples can also be primary or cultured cells or cell lines. Optionally, tissue or cell samples are obtained from diseased tissue / organ. Tissue samples may contain compounds that do not naturally mix with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0155] ALK inhibitors

[0156] The therapeutic significance of ALK gene alterations is primarily related to ALK gene fusions and can predict tumor response to ALK inhibitors. Crizotinib, ceritinib, and most recently alectinib have been approved by the FDA for the treatment of patients with ALK fusion-positive metastatic NSCLC. Early phase 1 studies showed that crizotinib produced a sustained response in patients with ALK fusion-positive metastatic NSCLC. Two phase 3 studies (which ultimately led to FDA approval of crizotinib) further confirmed that crizotinib is superior to standard first-line pemetrexed plus cisplatin chemotherapy in previously untreated advanced ALK-rearranged NSCLC patients.

[0157] Because the development of resistance leads to disease progression, durable responses remain uncommon in patients with ALK fusion-positive NSCLC, despite the high activity of crizotinib. The median progression-free survival (PFS) is 13 months. While researchers are still elucidating the mechanisms of resistance, acquired secondary mutations in ALK kinase domains (F1174L, F1174C, L1196M, I1171T, G1202R, S1206Y, G1269S, and G1269A) or ALK gene amplification are known to be associated with resistance. Resistance can also be mediated by activation of alternative ALK-independent survival pathways that impede the effectiveness of crizotinib, including the epidermal growth factor pathway, insulin-like growth factor pathway, RAS / SRC signaling, and AKT / mTOR signaling.

[0158] ALK inhibitors brigatinib and lorlatinib have shown inhibitory activity against several known resistance mutations, with lorlatinib demonstrating effective inhibition of the G1202R mutation. These results indicate a need to understand resistance mechanisms and select appropriate tyrosine kinase inhibitors (TKIs) to optimize treatment response.

[0159] In addition to direct ALK-targeting therapy, there are pharmacological strategies that allow for indirect targeting. Specifically, indirect inhibition of HSP90 (a chaperone protein that stabilizes a large number of proteins, including ALK) has been successfully achieved through targeting the lung cancer heat shock protein (HSP90). HSP90 inhibition has shown some preclinical efficacy against ALK fusions (EML4-ALK and NPM1-ALK) resistant to crizotinib, including in secondary resistance mutants in lung cancer models.

[0160] ALK rearranged tumors represent a specific subset of tumors that can be effectively targeted using currently available ALK inhibitors. Therefore, detecting ALK alterations in tumors known to have this molecular aberration has become an essential part of diagnosis. FISH, next-generation sequencing (NGS) of tumor tissue, and sequencing of circulating tumor cells provide alternative and often complementary approaches for detecting ALK-altered tumors. Resistance to TKIs is almost inevitable during treatment, necessitating repeat biopsies at relapse to identify resistance mechanisms that newer ALK inhibitors and other novel treatment strategies may target.

[0161] Crizotinib, with its aminopyridine structure, acts as a protein kinase inhibitor through competitive binding within the ATP-binding cassette of its target kinase. In approximately 4% of non-small cell lung cancer patients, chromosomal rearrangements result in a fusion gene between EML4 ('echinoderm microtubule-associated protein-like 4') and ALK ('anaplastic lymphoma kinase'), leading to constitutive kinase activity that contributes to carcinogenesis and appears to drive a malignant phenotype. The kinase activity of this fusion protein is inhibited by crizotinib. Patients with this gene fusion are typically young, non-smokers without mutations in either the epidermal growth factor receptor (EGFR) gene or the K-Ras gene. In approximately 15% of neuroblastoma cases, ALK mutations are considered a significant driver of the malignant phenotype; neuroblastoma is a rare form of peripheral nervous system cancer that occurs almost exclusively in very young children. Crizotinib inhibits c-Met / hepatocyte growth factor receptor (HGFR) tyrosine kinase, which is involved in the development of many other histological forms of malignancy.

[0162] Alectinib is a tyrosine kinase receptor inhibitor and anti-tumor drug used to treat selected forms of advanced non-small cell lung cancer. Alectinib is associated with a moderate rate of transient increases in serum transaminase levels during treatment and with very rare cases of clinically significant acute liver injury.

[0163] Alectinib is an oral inhibitor of anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase with antitumor activity. After administration, alectinib binds to and inhibits ALK kinase, the ALK fusion protein, and the gatekeeper mutant ALKL1196M, a mechanism known as acquired anti-small molecule kinase inhibitor. Inhibition leads to disruption of ALK-mediated signaling and ultimately suppresses tumor cell growth in ALK-overexpressing tumor cells. ALK belongs to the insulin receptor superfamily and plays a crucial role in the development of the nervous system. ALK dysregulation and gene rearrangements are associated with a range of tumors.

[0164] Alectinib is an organic isotetracyclic compound, consisting of 6,6-dimethyl-5,6-dihydro-11H-benzo[b]carbazole-11- with additional cyano groups, 4-(morpholino-4-yl)piperidin-1-yl, and ethyl substituents at positions 3, 8, and 9, respectively. It is used to treat patients with anaplastic lymphoma kinase-positive, metastatic non-small cell lung cancer (as hydrochloride). It has EC2.7.10.1 (receptor protein tyrosine kinase) inhibitory and antitumor effects. It is an organic isotetracyclic compound, a member of the morpholino group, a member of the piperidine group, a nitrile, and an aromatic ketone. It is the conjugate base of alectinib (1+).

[0165] Brigatinib is a next-generation ALK inhibitor that targets a broad range of ALK mutations and ROS1 rearrangements. Currently, it is also the only ALK inhibitor with cell line activity in which mutations occur in the gene encoding the epidermal growth factor receptor (EGFR). In the AP26113 (ALTA) lung cancer trial involving 222 patients with crizotinib-refractory disease, brigatinib was associated with high systemic and CNS response rates and a median progression-free survival of 16.7 months when administered at the recommended dosing regimen of 180 mg once daily (90 mg in 110 patients with a 7-day lead-in period) according to the recommended regimen. This regimen was also associated with similar progression-free survival (16.3 months) in patients receiving crizotinib in phase 1-2 trials. These median rates of progression-free survival in this group of patients are higher than those associated with other next-generation ALK inhibitors, including alectinib, ceritinib, ensartinib, and lorlatinib.

[0166] STAT3 (Signal Transducer and Activator of Transcription 3) is an oncogenic transcription factor that is active in many cancers and regulates the transcription of several genes involved in cell cycle progression, anti-apoptosis, cell survival, and angiogenesis.

[0167] STAT3 can be activated by EGFR, JAK2, and other tyrosine kinases, the activation of which can be mediated by EGF, leukemia inhibitory factor (LIF), and other cytokines. Therefore, STAT3 is a convergence point for many signaling pathways and plays an important role in tumorigenesis and metastasis. It has been suggested that STAT3 can be activated by various forms of mutant EGFR and may contribute to the oncogenic effects of these mutants on fibroblasts and human lung cancer cells.

[0168] Upon activation via ligand binding or mutation, EGFR initiates a series of signal transduction pathways that alter cell biology through transcriptional and post-translational mechanisms. Signaling pathways mediating these changes include Ras-Raf-mitogen-activated protein (MAP) kinase (MAPK), phosphatidylinositol 3-kinase-AKT, and the STAT3 and STAT5 signal transduction pathways. The STAT family of transcription factors is activated by phosphorylation at conserved tyrosine residues, leading to dimerization, nuclear translocation, and DNA binding. STAT1, STAT3, and STAT5 are also phosphorylated at their COOH-terminal serine residues; phosphorylation is less dependent on dimerization, nuclear translocation, and DNA binding, but maximal transcriptional activity of some genes requires phosphorylation.

[0169] Some non-small cell lung cancer (NSCLC) cell lines contain constitutively active STAT3. Recent studies have shown that STAT3 is activated by these EGFR mutants in genetically defined systems. It remains unclear which downstream signaling pathway of mutant EGFR is required for oncogenicity; however, given the role of STAT3 in a wide range of human malignancies and its activation by EGF in various cell types, it can be considered that STAT3 is essential for the oncogenic effect of somatic mutant EGFR. STAT3 has been reported to be activated in fibroblasts expressing mutant EGFR, as well as in two naturally occurring EGFR-mutant NSCLC cell lines, and this activation is required for transformation and survival in these cells.

[0170] STAT3 activation typically involves ligand-receptor interactions. STAT3 can be activated by a variety of cytokines, including interferons, EGF, G-CSF, and interleukin (IL-6) family cytokines. Cytokine binding to their homologous receptors leads to JAK phosphorylation, STAT3 dimerization, nuclear translocation, DNA binding, and gene activation (12, 13). Furthermore, STAT3 phosphorylation can also be induced by cytoplasmic tyrosine kinases such as Src family kinases (14). Elevated EGFR activity has been reported to be positively correlated with STAT3 activation in many primary tumor specimens and tumor cell lines, including NSCLC, breast cancer, and head and neck cancer.

[0171] Increased STAT3 activity has been observed in lung adenocarcinoma and cell lines expressing mutant EGFR. Without being bound by any particular theory, it can be assumed that mutant EGFR requires STAT3, and that STAT3 is essential for its downstream phenotypic effects. Inhibition of STAT3 function in fibroblasts cancels the conversion mediated by mutant EGFR. Unfortunately, targeted therapies (such as TKIs and ALK inhibitors) cannot completely eliminate STAT3 activity in NSCLC cell lines.

[0172] Previous studies have shown that mutant EGFR induces activation of the gp130 / JAK / STAT3 pathway through IL-6 upregulation. Tumor expression of IL-6 and its receptor components gp80 and gp130 has been found in NSCLC specimens (20). Studies have also observed that elevated levels of pro-inflammatory cytokines (such as IL-6 and IL-8) are associated with tumorigenesis and prognosis in NSCLC. This suggests that IL-6 and its downstream pathways may be targets for NSCLC patients with EGFR mutations. However, the mechanism by which oncogenic EGFR mutations induce IL-6 in NSCLC remains unclear; however, some researchers suggest that NF-κB and STAT3 signaling are responsible for regulating IL-6 autocrine secretion in lung cancer.

[0173] According to one aspect of the invention, an anti-EGF antibody is used to treat patients with cancer driven by an uncontrolled human epidermal growth factor receptor expressing an ELM4-ALK fusion gene, which is inhibited by a flexible and proactive regimen of administration to patients requiring such treatment, in combination with an anaplastic lymphoma kinase inhibitor (ALK inhibitor) and an anti-EGF antibody as described in the invention, in order to inhibit the pathway activated by EGF-EGFR binding (mAb), wherein the ALK inhibitor is administered according to a continuous regimen based on an average daily dose in the range of about 10 to 250 mg, and the EGF-PTI of the invention is co-administered according to the dosing regimen, achieving a therapeutically effective dose that is repeated three times, twice, or once a week, once every two weeks, once every three weeks, or at least once a month.

[0174] According to another aspect of the invention, anti-EGF antibodies are generated by vaccinating patients with uncontrolled human epidermal growth factor receptor-driven cancer expressing the ELM4-ALK fusion gene. An immune response to EGF is generated by administering a flexible and proactive regimen to patients requiring this treatment, in combination with an ALK inhibitor and a vaccine. The ALK inhibitor is administered according to a continuous regimen based on an average daily dose in the range of about 10 to 250 mg, and the vaccine of the invention is administered in combination according to the dosing regimen, achieving a therapeutically effective dose that is repeated three times a week, twice or once a week, once every two weeks, once every three weeks, or at least once a month.

[0175] The methods of this invention are not limited to the treatment of NSLC. Rather, it is readily understood that the biomolecular pathways addressed and ALK inhibitor resistance eliminated by the methods of this invention can be applied to the treatment of other disease symptoms; any disease symptom treated with ALK inhibitors will produce beneficial outcomes in the treated patient. “Beneficial outcomes” may include, but are not limited to, reducing the severity of disease symptoms, preventing disease symptom exacerbation, curing disease symptoms, or extending the patient’s lifespan or life expectancy. These disease symptoms may be related to, or regulated by, EGFR or any other kinase that may be clinically affected by the methods of this invention.

[0176] More specifically, the inventors’ experimental studies described in the following examples demonstrate the clinical activity of ALK inhibitors in routine dosing regimens and, in molecular studies of these tumors, demonstrate effective inhibition of the EGFR signaling cascade. The examples confirm that the molecular studies accurately reflect the behavior of these ALK inhibitors observed in other model systems. This invention has also unexpectedly demonstrated that the combined use of ALK inhibitors with anti-EGF antibodies (passively administered or actively generated by administration of vaccines that produce such antibodies) can effectively inhibit tumor growth in molecular models—even in tumors that have demonstrated resistance to conventional ALK inhibitor therapy.

[0177] In one illustrative embodiment, anti-EGF antibodies used in preclinical studies are actively generated through immunization with a BVN22E vaccine, as described in PCT application WO 2019 / 016597 A2 entitled "Synthetic Protein and Its Therapeutic Use Thereof". Within the scope of this invention, other vaccine formulations that elicit an immune response to EGF or EGFR can be used. Vaccines that elicit an immune response to other growth factors or their receptors can also be used within the scope of this invention. Specifically, the immunogenic protein BVN22E described in WO 2019 / 016597 A2 (the contents of which are incorporated herein by reference in their entirety) can be used to generate the anti-EGF antibodies of this invention.

[0178] BVN22E has a molecular weight of approximately 120 kDt, and its EGF domain includes regions that present or restrict β-loops, such as the region defined by approximately cysteine ​​6 to approximately cysteine ​​42, the region defined by approximately cysteine ​​6 to approximately cysteine ​​31, or the region defined by approximately cysteine ​​22 to approximately cysteine ​​33, or the region defined by approximately cysteine ​​22 to approximately cysteine ​​31, or the region defined by approximately cysteine ​​62 to approximately cysteine ​​14. Without being bound by any particular theory, it is conceivable that different regions or subregions between cysteine ​​6 and cysteine ​​42 may have beneficial effects when incorporated into synthetic proteins / molecules. It is thought that the following regions may have beneficial effects: the region between cysteine ​​6 and cysteine ​​14, the region between cysteine ​​6 and cysteine ​​20, the region between cysteine ​​6 and cysteine ​​31, the region between cysteine ​​6 and cysteine ​​33, and the region between cysteine ​​6 and cysteine ​​42. It is also conceivable that reverse asymptotic sequences may also be beneficial sequences. For example, the following regions may have beneficial effects: the region between cysteine ​​42 and cysteine ​​33, the region between cysteine ​​42 and cysteine ​​31, the region between cysteine ​​42 and cysteine ​​20, the region between cysteine ​​42 and cysteine ​​14, and the region between cysteine ​​42 and cysteine ​​6. Within the scope of this invention, it is further conceivable that specific intervals within the region between cysteine ​​6 and cysteine ​​42 (e.g., the region between cysteine ​​6 and cysteine ​​14, the region between cysteine ​​14 and cysteine ​​20, the region between cysteine ​​20 and cysteine ​​31, and the region between cysteine ​​33 and cysteine ​​42) may have beneficial effects when incorporated into the synthetic proteins / molecules of this invention.

[0179] The expression of BVN22E and its growth factor epitopes folds in a manner that allows for substantial preservation of their native conformation and presents them to various components of the host immune system in a way that elicits a robust host immune response to the epitopes. Examples of suitable native protein models for mimicking the supporting domains of synthetic proteins / molecular epitopes include, but are not limited to, cholera toxin B subunits, Escherichia coli thermostable LT and LT-II enterotoxin B subunits, enterotoxins, pertussis toxin, Campylobacter jejuni enterotoxin, Shiga toxin, Listeria toxin, tetanus toxoid, diphtheria toxoid, Neisseria meningitidis outer membrane protein, bacteriophage capsid protein, adenovirus, and other viral capsid proteins. Alternatively, the non-self components of the protein can be very small. The non-self sequence should contain at least 9, 10, 11, or longer amino acids and include all or part of at least one human T-cell epitope. Cholera toxin B subunits may be used to satisfy the requirement of conferring immunogenicity to the entire protein and allowing appropriate presentation of growth factors, receptors, tumor antigens, or their epitopes to the host immune system.

[0180] BVN22E can be used to treat chronic diseases such as breast cancer, lung cancer, bladder cancer, ovarian cancer, vulvar cancer, colon cancer, brain cancer, colorectal cancer, bowel cancer, head and neck cancer, and esophageal cancer. In these diseases, different tumor antigens may be expressed and multiple cellular receptors and growth factors may be overexpressed; therefore, the protein described below may contain one or more different tumor antigens, one or more different receptors or growth factors associated with one or more disease-related cellular pathways. These proteins are called multivalents.

[0181] BVN22E is a synthetic protein composed of a homogeneous synthetic protein / molecule expressing one or more epidermal growth factor (EGF) neutralizing domains (e.g., the TSP domain). This protein can exist in the form of a synthetic protein / molecule and can be used to treat chronic diseases such as breast cancer, lung cancer, bladder cancer, ovarian cancer, vulvar cancer, colon cancer, brain cancer, colorectal cancer, head and neck cancer, and esophageal cancer. BVN22E is a synthetic protein / molecule that expresses or contains a synthetic EGF sequence and a CT-B sequence. BVN22E contains a growth factor component of a synthetic protein sequence, which includes a sequence with less than 80% EGF identity. For example, the growth factor component may include an EGF sequence with 11 amino acid substitutions, which can increase the immunogenicity of the growth factor portion of the synthetic protein sequence. Without being bound by theory, it has been considered that EGF regions “presenting” or restricting the β-loop (e.g., the region defined by Cys6 to Cys31) may be important regions included in the synthetic protein and suitable as targets for amino acid modifications.

[0182] BVN22E includes one or more connectors or spacers. One or more of the above embodiments include sEGF fused to CT-B such that the sEGF portion of the synthetic molecule is separated from the CT-B portion via a GGSGGTSGGGGGSG connector. These resulting recombinant or chimeric proteins essentially consist of sEGF fused directly to CT-B. In other embodiments, the EGF and CT-B components of the chimeric protein are efficiently separated by 3 to 14 amino acids, forming a flexible spacer or connector between the two domains. It is conceivable that the connector length can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acids. In some cases where the growth factor has a large size (e.g., human growth factor), it may be useful to use a longer connector sequence. The following exemplary connectors may also be used, including but not limited to: SSG, SSGGG, SGG, GSSG, GGSGG, GGGGS, SSGGGSGG, SSGGGGSGG, TSGGGSG, TSGGGGSGG, SSGGGSGGSSG, GGSGGTSGGGSG, SGGTSGGGGSG, GGSGGTSGGGGSG, SSGGGGSGGGSSG, SSGGGSGGSGGSSGGG, and SSGGGGSGGSGGSSGGG. Those skilled in the art will understand that many other sequences / combinations (primarily “G” and “S”) will also be useful connector sequences.

[0183] Researchers believe that BVN22E has significant clinical benefits. For example, BVN22E can be expressed in commercially available and pure bacterial systems, producing correctly folded and functional stable peptides. Furthermore, BVN22E also has the advantageous property of requiring significantly lower protein levels for inoculation due to the significantly lower amount of carrier required compared to existing techniques (e.g., U.S. Patent No. 5,984,018, Davila et al.). In this respect, BVN22E can deliver more growth factors to patients with significantly lower vaccine doses.

[0184] While not wishing to be bound by any theory, researchers believe that these inhibitions of the STAT3 metabolic pathway (required to stimulate cell signaling pathways responsible for cell proliferation) are assisted by BVN22E. Researchers also believe that the additional inhibition of STAT3 using the combination dosing regimen of this invention can effectively suppress or downregulate cell signaling. Furthermore, since STAT3 is also inhibited, the combination dosing regimen of this invention can provide beneficial antitumor effects even in patients resistant to conventional ALK inhibitor therapy. The combination therapy of this invention may be related to barriers to disease symptoms where conventional ALK inhibitor therapy has failed. Therefore, by operating at the cellular and molecular levels in a manner different from conventional methods, the method of this invention can overcome resistance or unresponsiveness to ALK inhibitor therapy.

[0185] In specific embodiments, the combined dose of an ALK inhibitor and an anti-EGF antibody can effectively treat cancers, particularly lung, breast, and prostate cancers, in individuals resistant to conventional ALK inhibitor therapy. Other forms of cancer that can be treated with the methods of the present invention include, but are not limited to, gastric, colorectal, and ovarian cancers, as well as glioblastoma. Each form of cancer exhibits significant EGFR expression, making it a suitable target for treatment according to the methods of the present invention.

[0186] The ALK inhibitors suitable for use according to the methods of the present invention may include, but are not limited to, crizotinib, ceritinib, alectinib, brigatinib and lorlatinib, or pharmaceutically acceptable salts thereof, or equivalents thereof; all of these drugs are included in the scope of the term "ALK inhibitor".

[0187] The efficacy of a given cancer treatment can be determined by a skilled clinician. However, based on the use of the term "effective treatment" in this application, a treatment is considered "effective" if any or all signs or symptoms (e.g., tumor) change in a beneficial manner, or if other clinically accepted symptoms improve or even lessen, for example, by at least 10% improvement after treatment with the agents described in this application. Efficacy can also be determined by the absence of deterioration in an individual's condition (assessed through hospitalization or the need for medical intervention (i.e., cessation of disease progression)). Methods for measuring these indicators are known to those skilled in the art and / or described in this application.

[0188] According to the definition of the disease in this application, an effective dose for disease treatment refers to an amount sufficient to effectively treat the disease when administered to a mammal requiring that effective dose. The efficacy of a drug can be determined by assessing physical indicators, such as those for cancer, including tumor size, tumor mass, tumor density, angiogenesis, and tumor growth rate. Additionally, the efficacy of a drug can be determined by reducing the circulating MIC peptides or fragments thereof in subjects receiving treatment with a drug (including antibodies or their antigen-binding portions as described in this application, or nucleic acids encoding antibodies or their antigen-binding portions as described in this application).

[0189] The description of embodiments of the present invention is not intended to be exhaustive or to limit the invention to the precise forms disclosed. While specific embodiments and examples of the invention have been described in this application for illustrative purposes, those skilled in the art will recognize that various equivalent modifications may be possible within the scope of the invention. Where appropriate, the teachings of the invention provided herein can be applied to other procedures or methods. The various embodiments described herein can be combined to provide further embodiments. If necessary, various aspects of the invention can be modified to utilize the composition, function, and concepts of the foregoing references and applications to provide yet another embodiment of the invention. These and other changes can be made to the invention in accordance with the specification.

[0190] Specific elements of any of the above embodiments can be combined or substituted with elements from other embodiments. Furthermore, while advantages relating to certain embodiments of the invention have been described in the context of these embodiments, other embodiments may also exhibit these advantages, and it is not necessary for all embodiments to exhibit these advantages in order to fall within the scope of the invention.

[0191] Example

[0192] The present invention is further illustrated by the following embodiments, but should not be construed as limiting the present invention.

[0193] Example 1: The effect of combined use of ALK inhibitors and anti-EGF antibodies in non-small cell lung cancer cell lines.

[0194] Compared to EGFR-mutant patients treated with EGFR-TKIs, patients treated with ALK-TKIs achieved longer progression-free survival. Clinically, 20-30% of patients rapidly develop resistance, but it is unknown whether these patients will develop resistance or never respond. The role of circulating EGF in these patients is currently unclear. This invention proposes the role of EGF in TKI resistance using two cell lines containing major ALK rearrangements from NSCLC patients.

[0195] The combined effects of BVN22E antibody with the following ALK-TKIs—crizotinib, alectinib, and brigantinib—were investigated in two cell lines containing major ALK rearrangements from NSCLC patients. ALK "rearrangement" refers to a series of different mutations. These cell lines include... Figure 1 As shown: H3122 lung adenocarcinoma contains EML4-ALK fusion variant v1, and H2228 lung adenocarcinoma contains EML4-ALK fusion variant v3, together accounting for approximately 30-40% of ALK rearrangement patients.

[0196] All studies and the resulting examples used anti-BVN22E antibodies prepared and purified in rabbits. Initial serum collected from immunized rabbits was diluted approximately 10-fold after a two-step purification process. All experiments were further diluted, with 1-10 dilutions corresponding to 1-100 dilutions of the original titer.

[0197] In the presence of EGF, the effects of brigatinib and alectinib on the proliferation of H3122 cells were determined individually. Results (see...) Figure 2A -B) indicates that brigatinib and alectinib alone had a greater inhibitory effect on H3122 cell proliferation than brigatinib and alectinib in the presence of EGF. Similarly, in the presence of EGF, crizotinib had a greater inhibitory effect on H3122 cell proliferation than crizotinib alone, see [reference needed]. Figure 3 The effect of BVN22E antibody in H3122 cells in the presence of EGF was evaluated in a 72-hour cell proliferation assay. Figure 4 As shown in the figure. The data indicate that, compared with the control antibody, the BVN22E antibody can reduce the viability of H3122 cells.

[0198] Next, the effects of combining an ALK inhibitor with the BVN22E antibody were determined. The effects of alectinib alone or in combination with the BVN22E antibody on the 72-hour proliferation of H3122 cells were as follows: Figure 5 As shown in Table 6, the effects of crizotinib monotherapy or its combination with BVN22E antibody on the 72-hour proliferation of H3122 cells are illustrated. For both TKI inhibitors, the combination of BVN22E antibody and the inhibitor significantly reduced cell viability more than either inhibitor alone; however, the combination of alectinib and BVN22E antibody was more potent than crizotinib. The effects of brigatinib and alectinib monotherapy or their combination with BVN22E antibody on the viability of H31122 and H2228 cells are shown in Figure 7.

[0199] After incubation for 2 hours with BVN22E antibody at concentrations of 1 / 25, 1 / 50, and 1 / 250, the effect of BVN22E antibody in H3122 cells was evaluated using Western blotting. Figure 8 As shown. The study showed that the specific monoclonal antibody contained EGFR, AKT, STAT3, and ERK1 / 2. Phosphorylated forms of the antibody were also used. C represents control, untreated cell line samples. The second channel is the same control cells receiving EGF, and all other channels are dilutions of the control antibody and anti-BVN22E antibody. These data indicate that pEGFR was significantly inhibited, while pAkt, pStat3, and pErk1 / 2 levels remained unchanged. After incubation for 24 hours with 1 / 25, 1 / 50, and 1 / 250 concentrations of BVN22E antibody, the effect of BVN22E antibody in H3122 cells was evaluated by Western blotting, as shown. Figure 9 As shown in the figure. These data indicate that significant pEGFR inhibition was maintained 24 hours after incubation with the BVN22E antibody. After incubation for 2 hours with crizotinib at concentrations of 0.1, 0.25, 0.5, 1, 2.5, and 5%, the effect of crizotinib in H3122 cells was evaluated using Western blotting, as shown in the figure. Figure 10 As shown in the figures, these data indicate that significant inhibition of pStat3 and pAkt was observed after 2 hours of incubation with crizotinib.

[0200] In the presence of EGF, the effects of brigatinib and alectinib on the proliferation of H2228 cells were determined individually. The results showed that in the presence of EGF, brigatinib and alectinib alone had a greater inhibitory effect on the proliferation of H3122 cells than either brigatinib alone (Figure 11). Figure 12 The study demonstrated the effect of crizotinib alone on H3122 cell proliferation at 72 hours in the presence of EGF. The results showed that crizotinib alone had a greater inhibitory effect on H3122 cell proliferation than crizotinib in the presence of EGF. Figure 13 This study demonstrates the effect of BVN22E antibody on cell viability in H2228 cells in the presence of EGF. A significant decrease in cell viability was observed at higher concentrations of BVN22E antibody.

[0201] Next, the effects of combining ALK inhibitors with BVN22E antibody were determined. The effect of alectinib as monotherapy or in combination with BVN22E antibody on H2228 cell proliferation at 72 hours was evaluated. Although alectinib had the least effect on cell viability, higher concentrations of alectinib combined with BVN22E antibody significantly reduced cell viability (see [link to study]). Figure 15Even low concentrations of crizotinib, when used in combination with BVN22E antibody, significantly reduce cell viability. (See [link to relevant documentation]). Figure 15 The combined use of ALK tyrosine kinase inhibitors brigatinib and alectinib with BVN22E antibody significantly inhibited cell proliferation in H2228 cells, see [reference needed]. Figure 16A .

[0202] After incubation for 2 hours with BVN22E antibody at concentrations of 1 / 25, 1 / 50, and 1 / 250, the effect of BVN22E antibody in H2228 cells was evaluated using Western blotting. Figure 17 As shown in the figures, these data indicate that pEGFR was significantly suppressed, pERK1 / 2 was somewhat suppressed, while pAkt and pStat3 levels remained unchanged.

[0203] Next, after incubation for 24 hours with BVN22E antibody at concentrations of 1 / 25, 1 / 50, and 1 / 250, the effect of BVN22E antibody in H2228 cells was evaluated using Western blotting. Figure 18 As shown in the figures, these data indicate that significant pEGFR inhibition was maintained after 24 hours of incubation with the BVN22E antibody.

[0204] Next, after incubation for 2 hours with crizotinib at concentrations of 0.025, 0.05, 0.1, 0.5, 1, and 2.5 μM, the effect of crizotinib in H2228 cells was evaluated using Western blotting. Figure 19 As shown. These data indicate that pStat3 was significantly inhibited 2 hours after incubation with crizotinib. Similarly, brigatinib significantly inhibited pSTAT3, and high concentrations of brigatinib (1 and 2.5 μM) increased pEGFR and pAKT, see [reference needed]. Figure 20 Notably, the combination of BVN22E antibody and brigatinib prevented the activation of pEGFR and pERK in H2228 cells, see [link to relevant documentation]. Figure 21 .

[0205] Brigatinib significantly inhibited pSTAT3, and a slight inhibition of pERK1 / 2 was observed in H3122 cells. Figure 22 Higher concentrations of brigatinib also showed significant inhibitory effects on pSTAT3 and slight inhibitory effects on pERK1 / 2 in H3122 cells. Conversely, the combination of BVN22E antibody and brigatinib completely inhibited the activation of pEGFR and pERK in H2228 cells. Figure 24 .

[0206] It is well known that patients can develop resistance to monotherapy with ALK TKIs. Therefore, researchers investigated whether the combination of crizotinib or alectinib with a BVN22E antibody could delay the development of ALK TKI resistance in H2228 cells. In these studies, the combination of a TKI inhibitor and a BVN22E antibody was added to the cells each time the cell culture medium was changed (1-2 times per week). The studies were conducted in 96-well microplates, and microscopic imaging was used to determine the percentage of wells containing resistance colonies.

[0207] BVN22E antibody can delay the development of resistance to ALK-TKIs in vitro. Experiments were conducted using crizotinib (…). Figure 25 A), to a greater extent, experiments were conducted using alectinib ( Figure 25 (B) The results showed that combination therapy controlled cell proliferation for a longer period, indicating a delay in the development of resistance. With alectinib alone, all wells showed active replication after 10 weeks, while the combination therapy delayed replication for an even longer period. Notably, when the highest concentration of BVN22E antibody was used in combination with alectinib, 100% resistance was not achieved until week 25. Figure 25 C (crizotinib) and Figure 25 D (alectinib) presented the same experiment, but showed the percentage of tumor-free wells instead of positive wells; the results of this invention are presented in a manner similar to Kaplan Meyer curves in clinical trials, where the y-intercept corresponds to the zero patient survival point. Similarly, in H3122 cells, the BVN22E antibody delayed the emergence of resistance to 0.1 μM and 0.2 μM crizotinib in H3122 cells, see [reference needed]. Figure 26 and 27 .

[0208] Notably, in a 3-day proliferation assay, the combination of ALK-TKI and BVN22E antibody significantly improved cell proliferation inhibition and EGF signaling pathway regulation compared to ALK-TKI alone. The combination therapy enhanced the effects of ALK-TKI, neutralized its negative effects, and broadened the inhibition of all signaling molecules. Compared to ALK-TKI alone, the development of ALK-TKI resistance in H228 cells was significantly delayed by two-fold in the presence of both ALK-TKI and BVN22E antibody, supporting a stronger and broader inhibition of EGF signaling.

[0209] Example 2: Evaluation of the combined use of anti-EGF antibody and trametinib in BRAF and KRAS mutant cell lines.

[0210] Colorectal cancer (CRC) is not only one of the most common cancers, but also presents unique challenges in treatment. Clinically, CRC patients without KRAS mutations can receive EGF-targeted therapy, such as cetuximab and erlotinib. However, a large proportion of CRC patients have KRAS, BRAF, or PIK3CA mutations. Currently, there are no effective treatments for these patients. Chemotherapy and angiogenesis-targeted therapy are commonly used treatments, but they also have significant drawbacks.

[0211] To begin addressing the treatment needs of CRC patients with KRAS, BRAF, or PIK3CA mutations, in vitro assays were performed in cell lines with these mutations using a combination of EGF antibody and the MEK inhibitor trametinib. All experiments were conducted in conjunction with a BVN22E antibody.

[0212] The cell viability of HT29 (which is also a colorectal adenocarcinoma cell line, but with the BRAF V66E mutation) was investigated (see [link to study]). Figure 28 ).like Figure 29 As shown, EGF can enhance the viability of HT29 cells. The effects of BVN22E antibody and trametinib alone and in combination on cell viability in HT29 cells were evaluated, as shown in the following figures. Figure 30 and 31 As shown, the combined use of BVN22E antibody and trametinib significantly inhibited cell proliferation compared to trametinib alone.

[0213] To investigate the effects of BVN22E antibody and trametinib on signal transduction in HT29 cells, Western blotting was performed after 2 hours of incubation in HT29 cells. BVN22E antibody was used at a 1 / 10 dilution. (Immunoblotting...) Figure 32 and 33 The results showed that the combined use of trametinib and BVN22E antibody enhanced the EGF-induced inhibition of pEGFR and pERK1 / 2. These results confirm the significant combined effect of BVN22E antibody and trametinib in HT29 cells observed in cell proliferation assays.

[0214] Cell cycle and apoptotic status of individual cells were determined using propidium iodide staining and flow cell counting. (See...) Figure 34 This method has been used in KRAS mutant (A549 and DLD1) and ALK transposable (H2228 and H3122) cell lines.

[0215] In DLD1 cells, the percentage of S and G2 / M state cells induced by EGF alone was significantly higher than that induced by BVN22E antibody containing EGF or by the combination of BVN22E antibody containing EGF and trametinib. (See...) Figure 35 In A549 cells, only minor differences in cell cycle status were observed when BVN22E antibody was used in combination with EGF and trametinib. (See [link to relevant documentation]). Figure 36 Conversely, in H228 cells, the combination of BVN22E antibody with EGF and trametinib significantly reduced S and G2 / M cells compared to either BVN22E antibody alone or trametinib alone (see [link to relevant documentation]). Figure 37 In H3122 cells, only a few differences in cell cycle states were observed, see [link to relevant documentation]. Figure 38 These data suggest that the combined use of BVN22E antibody and TKI inhibitor may inhibit EGF-regulated cell cycle processes.

[0216] Figure 39 Four KRAS-mutated cell lines are shown. A549 is a lung adenocarcinoma cell line with a G12S mutation. H23 is a lung adenocarcinoma cell line with a G12C mutation. DLD1 is a colon adenocarcinoma cell line with a G13D mutation. LS174T is a colon adenocarcinoma cell line that also contains a G12C mutation.

[0217] Next, the effect of using BVN22E antibody alone was evaluated in the DLD1 cell line. Higher concentrations of BVN22E antibody significantly inhibited DLD1 cell viability compared to the control antibody (see [link to study]). Figure 40 Similarly, in DLD1 cells, the combination of trametinib and BVN22E antibody significantly inhibited cell viability compared to trametinib alone, as shown in [reference needed]. Figure 41 .

[0218] The role of BVN22E antibody in DLD1 cells was investigated using Western blotting. Figure 42 and 43 The effect of BVN22E antibody alone on the DLD1 cell line was limited. As expected, EGFR phosphorylation was strongly inhibited at all dilutions tested. AKT phosphorylation was only slightly inhibited, and no effect on ERK1 / 2 activation was observed.

[0219] For all concentrations of trametinib tested, cell viability was enhanced in the presence of EGF compared to trametinib alone. Higher cell viability indicates the presence of tumor cell replication, a process that trametinib is designed to inhibit; therefore, the efficacy of trametinib is lower in the presence of EGF. (See [link to relevant documentation]). Figure 44 .

[0220] In the DLD1 cell line, the efficacy of trametinib treatment was also evaluated using Western blotting, and the results are shown in […]. Figure 45 It had no effect on pEGFR or pAKT. Inhibition of pStat3 and pERK1 / 2 was observed, but their concentrations only exceeded the physiological concentrations of trametinib used in patients.

[0221] Next, the combination of BVN22E antibody and trametinib was studied in the DLD1 cell line, such as... Figure 46 As shown, cell viability when trametinib was used in combination with BVN22E antibody was significantly lower than when trametinib was used alone or in combination with the control antibody. These results indicate that inhibition of EGF with BVN22E antibody significantly enhanced the effect of trametinib.

[0222] To understand the mechanism behind the enhanced effect of trametinib combined with BVN22E antibody, Western blotting experiments were performed in the DLD1 cell line, such as... Figure 47 As shown. Besides inhibiting pERK1 / 2, trametinib monotherapy had no effect on the measured signaling molecules. BVN22E antibody monotherapy inhibited EGFR phosphorylation. Notably, the combination of BVN22E antibody and trametinib had a greater inhibitory effect on signal transduction than either treatment alone. In addition to pERK1 / 2 and pEGFR, BVN22E antibody combined with trametinib also inhibited pSTAT3 and pAKT. Furthermore, in the presence of trametinib, the development of trametinib resistance was delayed when combined with BVN22E antibody compared to trametinib monotherapy, see [reference needed]. Figure 48 .

[0223] In summary, the addition of BVN22E antibody enhanced the antiproliferative effect of trametinib in the DLD1-KRAS mutant cell line. The presence of EGF significantly inhibited the effect of trametinib alone on cell viability. Besides inhibiting pERK1 / 2, trametinib alone had no effect on the measured signaling molecules. BVN22E antibody alone inhibited EGFR phosphorylation. Notably, in DLD1 cells, the combination of BVN22E antibody and trametinib had a greater inhibitory effect on signal transduction than either treatment alone. In addition to pERK1 / 2 and pEGFR, the combination of BVN22E antibody and trametinib also inhibited pSTAT3 and pAKT.

[0224] Higher concentrations of BVN22E antibody can inhibit the viability of the A549 cell line, such as... Figure 49As shown. In A549 cells, the effects of incubation with BVN22E antibody for 2 hours on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473), and pERK1 / 2 (THR 202 / TYR 204) were as follows. Figure 50 As shown. A mild inhibitory effect on pERK1 / 2 was observed, and EGF completely inhibited the activation of pEGFR. In A549 cells, the effects on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473), and pERK1 / 2 (THR 202 / TYR 204) were as follows, in response to 24 hours of incubation with BVN22E antibody. Figure 51 As shown; after 24 hours, the effect on cell signaling was weakened in this cell line. The effects of trametinib on cell viability in the A549 cell line with and without EGF are as follows: Figure 52 As shown, EGF significantly enhanced cell viability. In A549 cells, the effects of EGF on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473), and pERK1 / 2 (THR 202 / TYR 204) as a response to trametinib alone for 2 hours were as follows: Figure 53 As shown; at this initial time point, complete suppression of pERK1 / 2 was observed. Figure 54 This indicates that, although the combination of BVN22E antibody and trametinib significantly reduced cell viability in A549 cells, the decrease was less severe compared to other cell types presented in this invention.

[0225] After 2 hours of incubation, the effects on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473), and pERK1 / 2 (THR 202 / TYR 204) were observed in A549 cells with and without EGF, as a response to the combination of BVN22E antibody and trametinib. The strongest effects on pEGFR and pERK1 / 2 signaling were observed in the presence of EGF when BVN22E antibody was used alone or in combination with trametinib, as detailed below. Figure 55 .

[0226] BVN22E antibody also significantly inhibited the cell viability of H23 cells, see [link to article]. Figure 56After 2 hours of incubation, the effects on pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473), and pERK1 / 2 (THR 202 / TYR 204) were observed in H23 cells as a response to 2 hours of BVN22E antibody incubation. (See [link to relevant documentation]). Figure 57 The BVN22E antibody completely extinguished the increase in pEGFR and pERK1 / 2 observed in H23 cells as a response to EGF. This inhibition of pEGFR and pERK1 / 2 persisted for 24 hours. Figure 58 As shown.

[0227] The presence of EGF in H23 cells significantly inhibited the inhibitory effect of trametinib on cell viability. Figure 59 After 2 hours of incubation, the effects of pEGFR (TYR 1068), pSTAT3 (TYR 705), pAKT (SER 473), and pERK1 / 2 (THR 202 / TYR 204) on the response to trametinib were observed in H23 cells. At higher trametinib concentrations, pERK1 / 2 was significantly inhibited (see [reference needed]). Figure 60 Compared with trametinib alone, the combination of BVN22E antibody and trametinib significantly inhibited the cell viability of H23 cells. (See attached image) Figure 61 In H23 cells, a significant inhibition of pEGFR was observed as a response to the combination of BVN22E antibody and trametinib followed by incubation for 2 hours.

[0228] Compared with the control antibody, the BVN22E antibody significantly inhibited the viability of LS174T cells, see [link to relevant documentation]. Figure 63 The BVN22E antibody also inhibited pEGFR and pAKT signaling in LS174T cells, see [link to relevant documentation]. Figure 64 and 65 Trametinib alone inhibited the proliferation of LS174T cells; this effect was attenuated in the presence of EGF. Figure 66 The effect of trametinib in the LS174T cell line was evaluated by a 2-hour incubation immunoblotting assay, see [link to relevant documentation]. Figure 67 Within the physiological range of 1-10 nM, trametinib did not affect the phosphorylation of any of the tested molecules. At higher concentrations (not clinically relevant), effects on pERK1 / 2 were observed. However, trametinib in combination with anti-BVN22E inhibited pAKT, pEGFR, and pERK1 / 2, and also inhibited pSTAT3 (see [link to relevant documentation]). Figure 68 Therefore, the BVN22E antibody broadens the inhibitory effect of trametinib on signal transduction.

[0229] The effect of combined use of BVN22E antibody and trametinib on the viability of LS174T cells. Figure 69 As shown, compared to trametinib alone, the combination of BVN22E antibody and trametinib significantly increased cell viability within the 1-10 nM trametinib range, i.e., the physiological level of trametinib in patients. However, the effect of the combination of BVN22E antibody and trametinib in LS174T cells was less pronounced compared to the effect in DLD1 cells.

[0230] In the LS174T KRAS mutant cell line, the BVN22E antibody enhanced the antiproliferative effect of trametinib. The presence of EGF significantly reduced the inhibitory effect of trametinib on cell viability. BVN22E antibody alone inhibited both pEGFR and pAKT. However, the combination of trametinib and anti-BVN22E inhibited pAKT, pEGFR, and pERK1 / 2, while also inhibiting pSTAT3. Therefore, in both KRAS mutant cell lines, the BVN22E antibody enhanced the inhibitory effect of trametinib on cell proliferation and broadened its signal transduction inhibition pathway.

[0231] Example 3: Serum evaluation of patients vaccinated with EGF cancer vaccine in SW900 cells.

[0232] In the presence of EGF, the effect of anti-EGF serum on pEGFR in SW900 wild-type cells from human patient (22180004) was observed. 2a = patients before vaccination, 3d = patients after vaccination. In the presence of anti-EGF serum, complete inhibition of pEGFR activation was observed, along with a certain degree of decrease in pERK1 / 2. Figure 70 In the presence of EGF, the effects of anti-EGF serum from two other patients on pEGFR were observed in SW900 wild-type cells. (See attached image.) Figure 71 In patient 27030004, significant inhibition of pEGFR and pERK1 / 2 activation was observed, while in patient 29080005, the inhibitory effect on pEGFR and pERK1 / 2 was less pronounced. Anti-EGF serum results for the other three patients are as follows... Figure 72 As shown. Significant inhibition of pEGFR and pERK1 / 2 signaling was observed in two of the patients (29040007 and 3308020). Minimal changes were observed in patient 31070004. Figures 70-72 The results are summarized in Figures 73 and 74.

[0233] References

[0234] 1.Gandhi J,Zhang J,Xie Y,et al.Alterations in genes of the EGFRsignaling pathway and their relationship to EGFR tyrosine kinase inhibitorsensitivity in lung cancer cell lines.PLoS ONE.2009;4:e4576.

[0235] 2.Rosell R,Moran T,Queralt C,et al.Screening for epidermal growthfactor receptor mutations in lung cancer.N Engl J Med.2009;361:958-967.

[0236] 3.Rosell R,Carcereny E,Gervais R,et al.Erlotinib versus standardchemotherapy as first-line treatment for European patients with advanced EGFRmutation-positive non-small-cell lung cancer(EURTAC):a multi-centre,open-label,randomised phase 3 trial.Lancel Oncol.2012;13:239-246.

[0237] 4.Mok TS,Wu YL,Thongprasert S,et al.Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma.N Engl J Med.2009;361:947-957.

[0238] 5.Maemondo M,Inoue A,Kobayashi K,et al.Gefitinib or chemotherapy fornon-small-cell lung cancer with mutated EGFR.N Engl J Med.2010;362:2380-2388.

[0239] 6.Bradley JD,Paulus R,Komaki R,et al.Standard-dose versus high-doseconformal radiotherapy with concurrent and consolidation carboplatin pluspaclitaxel with or witbout cetuximab for patients with stage IIIA or IIIBnon-small-cell lung cancer(RTOG 0617):a randomised,two-by-two factorial phase3 study.Lancet Oncol.2015;16:187-199.

[0240] 7.Lynch TJ,Patel T,Dreisbach L,et al.Cetuximab and first-line taxane / carboplatin chemotherapy in advanced non-small-cell lung cancer:results ofthe randomized multicenter phase III trial BMS099.J Clin Oncol.2010;28:911-917.

[0241] 8.Herbst R,Redman M,Kim E.A randomized,phase III study comparingcarboplatin / paclitaxel or carboplatin / paclitaxel / bevacizumab with our withoutconcurrent cetuximab in patients with advanced non-small cell lung cancer(NSCLC).SWOG S0819.Presented at the 2015 World Conference on Lung Cancer,Denver,Colorado.

[0242] 9.Janjigian YY,Smit EF,Groen HJ,et al.Dual inhibition of EGFR withafatinib and cetuximab in kinase inhibitor-resistant EGFR-mutant lung cancerwith and without T790M mutations.Cancer Discov.2014;4:1036-1045.

[0243] 10.Horn L,Gettinger S,Camidge DR,et al.Continued use of afatinib withthe addition of cetuximab after progression on afatinib in patients with EGFRmutation-positive non-small-cell lung cancer and acquired resistance togefiti-nib or erlotinib.Lung Cancer2017;113:51-58.

[0244] 11.Rodriguez PC,Popa X,Martinez O,et al.A phase III clinical trial ofthe epidermal growth factor vaccine CIMAvax-EGF as switch maintenance therapyin advanced non-small cell lung cancer patients.Clin Cancer Res.2018;22:3782-3790.

[0245] 12.Rosell R,Neninger E,Nicolson M,et al.Pathway targetedimmunotherapy:rationale and evidence of durable clinical responses with anovel,EGF-directed agent fot advanced NSCLC.J Thorac Oncol.2016;11:1954-1961.

[0246] 13.Jacobsen K,Bertran-Alamillo J,Molina MA,et al.Convergent Aktactivation drives acquired EGFR inhibitor resistance in lung cancer.NatCommun.2017;8:410.

[0247] 14.Tricker EM,Xu C,Uddin S,et al.Combined EGFR / MEK inhibitionprevents the emergence of resistance in EGFR-mutant lung cancer.CancerDiscov.2015;5:960-971.

[0248] 15.Chaib I,Karachaliou N,Pilotto S,et al.Co-activation of STAT3 andYES-associated protein1(YAP1)pathway in EGFR-mutant NSCLC.J Natl CancerInst.2017;109(9).

[0249] 16.Borghaei H,Paz-Ares L,Horn L,et al.Nivolumab versus docetaxel inadvanced nonsquamous non-small-cell lung cancer.N Engl J Med.2015;373:1627-1639.

[0250] 17.Herbst RS,Baas P,Kim DW,et al.Pembrolizumab versus docetaxel forpreviously treated,PD-L1-positive,advanced non-small-cell lung cancer(KEYNOTE-010):a randomised controlled trial.Lancet.2016;387:1540-1550.

[0251] 18.Rittmeyer A,Barlesi F,Waterkamp D,et al.Atezolizu-mab versusdocetaxel in patients with previously treated non-small-cell lung cancer(OAK):a phase 3,open-label,multicentre randomised controlledtrial.Lancet.2017;389:255-265.

[0252] 19.Lee CK,Man J,Lord S,et al.Checkpoint inhibitors in metastaticEEGFR-mutated non-small cell lung cancer-a meta-analysis.J Thorac Oncol.2017;12:403-407.

[0253] 20.Dong ZY,Zhang JT,Liu SY,et al.EGFR mutation correlates withuninflamed phenotype and weak immunoge-nicity,causing impaired response toPD-1 blockade in non-small cell lung canccr,Oncoimmunology.2017;6:c1356145.

[0254] 21.Neninger Vinageras E,de la Torre A,Osorio Rodriguez M,et al.PhaseII randomized controlled trial of an epidermal growth factor vaccine inadvanced non-small-cell lung cancer.J Clin Oncol.2008;26:1452-1458.

[0255] 22.Garcia B,Neninger E,de la Torre A,et al.Effective inhibition ofthe epidermal growth factor / epidermal growth factor receptor binding by anti-epidermal growth factor antibodies is related to better survival in advancednon-small-cell lung cancer patients treated with the epidermal growth factorcancer vaccine.Clin Cancer Tes.2008;14:840-846.

[0256] 23.Joh T,Itoh M,Katsumi K,et al.Physiological concentrations of humane[idermal growth factor in biological fluids:use of a sensitive enzymeimmunoassay.Clin Chim Acta.1986;158:81-90.

[0257] 24.Wakeling AE,Guy SP,Woodburn JR,et al.ZD1839(lre-ssa):an orallyactive inhibitor of epidermal growth factor signaling with potential forcancer therapy.Cancer Res.2002;62.5749-5754.

[0258] 25.Vollebergh MA,Kappers I,Klomp HM,et al.Ligands of epidermal growthfactor receptor and the insulin-like growth factor family as serum biomarkersfor response to epidermal growth factor receptor inhibitors in patients withadvanced non-small cell lung cancer.J Thorac Oncol.2010;5:1939-1948.

[0259] 26.lshikawa N,Daigo Y,Takano A,et al.Increases of amphiregulin andtransforming growth factor-alpha in serum as predictors of poor response togefitinib among patients with advanced non-small cell lung cancers.CancerRes.2005;65:9176-9184.

[0260] 27.Romero-Ventosa EY,Blanco-Prieto S,Gonzalez-Pineiro AL,etal.Pretreatment levels of the serum biomarkers CEA,CYFRA 21-1,SCC and thesoluble EGFR and its ligands EGFTGF-alpna,HB-EGF in the prediction of outcomein erlo-tinib treated non-smallcell lung cancer patients.Spring-erplus.2015;4:171.

[0261] 28.Perez-Torres M,Guix M,Gonzalez A,et al.Epidermal growth factorreceptor(EGFR)antibody down-regulates mutant receptors and inhibits tumorsexpressing EGFR mutations.J Biol Chem 2006;281:40183-40192.

[0262] 29.Mukohara T,Engelman JA,Hanna NH,et al.Differential effecis ofgefitinib and cetuximab on non-small-cell lung cancers bearing epidermalgrowth factor receptor mutations.J NatlCancer Inst.2005;97:1185-1194.

[0263] 30.Huang S,Armstrong EA,Benavente S,et al.Dual-agent moleculartargeting of the epidermal growth factor receptor(EGFR):combining anti-EGFRantibody with tyrosine kinase inhibitor.Cancer Res.2004;64:5355-5362.

[0264] 31.Pirazzoli V,Ayeni D,Meador CB,et al.Afatinib plus cetuximab delaysresistance compared to single-agent erlotinib or afatinib in mouse models ofTKI-anive EGFR 1.858R-induced lung adenocarcinoma.Clin Cancer Res,2016;22:426-435.

[0265] 32.Sen M,Joyce S,Panahandeh M,et al.Targetimg Stat3 abrogates EGFRinhibitor resistance in cancer.Clin Cancer Res.2012;18:4986-4996.

[0266] 33.Zhang Z,Lee JC,Lin L,et al.Activation of the AXL kinase causesresistance to EGER-targeted therapy in lung cancer.Nat Genet.2012;44:852-860.

[0267] 34.Schulz D,Wirth M,Piontek G,et al.HNSCC cells resistant to EGFRpathway inhibitors are hypermutated and sensitive to DNA damagingsubstances.Am J Cancer Res.2016;6:1963-1975.

[0268] 35.Codony-Servat C,Codony-Servat J,Karachaliou N,et al.Activation ofsignal transducer and activator of transcription 3(STAT3)signaling in EGFRmutant non-small-cell lung cancer (NSCLC).Oncotarget.2017;8:47305-47316.

[0269] 36.Hashida S,Yamamoto H,Shien K,et al.Aequisition of cancer stemcell-like properties in non-small cell lung cancer with acquired resistanceto afatinib.Cancer Sci.2015,106:1377-1384.

[0270] 37. Vouri M, Croucher DR, Kennedy SP, et al. Axl-EGFR receptor tyrosinekinase hetero-interaction provides EGFR with access to pro-invasive signaling in cancer cells. Oncogenesis. 2016;5:e266

[0271] 38. Brand TM, Iida M, Stein AP, et al. AXL mediates resistance tocetuximab therapy. Cancer Res. 2014;74:5152-5164.

[0272] 39, Arasada RR, Amann JM, Rahman MA, et al. EGFR blockade enriches for lung cancer stem-like cells through notch3-dependent signaling CancerRes. 2014;74:5572-5584. sequence list <110> IN3BIO LTD. <120> Methods and compositions for inhibiting the EGF / EGFR pathway by combining anaplastic lymphoma kinase inhibitors <130> 48199-105PCT <140> PCT / IB19 / 00905 <141> 2019-08-07 <150> US 62 / 715,351 <151> 2018-08-07 <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 681 <212> DNA <213> artificial <220> <223> synthesis <400> 1 aataccgaaa acgattgccc tctgtctcat gaagcgtatt gtctgcacga cggcgtgtgt 60 atgtacattg aagccctgga caaatatgca tgtaactgtg tcgtgggcta cgtgggggag 120 cgatgtcagt ttcgagacct gcgttggtgg gatgcgcgcg gctcgagcgg taataccgaa 180 aacgattgcc ctctgtctca tgaagcgtat tgtctgcacg acggcgtgtg tatgtacatt 240 gaagccctgg acaaatatgc atgtaactgt gtcgtgggct acgtggggga gcgatgtcag 300 tttcgagacc tgcgttggtg ggatgcgcgc ggcgggtctg gaggtactag tggcggcggt 360 ggagggtcgg gtaccccgca gaacatcacc gacctgtgcg ccgagtacca caacacccag 420 atccacaccc tgaacgacaa gatcttctcg tacaccgaga gcctggccga taagcgtgaa 480 atggccatca tcaccttcaa gaacggtgcg accttccagg tggaggtccc gggtagccag 540 cacatcgatt cacagaagaa ggccatcgag cgtatgaagg acaccctgcg tatcgcctac 600 ctgaccgaag ccaaggtgga aaagctgtgc gtctggaaca acaagacgcc gcacgccatc 660 gccgccatca gcatggccaa t 681 <210> 2 <211> 227 <212> PRT <213> Artificial <220> <223> Synthesis <400> 2 Asn Thr Glu Asn Asp Cys Pro Leu Ser His Glu Ala Tyr Cys Leu His 1 5 10 15 Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30 Cys Val Val Gly Tyr Val Gly Glu Arg Cys Gln Phe Arg Asp Leu Arg 35 40 45 Trp Trp Asp Ala Arg Gly Ser Ser Gly Asn Thr Glu Asn Asp Cys Pro 50 55 60 Leu Ser His Glu Ala Tyr Cys Leu His Asp Gly Val Cys Met Tyr Ile 65 70 75 80 Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Val Gly 85 90 95 Glu Arg Cys Gln Phe Arg Asp Leu Arg Trp Trp Asp Ala Arg Gly Gly 100 105 110 Ser Gly Gly Thr Ser Gly Gly Gly Gly Gly Ser Gly Thr Pro Gln Asn 115 120 125 Ile Thr Asp Leu Cys Ala Glu Tyr His Asn Thr Gln Ile His Thr Leu 130 135 140 Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu Ala Asp Lys Arg Glu 145 150 155 160 Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Thr Phe Gln Val Glu Val 165 170 175 Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys Ala Ile Glu Arg Met 180 185 190 Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu Ala Lys Val Glu Lys 195 200 205 Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala Ile Ala Ala Ile Ser 210 215 220 Met Ala Asn 225 <210> 3 <211> 14 <212> PRT <213> Artificial <220> <223> Synthetic <400> 3 Gly Gly Ser Gly Gly Thr Ser Gly Gly Gly Gly Gly Ser Gly 1 5 10 <210> 4 <211> 3 <212> PRT <213> Artificial <220> <223> Synthetic <400> 4 Ser Ser Gly 1 <210> 5 <211> 5 <212> PRT <213> artificial <220> <223> synthesis <400> 5 Ser Ser Gly Gly Gly 1 5 <210> 6 <211> 3 <212> PRT <213> artificial <220> <223> synthesis <400> 6 Ser Gly Gly 1 <210> 7 <211> 4 <212> PRT <213> artificial <220> <223> synthesis <400> 7 Gly Ser Ser Gly 1 <210> 8 <211> 5 <212> PRT <213> artificial <220> <223> synthesis <400> 8 Gly Gly Ser Gly Gly 1 5 <210> 9 <211> 5 <212> PRT <213> artificial <220> <223> synthesis <400> 9 Gly Gly Gly Gly Ser 1 5 <210> 10 <211> 8 <212> PRT <213> artificial <220> <223> synthesis <400> 10 Ser Ser Gly Gly Gly Ser Gly Gly 1 5 <210> 11 <211> 10 <212> PRT <213> artificial <220> <223> synthesis <400> 11 Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 1 5 10 <210> 12 <211> 7 <212> PRT <213> artificial <220> <223> synthesis <400> 12 Thr Ser Gly Gly Gly Ser Gly 1 5 <210> 13 <211> 9 <212> PRT <213> artificial <220> <223> synthesis <400> 13 Thr Ser Gly Gly Gly Gly Ser Gly Gly 1 5 <210> 14 <211> 11 <212> PRT <213> artificial <220> <223> synthesis <400> 14 Ser Ser Gly Gly Gly Ser Gly Gly Ser Ser Gly 1 5 10 <210> 15 <211> 12 <212> PRT <213> artificial <220> <223> synthesis <400> 15 Gly Gly Ser Gly Gly Thr Ser Gly Gly Gly Ser Gly 1 5 10 <210> 16 <211> 12 <212> PRT <213> artificial <220> <223> synthesis <400> 16 Ser Gly Gly Thr Ser Gly Gly Gly Gly Ser Gly Gly 1 5 10 <210> 17 <211> 14 <212> PRT <213> artificial <220> <223> synthesis <400> 17 Gly Gly Ser Gly Gly Thr Ser Gly Gly Gly Gly Ser Gly Gly 1 5 10 <210> 18 <211> 13 <212> PRT <213> artificial <220> <223> synthesis <400> 18 Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Ser Gly 1 5 10 <210> 19 <211> 13 <212> PRT <213> artificial <220> <223> synthesis <400> 19 Ser Ser Gly Gly Gly Ser Gly Gly Ser Ser Gly Gly Gly 1 5 10 <210> 20 <211> 15 <212> PRT <213> Artificial <220> <223> Synthetic <400> 20 Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Ser Gly Gly Gly 1 5 10 15

Claims

1. Use of a BVN22E peptide and an anaplastic lymphoma kinase (ALK) inhibitor in a pharmaceutical composition for treating a patient with non-small cell lung cancer (NSCLC) expressing the EML4-ALK fusion gene, wherein the BVN22E peptide is SEQ ID NO:2, and the ALK inhibitor is selected from crizotinib, alectinib, and brigatinib, wherein the patient has a tumor expressing a mutated form of epidermal growth factor receptor (EGFR), the method comprising: The patient is given the ALK inhibitor and the BVN22E peptide, wherein the ALK inhibitor is administered at a therapeutically effective daily dose and the BVN22E peptide is administered in combination at a therapeutically effective dose that is repeated three times a week, twice or once a week, once every two weeks, once every three weeks, or at least once a month.

2. The use according to claim 1, wherein the ALK inhibitor is administered according to a flexible and proactive regimen, and the BVN22E peptide is administered in combination with a therapeutically effective dose that is repeated three times, twice, or once a week, once every two weeks, once every three weeks, or at least once a month.

3. The use according to claim 1, wherein the ALK inhibitor is administered according to a continuous regimen of an average daily dose ranging from 10 mg to 250 mg, and the BVN22E peptide is co-administered according to a therapeutically effective dose repeated three times, twice, or once a week, once every two weeks, once every three weeks, or at least once a month.

4. The use according to claim 1, wherein the tumor expressing the EGFR mutation has acquired resistance to ALK inhibitor treatment, and wherein the method results in overcoming resistance to ALK inhibitor treatment.

5. The use according to claim 1, wherein the NSCLC expressing the ELM4–ALK fusion gene is a metastatic form of NSCLC.

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