Anti-HDGF antibody methods for overcoming acquired resistance in EGFR-targeted therapy of lung cancer

Abstract

Targeted therapy using osimertinib (an EGFR tyrosine kinase inhibitor), is the first choice for patients with metastatic or recurring lung cancer. However, most patients develop resistance. Because the options after failure of TKls such as osimertinib are limited, improving EGFR-targeted therapy is an unmet need. Here, EGFR mutant patient-derived xenograft tumors responded partially to osimertinib despite near complete inhibition of EGFR activation. Many tumor cells escaped drug killing and regained growth following about 35 days of continuous osimertinib dosing. However, when an antibody to hepatoma-derived growth factor was given concurrently with osimertinib, tumors showed complete or near complete responses with significant prolongation of progressionfree survival of tumor bearing mice. The data support the idea that increased suppression of the AKT / mTOR and MAPK pathways is a mechanism that enhances efficacy of osimertinib when it is combined with an anti-HDGF antibody.

Description

Attorney Docket No. 15024-395PC0 ANTI-HDGF ANTIBODY METHODS FOR OVERCOMING ACQUIRED RESISTANCE IN EGFR-TARGETED THERAPY OF LUNG CANCER CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a United States utility application which claims the benefit of United States provisional application serial no. 63 / 559,451, filed 29 February 2024. The entire contents of the aforementioned application(s) is / are hereby incorporated by reference as if fully set forth herein. GOVERNMENT FUNDING SUPPORT

[0002] This invention was made with government support under grant no. TR003098, awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] This invention relates to the general field of medicine and more specifically to lung cancer therapy. Epidermal growth factor receptor (EGFR) is one of the major oncogenic drivers in non-small cell lung cancer (NSCLC). The present invention relates to methods for attenuating the rise of acquired resistance in targeted therapies for treating lung cancer, using an anti-HGDF antibody in combination with chemotherapy agents. This specification describes a method to overcome or attenuate the emergence of acquired resistance to TKl by systemically blocking hepatoma-derived growth factor (HDGF).

[0005] 2. Background

[0006] Lung cancer remains a major disease and leads in cancer-related death worldwide. Among non-small cell lung cancers, the major form of lung cancer, an activating mutation of EGFR is an important oncogenic driver. In patients of European origin, theAttorney Docket No. 15024-395PC0 incidence rate of NSCLC with EGFR mutation ranges from 10% to 15%. In East Asian patient populations, the incidence rate of NSCLC with EGFR mutation ranges from 30% to 60%. Lung cancer patients with an EGFR mutant are often non-smokers, younger than the median age of average lung cancer patients, and a higher percentage of them are women.

[0007] EGFR is a member of the ERBB family receptor tyrosine kinase vital for the survival, growth, migration and differentiation of epithelial cells. Structurally, EGFR consists of an amino terminal extracellular ligand binding domain linked to a transmembrane region, followed by the kinase domain and a carboxyl terminal regulatory domain. Physiological activation of EGFR requires engagement of EGFR ligands, such as EGF or amphiregulin, to induce receptor dimerization, followed by autophosphorylation of several tyrosine residues in the c-terminal regulatory domain, leading to release of inhibition on the kinase. The activated kinase in turn recruits and initiates the activation of a number of downstream pathways, including Ras-MAPK, Pl3 / AKT / mTOR, that are major drivers of survival and proliferation. Dysregulated EGFR activation in the absence of proper physiological signal thus plays a crucial role in oncogenesis and in maintaining malignancy of lung cancer.

[0008] Gain-of-function mutation is a common mechanism that can result in abnormal EGFR activation. More than 30 activating mutations in EGFR kinase domain have been identified in treatment naive tumors. Among them, exon 19 in-frame deletion and exon 21 L858R mutation account for about 90% of the incidences. Exon 20 in frame deletion and exon 19 mutations account for about 10%. EGFR with exon 19 deletion, L858R and G719X mutations can be inhibited by several classes of EGFR tyrosine kinase inhibitors (TKls) at low concentration. Therefore, these mutations are often referred to as the classical sensitizing EGFR mutation. Inhibition of EGFR activity in NSCLC cells with sensitizing mutation cause growth arrest and cell death, typically at nanomolar concentration in cell culture conditions. Clinically approved EGFR inhibitors for NSCLC treatment include the first-generation competitive inhibitors, such as erlotinib, icontinib, and gefitinib, the second-generation irreversible pan-ERBB family inhibitors such as afatinib and dacomitinib, and the third-generation mutation specific inhibitors such as osimertinib and lazertinib.Attorney Docket No. 15024-395PC0

[0009] EGFR targeted therapy has achieved great clinical success. In NSCLC patients with metastatic or recurring diseases harboring sensitizing EGFR mutation, targeted therapy using these small molecule TKls resulted in significantly increased progression- free survival (PFS) comparing to platinum doublet chemotherapy (10 to 19 months vs.5.4 months), and extension of OS (31 to 38 months vs. 26 months), thus became thepreferred first-line choice for these patients. However, despite the high response rate (80%) and significantly extended PFS, few patients have radiographic complete responses. In most patients, the disease will eventually progress as clones with acquired drug resistance emerge.

[0010] Extensive studies have identified complex mechanism of resistance that cause treatment failure. About 50% of the resistance to the 1st or 2nd generation TKls occur because of secondary T790M mutation in EGFR. Other mechanisms that confer resistance include amplification of EGFR or activation of other receptor tyrosine kinases, such as ERBB2 and MET, mutation of EGFR downstream signaling effectors, and alteration in cell differentiation status, such as SCLC transformation, epithelial-to- mesenchymal transition (EMT), or acquisition of stem-cell like state. Mutation in C797S or loss of T790M can result in resistance to osimertinib in addition to other target-independent alternations. Many of these secondary alternations bestow acquired resistance by reactivating the major prosurvival / proliferative signaling pathways.

[0011] In contrast to the mechanism of resistance, the mechanism that drives the transition from sensitive tumor to resistant tumor is less clearly understood. In most patients, this transition probably happened during the period of PFS that lasts for months among the pool of cells with decreased sensitivity to TKI, often referring as drug tolerant persister cells. In cultured cells harboring sensitizing EGFR mutation, most of the cells can be eliminated by physiologically achievable plasma concentration of TKI within a few days. A long-term exposure to escalating concentration of TKI is typically used to induce acquired resistant clones. Many of these in vitro established resistant clones have MET amplification, demonstrating MET signaling as an effective mechanism to bypass EGFR blockage.

[0012] Cell lines with high level MET expression, such as Hcc827 or PC-9, are theAttorney Docket No. 15024-395PC0 easiest to induce TKI resistant clones in cell culture by MET gene amplification. Clinically, about 5% to 20% of the tumors in patients relapsed on TKI have MET amplification. This suggests other mechanisms probably exist in vivo to promote tolerance and survival of tumor cells during the PFS where steady state plasma concentration can be achieved in 1 to 2 weeks.

[0013] Hepatoma-derived growth factor (HDGF) is a heparin-binding protein first isolated from hepatoma cell line Hu-7 conditioned medium base on its ability to stimulate Swiss 3T3 cell proliferation. It is distinct from hepatocyte growth factor (HGF), the ligand of MET, and there is no report of direct interaction of HDGF with MET. In several tumor cell lines, HDGF has been shown to activate Pl3K / Akt signaling through interaction with cell surface receptor or activate MAPK / ERK signaling by enhancing KRAS expression. Dysregulated over-expression of HDGF had been identified in multiple solid tumors and is associated with inferior clinic outcome of cancer patients.

[0014] There is a strong association of HDGF overexpression with poor performance in patients with NSCLC. Reducing the expression of HDGF in NSCLC cells by siRNA suppresses the tumorigenicity and malignancy of NSCLC cells. Furthermore, anti-HDGF antibody could enhance the treatment in cell line derived or patient tumor derived xenograft tumor models in combination with chemotherapy agents. SUMMARY OF THE INVENTION

[0015] There is a need in the art for treatment modalities for metastatic or recurring lung cancer, or lung cancer generally. The invention described herein thus provides embodiments related to this field. In particular, the invention relates to a method of treating lung cancer in a subject in need, comprising: administering to the subject a therapeutically effective amount of HDGF antibody and an EGFR inhibitor chemotherapeutic agent. Preferably, the EGFR inhibitor chemotherapeutic agent is selected from the group consisting of erlotinib, gefitinib, afatinib, dacomitinib, lazertinib, icotinib, and osimertinib. Most preferably, the EGFR inhibitor chemotherapeutic agent is osimertinib. In preferred embodiments, the HDGF antibody is H3.

[0016] In preferred embodiments, the lung cancer is primary lung cancer, recurrent lungAttorney Docket No. 15024-395PC0 cancer, or metastatic lung cancer. More preferably, the lung cancer is non-small cell lung cancer.

[0017] In other embodiments, the invention relates to a method for overcoming acquired resistance to EGFR-targeted therapy of lung cancer in a subject in need, comprising: administering to the subject a therapeutically effective amount of a therapeutically effective amount of HDGF antibody and an EGFR inhibitor chemotherapeutic agent. More preferably, the EGFR inhibitor chemotherapeutic agent is selected from the group consisting of erlotinib, gefitinib, afatinib, dacomitinib, lazertinib, icotinib, and osimertinib. Most preferably, the EGFR inhibitor chemotherapeutic agent is osimertinib. In preferred embodiments, the HDGF antibody is H3.

[0018] In certain embodiments of the invention, the lung cancer is primary lung cancer, recurrent lung cancer, or metastatic lung cancer. Preferably, the lung cancer is non-small cell lung cancer.

[0019] In certain other embodiments, the invention relates to a pharmaceutical composition comprising an anti-HDGF antibody for the treatment of lung cancer in combination with an EGFR inhibitor chemotherapeutic agent. Preferably, the EGFR inhibitor chemotherapeutic agent is osimeritinib. Preferably, the anti-HDGF antibody is H3. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 is a spider plot which shows the effect of erlotinib on Hcc827 xenograft tumor.

[0021] FIG. 2 is a schematic drawing showing the progression of tumors with treatment.

[0022] Certain embodiments are illustrated by way of example, and not by way of limitation in the figures of the accompanying drawings.

[0023] FIG. 3A is a western blot demonstrating the specific binding of HDGF by humanized HDGF antibody H3. FIG. 3B is a western blot showing the specificity of the H3-reactive bands. FIG. 3C is a surface plasmon resonance (SPR) sensorgram showing the binding affinity of H3 to recombinant HDGF. FIG. 3D is a western blot showing H3 epitope mapping.Attorney Docket No. 15024-395PC0

[0024] FIG. 4A and FIG. 4B are spider plots of tumor volume changes in mice treated with osimertinib or osimertinib plus anti-HDGF antibody. FIG. 4C and FIG. 4D are waterfall plots showing the best percentage change from baseline of each tumor in the two treatment arms. FIG. 4E and FIG. 4F are plots showing the mean percentage change from baseline in the two treatment arms. FIG. 4G and FIG. 4H are Kaplan-Meier plots of the PFS of mice in the two treatment arms.

[0025] FIG. 5A and FIG. 5B are plots showing the tumor volume after treatment with PBS control (FIG. 5A) and anti-HDFG antibody H3 (FIG. 5B).

[0026] FIG. 6A and FIG. 6B are spider plots showing tumor volumes after osimertinib plus H3 treatment and osimertinib monotherapy for comparison, respectively.

[0027] FIG. 7A shows FFPE sections stained with P-EGFR. FIG. 7B shows FFPE sections stained with P-Erk ½ (T202 / Y204), FIG. 7C shows FFPE sections stained with P-Aktl (S473). FIG. 7D shows FFPE sections stained with P-PRAS40 (T245).

[0028] FIG. 7E is a PVDF membrane blot probed with the indicated antibodies.

[0029] FIG. 8A, FIG. 8B, FIG. 8C, and FIG. 8D are photomicrographs showing naïve, osimertinib-treated, and osimertinib plus H3-treated cells.

[0030] FIG. 9A shows the expression of MET and P-MET in TM00219 tumors and Hcc827 cells, examined by western blot. FIG. 9B shows the C797X secondary mutation status, examined by sequencing genomic DNA in TM00219 tumors.

[0031] FIG. 10A shows representative slides (10x) of staining in naïve, osimertinib- treated, or osimertinib-treated plus H2-treated cells. FIG. 10B and FIG. 10C present representative slides from naïve (FIG. 10B) and Osimertinib-treated (FIG. 10C) tumor, viewed under different power. DETAILED DESCRIPTION OF THE INVENTION

[0032] 1. Overview

[0033] The present invention relates to results that indicate that HDGF is involved in promoting acquired resistance to TKI in NSCLC PDX tumors. Blocking HDGF signaling could be a potential means to enhance EGFR targeted therapy of NSCLC.

[0034] 2. DefinitionsAttorney Docket No. 15024-395PC0

[0035] Unless defined otherwise, all technical and scientific terms use herein have the same meaning as commonly understood by one of ordinary skill in the art. Although various methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials used are described below. However, the skilled artisan understands that the methods and materials used and described are examples and may not be the only ones suitable for use in the invention. Moreover, as measurements are subject to inherent variability, any temperature, weight, volume, time interval, pH, salinity, molarity or molality, range, concentration, and any other measurements, quantities, or numerical expressions given herein are intended to be approximate and not exact or critical figures unless expressly stated to the contrary.

[0036] In the foregoing specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. Throughout this specification and the claims, unless the context requires otherwise, the word “comprise” and its variations, such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated item, element or step or group of items, elements or steps but not the inclusion of any other item, element, step, or group of items, elements, or steps. Furthermore, the indefinite article “a” or “an” is meant to indicate one or more of the item, element, or step modified by the article.

[0037] As used herein, the term “about” means plus or minus 20 percent of the recited value so that, for example, “about 0.125” means 0.125 ± 0.025, and “about 1.0” means 1.0 ± 0.2. Notwithstanding that the numerical ranges and parameters setting for the broad scope of the invention are approximations, the numerical values set forth in specific non- limiting examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements at the time of this writing.

[0038] As used herein, the term “lung cancer” refers to any of the malignant diseases that develop in the lungs characterized by uncontrolled growth of abnormal cells in the lungAttorney Docket No. 15024-395PC0 tissue. Lung cancers, include but are not limited to squamous cell carcinoma, adenocarcinoma, large cell carcinoma, small cell carcinoma, mesothelioma, bronchioalveolar carcinoma, and the like, of any stage.

[0039] As used herein, the term “non-small cell lung cancer” refers to the most common type of lung cancer, and includes squamous cell carcinoma, adenocarcinoma, and large cell carcinoma.

[0040] As used herein, the term “HDGF antibody” refers to any antibody that binds specifically to hepatoma-derived growth factor.

[0041] As used herein, the term “H3” refers to histone H3 antibody, which is an anti- HDGF antibody.

[0042] As used herein, the term “EGFR inhibitor chemotherapeutic agent” refers to a cancer chemotherapeutic compound typically used for non-small cell lung cancer that has an epidermal growth factor receptor mutation. This class of chemotherapeutic agents includes afatinib, erlotinib, gefitinib, osimertinib, lapatinib, neratinib, cetkuximab, panitumumab, amivatabmab, and the like.

[0043] As used herein, the term “EGFR-targeted therapy” refers to cancer chemotherapy that targets EGFR.

[0044] As used herein, the term “therapeutically effective amount” refers to an amount of a therapeutic agent that produces a therapeutic effect in a subject, in one dose or in a course or regimen of doses. A “therapeutic effect” is an effect in the subject that produces a physiological effect in the subject that tends to ameliorate a disease condition or symptom thereof.

[0045] As used herein, the term “subject” refers to any animal, and can include simians, humans, avians, felines, canines, equines, rodents, bovines, porcines, ovines, caprines, mammalian farm animals, mammalian sport animals, and mammalian pets. A suitable subject for the invention preferably is a human that is suspected of having, has been diagnosed as having, or is at risk of developing a lung cancer that can be ameliorated, treated or prevented by the treatment discussed and claimed herein. Conditions amenable to treatment by the invention which define an appropriate subject or patient will be discerned easily by the person of skill in the art based on the disclosures herein.Attorney Docket No. 15024-395PC0

[0046] As used herein, the term "administering" to a subject refers to introducing an agent to a subject or contacting the agent with a subject, and can be performed using any of the various methods or delivery systems for administering agents, pharmaceutical compositions or other agents known to those skilled in the art. Modes of administering include, but are not limited to oral administration or intravenous, subcutaneous, intramuscular or intraperitoneal injections, rectal administration by way of suppositories or enema, or local administration directly into or onto a target tissue (such as the lung or bronchi), or administration by any route or method that delivers a therapeutically effective amount of the drug or composition to the cells or tissue to which it is targeted.

[0047] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. "Treatment," includes: (a) preventing the condition or disease or symptom thereof from occurring in a subject which may be predisposed to the condition or disease but has not yet been diagnosed as having it; (b) inhibiting the condition or disease or symptom thereof, such as, arresting its development; and (c) relieving, alleviating or ameliorating the condition or disease or symptom thereof, such as, for example, causing regression of the condition or disease or symptom thereof.

[0048] 3. Summary of Results

[0049] Using EGFRm+ NSCLC PDX tumor models, we have demonstrated in this study that HDGF-mediated reactivation of pro-survival and pro-proliferation signaling pathways could be one of the major mechanisms that protect tumor cells at the early phase of EGFR targeted therapy using TKI. The surviving tumor cells at this stage are mainly composed of drug-tolerant cells which can be sensitized to TKI with concurrent blocking of HDGF, leading to enhanced tumor regression and extended survival of tumor bearing mice. This study suggests targeting drug-tolerant cells at the early stage could be a feasible strategy for improving EGFR targeted therapy in lung cancer.

[0050] Anti-HDGF antibody can significantly enhance the efficacy of EGFR-targeted tyrosine kinase inhibitor in EGFRm+ NSCLC PDX models. In addition, anti-HDGF antibody attenuates reactivation of pro-survival and pro-proliferative signaling pathways in EGFR TKI-treated tumors. These results suggest HDGF mediates a novel bypassAttorney Docket No. 15024-395PC0 pathway in vivo upon EGFR blockade, thus a potential target in NSCLC treatment.

[0051] 4. Embodiments of the Invention

[0052] Dysregulated activation of receptor coupled tyrosine kinases (RTK) are some of the most common oncogenic drivers in NSCLC, with EGFR mutation accounting for the largest percentage of them worldwide. RTK targeted therapy, typically through the use of small molecule TKls, has been used in the treatment of NSCLC in patients with these mutations. EGFR targeted therapy of NSCLC has resulted in significant extension of progression free survival in patients with the classical sensitizing EGFR mutation, mainly Exon 19 deletion and L858R mutation, compared to platinum doublet chemotherapy.

[0053] However, the extension in PFS hardly translated to extension in OS. Most of these patients will eventually develop resistance to TKI, leading to treatment failure. In general, less than 10% of the patients treated with EGFR TKI achieved a radiographic complete response; most patients had only partial response with best change from base line range from -20% to -80% during the PFS period despite the high response rate and long duration of response.

[0054] In contrast to the limited tumor shrinkage in these patients, most NSCLC cells carrying sensitizing EGFR mutation without concurrent bypassing mutation in culture can be eliminated by TKI drugs at a fraction of pharmacologically achievable plasma concentration, typically in the sub-micromolar to nanomolar range within days. The resistant cells were either presented at very low frequency (1 in 105 to 1 in 107) or needed to be induced through escalating concentration of TKI over multiple passages. In addition, xenograft tumors derived from cell lines sensitive to TKI in vitro, such as Hcc827, are partial responsive to oral TKI treatment with BCB about -70% to -80%. This suggests that in vivo environment can promote tumor cells survival when they are exposed to TKI. See FIG. 1. The surviving tumor cells, often called "drug-tolerant persister" (DTP), possess transient, reversible drug tolerance. The ability of tumor cells to adapt rapidly to the drug-tolerant state upon initiation of TKI administration may be important for the survival of tumor cells during the early stage of EGFR-targeted therapy. Subsequent evolution and acquisition of resistant drivers within the drug-tolerant cells ultimately can give rise to bona fide resistant clones, leading to disease progression. TheAttorney Docket No. 15024-395PC0 incomplete response and the duration of PFS in many patients undergoing TKI therapy suggest that this drug-tolerance phase may exist prior to overt disease progression.

[0055] Recently, there has been heightened interest in understanding the mechanisms and vulnerabilities of these cells. Among the commonly recognized mechanisms, acquisition of sternness is often associated with tolerance to chemotherapy, radiation therapy and targeted therapy. Activation of RTK bypassing signaling pathways is frequently observed in drug-tolerant persisters due to the highly convergent nature of RTK signaling, such as in other ERBB family of kinases, MET and AXL.

[0056] Secondary mutations or amplifications of these RTKs found in relapsed tumor demonstrated their abnormal activation could functionally substitute EGFR activation. Therefore, co-targeting these RTKs was considered as an approach to break tumor cell tolerance, either by using inhibitors with a wilder specificity (e.g., afatinib) or a combination of inhibitors (e.g., EGFR TKI plus crizotinib, or MET inhibitors). So far, these strategies have achieved varying degrees of success. Afatinib, a pan-ERBB inhibitor, has a similar efficacy profile as the first generation of TKls. Dacomitinib, another pan-ERBB inhibitor had extended PFS and some extension in OS. Whereas amivantamab, a bi-specific antibody targeting EGFR and MET, has shown significantly improved efficacy in combination with lazertinib, a third-generation brain-permeable EGFR TKI, in the first-line treatment of EGFR-mutant non-small-cell lung cancer. These results suggest complex mechanisms may exist to promote tumor cell tolerance and drive the transition from TKI sensitive to resistant.

[0057] In this study, we investigated the role of HDGF in promoting tumor tolerance to osimertinib using two PDX tumor models of NSCLC: TM00199 and TM00219. These models were derived from patients who had progressed on single-agent or combination erlotinib treatment. TM00199 has the 0L858R mutation, modest EGFR amplification and MET deletion, while TM00219 has an exon 19 deletion and the T790M mutation and very low level of MET amplification. See Table 1, below. We showed in this study that the xenograft tumors from both models are suspectable to EGFR inhibition. Oral osimertinib administration produced rapid tumor shrinkage in both PDX tumor models.

[0058] On the molecular level, osimertinib induced profound inhibition of EGFR phosphorylation at Y1068 and suppression of downstream phosphorylation inAttorney Docket No. 15024-395PC0 AKT / MTOR and MAPK pathways. These pathways represent the major proliferative signaling pathways that are essential for cell survival and when inhibited together can lead to rapid cell death. Secondary activating mutations in these pathways are often the cause of resistance to various anti-cancer therapies. The observed swift inhibition of phosphorylation after osimertinib administration in these pathways explained the rapid tumor shrinkage.

[0059] However, the response of these PDX tumors to osimertinib monotherapy was incomplete, with maximum tumor shrinkage ranging from 50% to 70% from the baseline that usually achieved around 30 days after initiation of treatment, followed by tumor progression. Upon examining of the cell signaling pathways, even at the early stage of treatment with about 30% tumor shrinkage from baseline, there was significant recovery of phosphorylation in AKT / MTOR and MAPK pathways. In samples serially collected from the start of osimertinib treatment, near maximum inhibition of EGFR Y1068 and downstream phosphorylation happened in as short a time as 6 hours, yet recovery of phosphorylation in these two pathways can be detected as early as 24 hours. The swift change from initial suppression to partial recovery seen in both IHC and western analysis suggest factors other than drug penetration or emergence of a novel mutation is likely responsible for this phenomenon. In studies of others using cell line and patient-derived xenograft tumors, reactivation of EGFR downstream pathways was also observed.

[0060] Surprisingly, when an anti-HDGF antibody was given concurrently at the start of osimertinib treatment, tumor shrinkage was able to continue until over 98% of the tumor mass disappeared in most of the tumors. On average, the progression-free time could last four to five times longer than with osimertinib monotherapy. This suggests that most of the tumor cells in the persistent tissue prior to progression were only transiently tolerant to TKI, as opposed to stable resistance. This tolerance can be broken by an anti-HOGF antibody. IHC staining of tumors harvested around 10 days after initiation of treatment indicates there were significantly lower levels of activating phosphorylation of the AKT / MTOR and MAPK pathways in the combination therapy arms compared to in the osimertinib monotherapy arm. This suggests that HDGF-driven signaling participated in reactivating the major prosurvival pathways that confer tolerance to TKI. Anti-HDGF antibody could attenuate reactivation of these pathways during osimertinib treatment inAttorney Docket No. 15024-395PC0 the tumor, thus enhancing the efficacy of EGFR targeted therapy. However, when administrated alone, anti-HDGF antibody did not affect the growth of osimertinib-naive or post-progression tumors, suggesting that HDGF signaling is relatively weak compared with the dominant oncogenic driver.

[0061] HDGF was originally identified as a secreted mitogen for 3T3 cells from the conditioned media of human hepatoma cell line Huh-7. Since then, the mitogenic effects of HDGF were reported in other cells. The expression of HDGF is developmentally regulated. The highest expression of HDGF is in the early embryonic stage, subsiding by the time of birth. Although the expression of HDGF in adult tissue remains medium to medium-high, injury could trigger an increase in HDGF expression.

[0062] HDGF protein, identified as a mitogen for several cell types, contains two nuclear localization signals. In most cell types, HDGF is predominately localized to the nucleus, where it likely functions as transcription factor or participates in RNA biogenesis. An interesting exception is HDGF expressed in neurons which show predominantly perinuclear distribution. In this study, we observed that in treatment naive NSCLC PDX tumor, the intensity of HDGF staining in tumor cells ranged from negative to strong nuclear positive with weak cytoplasmic staining. In osimertinib-treated tumors however, all the surviving tumor cells showed strong nuclear staining, suggesting that high level of HDGF expression is associated with increase tolerance. In another study, biopsy samples of patients with NSCLC treated with EGFR TKI demonstrated a considerable increase in HDGF staining intensity after relapse.

[0063] Furthermore, HDGF expression in cultured cells with EGFR mutation and in xenograft tumors derived from them significantly increased after gefitinib or osimertinib treatment. These findings suggest that higher levels of HDGF expression are associated with increased TKI tolerance. Since HDGF has been shown to promote the expansion of hematopoietic stem cells or cancer stem cells, it is possible that tumor cells with higher HDGF expression had more stem cell-like features and thus were more tolerant to TKI. This is particularly interesting considering that chemotherapy could extend the benefit of osimertinib, and stem-like cancer cells tend to be more resistant to chemotherapy. Since anti-HDGF antibody did not significantly alter the staining intensity in the osimertinibAttorney Docket No. 15024-395PC0 plus H3 treatment arm compared to the osimertinib only arm, the origin of this increased HDGF nuclear staining is likely endogenous. Targeting HDGF could potentially suppress resistance to chemotherapy too and further enhance the benefit of osimertinib plus chemotherapy. In a previous study, the administration of an anti-HDGF antibody resulted in reduced stem cell features in tumors and enhanced the efficacy of combination chemotherapy in NSCLC tumors.

[0064] As a protein lacking classical secretion signals, HDGF is readily detected in cell culture media of various cancer cells, including NSCLC cell cultures. The medium level of HDGF in abnormal fibroblast culture is also elevated. It is not entirely clear how HDGF enters the extracellular media. Studies have suggested that the N-terminal sequence is involved in HDGF secretion. It is possible that stress or apoptosis of cells can increase the level of HDGF released into the media, a condition that is relevant to cancer therapy. In patients with NSCLC treated with gefitinib or osimertinib, there were statistically significant increases in plasma concentrations of HDGF.

[0065] Exogenous HDGF could bind to cell surface receptor and activate several intracellular signaling pathways, including MAPK / Erk and Pl3K / AKT pathways. In hepatoma cells, nucleolin was reported to function as a cell surface HDGF receptor in mediating activation of Pl3 / AKT signaling. In cultured NSCLC cells or xenograft tumor, the expression of HDGF is positively correlated with the activation of these pathways and the resistance to EGFR TKI; knocking down HDGF diminishes the activation of these pathways and sensitizes the cells to TKI inhibition.

[0066] Beside the malignant cells, tumors also contain many non-malignant cells (such as vasculature cels, mesenchymal-derive cells such as fibroblasts, and immune cells in immune-competent animals and human patients) that constitute the tumor microenvironment. In immune deficient mice, this would mainly include vascular cells and mesenchymal-derived cells such as cancer associated fibroblast (CAF). In immune competent animals and human patients, immune cells are also an important component of tumor. These cells interact with each other through secreted growth factors, cytokines or surface displayed ligands and their receptors. HDGF released by tumor cells could signal to the non-malignant cells in tumor micro-environment to promote the survival and malignancy of tumor cells.Attorney Docket No. 15024-395PC0

[0067] Taken together, the present study demonstrates that during the early stage of EGFR targeted therapy, some tumor cells could gain tolerance to TKI without acquisition of new mutation or activating MET in a process likely mediated by HDGF signaling, either through tumor cell surface receptor or through interaction with non-malignant cells in tumor micro-environment. See FIG. 2. Emergence of robust bona fide drug-resistant driver alterations from this pool of DTP is likely the reason for progression while continuing on EGFR TKI. The drug tolerant cells thus serve as a link in the transition of tumor from sensitive to resistant. In this process, HDGF plays a role by inducing tumor tolerance, partly through the activation of MAPK / ERK and PI3K / AKT pathways. Blocking HDGF promotes tumor regression and significantly extends survival in tumor- bearing mice.

[0068] Although HDGF-dependent osimertinib-tolerant cells constitute 20% to 30% of the mass in tumors of the osimertinib arm prior to progression, analysis of TM00219 tumors collected approximately 13 days after treatment initiation revealed a low level of MET expression compared with Hcc827 and an insignificant level of P-MET expression. Notably, there were no meaningful differences in the expression of MET and P-MET between osimertinib-naive and osimertinib-treated tumors. Furthermore, no secondary mutations at C797 were detected after sequencing EGFR exon 20 in these tumors. These data suggest that MET amplification / activation and EGFR C797X secondary mutation, which are commonly associated with acquired resistance to osimertinib in post- progression tumor in human patients, were unlikely the primary mechanism of osimertinib tolerance at the early stage of osimertinib treatment.

[0069] Reducing the size of the DTP pool could extend the PFS. In this process, HDGF plays a crucial role by inducing tumor tolerance through activation of MAPK / ERK and Pl3K / AKT pathways. The studies presented here also exposed the complexity of tumor cell signaling under TKI inhibition. For example, near complete inhibition of EGFR Y1068 phosphorylation was observed in the first 72 hours of osimertinib treatment. Considerable phosphorylation at Y1068 was observed in samples collected at 10 to 13 days with about 30% tumor shrinkage. Surprisingly, however this increase in Y1068 phosphorylation did not consistently correlate with downstream activation in the MAPK / ERK and PI3K / AKT pathways. This phenomenon may be explained by cross-Attorney Docket No. 15024-395PC0 talk between EGFR and other kinases. Furthermore, anti-HDGF antibody could not completely prevent the emergence of osimertinib resistance, suggesting that other bypass pathways could co-exist.

[0070] In conclusion, the present studies demonstrate that tumor tolerance to TKIs can be a crucial mechanism that contributes to incomplete responses during the early phase of EGFR-targeted therapy. HDGF plays a role in this process. Concurrent inhibition of HDGF with the initiation of TKI treatment can resensitize drug-tolerant cells to TKI, resulting in enhanced tumor regression and delayed disease progression. These findings suggest that targeting drug tolerant cells early in the treatment process is a promising strategy for improving EGFR-targeted therapy in NSCLC.

[0071] Treatment regimens for use with the invention include a single administration or a course of administrations lasting two or more days, including a week, two weeks, several weeks, a month, two months, several months, a year, or more, including administration for the remainder of the subject’s life. The regimen can include multiple doses per day, one dose per day or per week, for example, or a long infusion administration lasting for an hour, multiple hours, a full day, or longer.

[0072] Dosage amounts per administration include any amount determined by the practitioner, and will depend on the size of the subject to be treated, the state of the health of the subject, the route of administration, the condition to be treated or prevented, and the like. In general, it is contemplated that for the majority of subjects, a dose in the range of about 0.01 mg / kg to about 100 mg / kg is suitable, preferably about 0.1 mg / kg to about 50 mg / kg, more preferably about 0.1 mg / kg to about 10 mg / kg, and most preferably about 0.2 mg / kg to about 5 mg / kg are useful. This dose can be administered weekly, daily, or multiple times per day or according to any regimen as determined by the practitioner. A dose of about 0.1 mg, 0.2 mg, 0.25 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 20 mg, 40 mg, 80 mg, 100 mg, 250 mg, 500 mg, or 1000 mg can be administered.

[0073] 5. Examples

[0074] This invention is not limited to the particular processes, compounds, compositions, or methods described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only,Attorney Docket No. 15024-395PC0 and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, systems, compounds, compositions and materials are now described.

[0075] Example 1: General Methods and Materials.

[0076] A. Drugs and Antibodies

[0077] Osimertinib methanesulfonate (>99%) was obtained from LC LaboratoriesTM. HDGF was purified according to known methods. Briefly, the cDNA sequence encoding human HDGF was amplified and cloned into pET7 expression vector. Recombinant GST-HDGF fusion protein was expressed in BL21(DE3) and purified by conventional chromatographic methods.

[0078] The development of humanized anti-HDGF antibody has been described previously. Briefly, cDNA of anti-HDGF clone H3 was prepared from hybridoma RNA. The variable domain of the murine antibody was amplified using a NovagenTMmouse lg-Primer set, then cloned into pcDNA5 and sequenced. The complementary determinant regions (CDRs) were identified.

[0079] To construct the humanized anti-HDGF antibody, the CDRs of murine antibody H3 were grafted onto a human lgG1 framework, synthesized and cloned into a mammalian expression vector driven by CMV promoter (lnvitrogenTM). Plasmids encoding the heavy and light chains of the humanized antibody were used to co- transfect Expi293 cells transiently.

[0080] Recombinant anti-HDGF antibody H3 was purified by protein G affinity chromatography. The purified antibody was furthered passed through a high-capacity endotoxin removal resin (ThermoScientificTM) to reduce the endotoxin level to < 0.02 EU / mg. The binding affinity of the humanized antibody to recombinant HDGF was determined by surface plasmon resonance (SPR) using GST-HDGF as analyte.

[0081] Antibodies to the following were obtained from Cell Signaling Technology: phospho-EGFR (Tyr1068; #3777, RRID: AB_2096270 and #2234, RRID: AB_331701), phospho-MEK1 / 2 (Ser217 / 221; #9121, RRID: AB_331648), MEK1 / 2 (#9122, RRID:Attorney Docket No. 15024-395PC0 AB_823567), phospho-p44 / 42 MAPK (Erk1 / 2; Thr202 / Tyr204; #9101, RRID: AB_331646 and #4307, RRID: AB_2315112), p44 / 42 MAPK (Erk1 / 2; #9102, RRID: AB_330744), phospho-Akt (Ser473; #4060, RRID: AB_2315049 and #9271, RRID: AB_329825), Akt (#9272, RRID: AB_329827), phospho-PRAS40 (Thr246; #2997, RRID: AB_2258110), PRAS40 (#2610, RRID: AB_916206), phospho-p70 S6 kinase (Thr389; #9234, RRID: AB_2269803), p70 S6 kinase (#2708, RRID: AB_390722), phospho-4EBP1 (Thr37 / 46; #2855, RRID: AB_560835), 4EBP1 (#9644, RRID: AB_2097841), Met (#8198, RRID: AB_10858224), and phospho-Met (Tyr1234 / 1235; #3077, RRID: AB_2143884). Anti-EGFR was obtained from Santa Cruz Biotechnology (Cat#sc-03, RRID: AB_631420). Anti-β-actin was obtained from Sigma (Cat#A1978, RRID: AB_476692).

[0082] B. Expression of Recombinant HDGF Protein for Epitope Mapping.

[0083] The DNA sequence encoding GST-HDGF fusion proteins and a series of C- terminal truncations were cloned into pBiEx-1 (NovagenTM), expressed in BL21(DE3) cells, fractionated by SDS-PAGE, and blotted and probed with recombinant anti-HDGF antibody H3.

[0084] C. Generation of HDGF-knockout Cells

[0085] HDGF single-guide RNA CRISPR / Cas9-targeting constructs (Applied Biological Materials Inc.TM) were packaged in lentivirus and used to transduce HEK293 cells. The sequences targeted were targets 1 to 3: GTC GCG ATC CAA CCG GCA GA (SEQ ID NO:1); targets 2 to 159: TGG CCT CCA CTC ACG TCT CG (SEQ ID NO:2); and targets 3 to 195: TCT CCT TGG ATT CCT CGT AA (SEQ ID NO:3). The transduced cells were selected using puromycin to generate polyclonal (pooled) cell lines.

[0086] D. Tumor Models and Dosing.

[0087] NSCLC PDX tumor models, TM00219 and TM00199 (Table 1), were obtained from Jackson LaboratoryTMand propagated per specified in immunodeficient NOD.Cg- Prkdcscidll2rgtm1Wjl / SzJ mice. The initial engraftment by the vender, designated as PO, was harvested and cryopreserved. Low passage tumors (P2 to P4) were used to prepare treatment cohorts. Briefly, a fresh tumor was cut into approximately 2-mm pieces and implanted into the flank of recipient animal. Female mice were used for both PDX models to match the sex with the original human donors. When tumors reached 250 mm3Attorney Docket No. 15024-395PC0 to 300 mm3, the mice were randomized into two arms to receive osimertinib monotherapy or osimertinib plus H3 treatments. Osimertinib was given at 10 mg / kg per os in a PEG / TW80 suspension. The dosing was repeated every 24 hours for five days a week. H3 was given intraperitoneally every three days at 13 mg / kg. Tumor sizes were measured with a caliper every three days and the approximate volume was calculated by the following formula: V = L x 2W x H.

[0088] Table 1. Selected Characteristics of PDX Models. Model Ploidy EGFR EGFRCNVMETCNVTumor Prior Donor Mutation Type Treatment? Sex

[0089] E. IHC staining and evaluation.

[0090] Tumors dissected from mice were fixed in 4% formaldehyde-PBS, embedded in paraffin, cut, and mounted on SuperfrostTMplus slides. The sections were deparaffined in HistoclearTMand an alcohol gradient. Antigen retrieval was performed in Tris-EDTA buffer, pH 8.5. Duplicate sections on one slide were stained with antibody diluted in SignalStainTMantibody diluent at 4 °C overnight. Chromogenic visualizations were performed using a VectorTMElite ABC kit (Vector LabsTM) with DAB substrate with hematoxylin counterstain. The mounted slides were scanned using OlympusTMVS 120 or ApriaTMCS2 Pathology Slice Scanner. The staining intensity was semi-quantitatively evaluated by two team members and a trained pathologist under an OlyVIATMor an lmageScopeTMslide viewer where each tumor section was giving a score as follows: negative or weak (1), moderate (2), and strong (3). Images were not adjusted or edited in any way.

[0091] F. Western analysis.

[0092] Fresh tumors were snap frozen in liquid nitrogen and stored in -80 °C until processed. Soluble proteins were extracted by homogenizing about 25 to 50 mg of tumor tissue in 6 volumes of lysis buffer containing 1% Triton X-100 supplemented withAttorney Docket No. 15024-395PC0 protease and phosphatase inhibitors. Clarified tissue lysates were heat denatured in SOS sample buffer, separated on 4% - 12% Bis-Tris gel (lnvitrogenTM), blotted to nitrocellulose or PVDF membrane and probed sequentially with 5 to 7 antibodies. When staining with antibodies with similar molecular weight, the membrane was stripped with RestoreTMWestern Blot Stripping buffer (ThermoScientificTM) between staining. Antibody reactivity was detected using electrochemiluminescence substrate and exposure to X-ray film. The films were scanned using the CanoScanTM9000F, and the intensity of the bands was determined using Image JTM. Images were not adjusted or edited in any way except automatic adjustments made by the scanner.

[0093] G. EGFR exon 20 sequence analysis.

[0094] Genomic DNA was purified from pellet after tumor protein extraction with proteinase K digestion and silica membrane binding. A 1,320-bp fragment in EGFR gene flanking exon 20 was amplified using high-fidelity PCR and primers in introns 19 to 20 (50-CACAGCACAGAGAGACCACT-30 (SEQ ID NO:4)) and introns 20 to 21 (50- CAAGGTAAGCAAGCCAGGCC-30 (SEQ ID NO:5)) and purified and sequenced using a primer in exon 20 (50-GAAGCCTACGTGATGGCCA-30 (SEQ ID NO:6)).

[0095] H. Cell lines

[0096] HEK293 (CRL-1573) and Hcc827 (CRL-2868) were obtained from ATCC. Expi293F was part of Invitrogen Cat#A14635. The cell lines were expanded upon arrival, and multiple aliquots of low-passage cells (P2–P3) were cryopreserved. Upon recovery, the cells were cultured in media containing tiamulin fumarate, followed by minocycline (Roche, BM-Cyclin). All cells were tested negative for Mycoplasma contamination monthly using the MycoFluorTMKit (InvitrogenTM). Cells within passage 6 were used for all experiments.

[0097] I. Statistical Analysis. Differences in progression-free survival were analyzed by Kaplan Meier (KM) survival curves using an online analysis tool (SRplot, bioinformatics.com.cn / srplot). Significance was tested by log-rank test. Quantitative data of IHC and Western blots were analyzed by t- test (one tailed homoscedastic).

[0098] Example 2: Humanized Anti-HDGF Antibody Retains High Affinity HDGF Binding.Attorney Docket No. 15024-395PC0

[0099] Western blot data shown in FIG. 3 demonstrates the specific binding of HDGF by humanized anti-HDGF antibody H3. Recombinant H3 in culture medium was immobilized on protein G agarose beads and used to capture HDGF in HEK293 lysate. Lane 1, H3 loaded-beads; lane 2, H3-beads plus HEK292 lysate; lane 3, captured protein probed with rabbit anti–HDGF antibody T221; lane 4 the same blot was stripped and probed with goat anti-human lgG antibody. The position of lgG heavy chain, light chain and HDGF were marked. HDGF migrates at 37–42 kDa, larger than the calculated molecular weight of 26.7 kDa. Thus, the recombinant humanized anti-HDGF antibody H3 binds HDGF in its native form, as shown in its ability to capture HDGF from HEK293 cell lysate (see FIG. 3A). The captured HDGF migrated on SDS-PAGE with an apparent molecular weight of about 37 kDa (major) and 42 kDa (minor).

[0100] The specificity of the H3–HDGF interaction was further validated by co-reduction in the intensity of H3-reactive bands in Western blot of HEK293 cells with CRISPR- Cas9–mediated HDGF knockout. See FIG. 3B. This figure shows western blots of HEK293 parental (ctrl) or pooled HDGF knockout (T1, T2, and T3) cells probed with H3 or T221. The intensity of HDGF staining by H3 or T221 is reduced in the T1 and T2 knockout cells.

[0101] The surface plasmon resonance (SPR) sensorgram shows the binding affinity of H3 to recombinant HDGF. See FIG. 3C. The binding affinity of H3 to recombinant HDGF was determined by SPR on BiacoreTM3000. The recombinant antibody wasimmobilized on BiacoreTM sensor chip and GST-HDGF w a s expressed in E. coli foruse as analyte. The measured dissociation constant K0for H3-HDGF binding is 6.14 nM. Thus, the H3–HDGF interaction is strong.

[0102] FIG. 3D presents data on H3 epitope mapping. Bacteria expressing GST-HDGF fusion proteins were lysed, separated by SDS-PAGE, and probed with H3, followed by A2, an antibody that recognizes an epitope in HDGF PWWP domain. The full-length GST-HDGF fusion protein migrated with a molecular weight of 75 kDa (lane m), larger than the predicted size of 52 kDa. The GSTHDGF99 fusion protein (lane a) migrated at the predicted size of 37 kDa. The transition of H3 reactivity happened between constructs ending at residues 160 and 170, indicating that the H3 recognition epitope is in this region.Attorney Docket No. 15024-395PC0

[0103] By probing a set of GST-HDGF fusion proteins with a sequential C-terminal deletion, the epitope recognized by H3 was mapped to between amino acid residue 160 and 170 in a region predominated by an unfolded sequence. See Table 2, below. This stretch of sequences is highly homologous between the human and mouse. See Table 3, below.

[0104] Table 2. Epitope Results. Lane Ending H3 residue reactivity

[0105] Table 3. HDGF Mouse / Human homology. SEQ ID NO

[0106] Example 3: Enhanced Response of EGFRm+ NSCLC PDX Tumors to Osimertinib plus Anti-HDGF Antibody.

[0107] For this study, the response of EGFRm+ NSCLC tumor to osimertinib was evaluated in two established PDX models, TM00199 (L858R) and TM00219 (T790M, E746_A750del). Whether anti-HDGF antibody can alter the tumor response to osimertinib was studied. Mice bearing established TM00219 tumors (257 mm3to 427Attorney Docket No. 15024-395PC0 mm3) were administered the humanized anti-HDGF antibody, hH3, at 13 mg / kg by intraperitoneal injection concurrently with the start of oral osimertinib dosing, and subsequently maintained at twice per week until tumor relapse or up to 200 days.

[0108] Mice with established NSCLC PDX tumors were randomized into two arms (n = 6 each) to receive osimertinib (AZD, 10 mg / kg per os) or osimertinib plus anti-HDGF antibody (13 mg / kg intraperitoneally, twice per week). Tumor volumes were measured twice a week.

[0109] In established TM00219 tumors (250 mm3to 329 mm3, continuous osimertinib dosing at 10 mg / kg induced rapid tumor regression. See FIG. 4A and FIG. 4B, which present spider plots of tumor volume changes in mice treated with osimertinib or osimertinib plus anti-HDGF antibody. However, the response was only partial, with the best percent change from baseline (BCB) ranging from 53% to 72% in TM00219 and 43% to 70% in TM000199. See FIG. 4C and FIG. 4D, which present waterfall plots of the best percent change from baseline of each tumor in the two treatment arms. Thus, osimertinib dosing of mice bearing TM00219 (250–329 mm3) or TM00199 (228–410 mm3) tumors induced rapid tumor regression.

[0110] Continuous dosing of established TM00199 tumor (228 mm3to 410 mm3) with osimertinib also induced rapid but partial tumor regression (see FIG. 4E) with BCB ranging from 43% to 70% (see FIG. 4F). FIG. 4E and FIG. 4F show the mean percent change from baseline in the two treatment arms. Asterisks mark the data point in which there is a significant difference between the two treatment arms. FIG. 4G and FIG. 4H are Kaplan–Meier plot of the PFS of mice in the two treatment arms, showing progression-free survival. The graphs in FIG. 4 show the results of TM00219 (FIG. 4A. FIG. 4C, FIG. 4E and FIG. 4G) and TM00199 (FIG. 4B, FIG. 4D, FIG, 4F, and FIG. 4H).

[0111] In both models, tumor growth resumed after about 24-31 days (median 25.8 days) for TM00219, or 17-38 days (median 28 days) for TM00199 even with continued osimertinib dosing.

[0112] Similarly, xenograft tumors derived from Hcc827, an EGFR-mutant cell line sensitive to erlotinib in vitro, also had incomplete response to erlotinib in vivo. See FIG. 1. For this study, Hcc827 cell (1 x 106) was subcutaneously injected on the flank of anAttorney Docket No. 15024-395PC0 NSG mouse to produce xenograft tumor. Once the tumor was established, the mouse bearing the tumor was given erlotinib hydrochloride (LC LaboratoryTM, E4007) daily at 100 mg / kg per os. Tumor volumes were measured every three days. FIG. 1 shows the tumor volume change over the treatment course.

[0113] To summarize these data, the combination treatment induced rapid tumor regression (FIG. 4A) at a pace similar to or slightly faster than osimertinib monotherapy as shown in the mean percent change from baseline in tumor volume (FIG. 4E). More importantly, tumor regression continued to near completion with the BCB reaching 96% to 99.8% (FIG. 4C). Similarly, in established TM00199 tumor (229 mm3to 410 mm3, the osimertinib plus hH3 combination also induced rapid tumor regression with BCB ranging from 91% to 99% (FIG. 4B and FIG. 4D) at a pace of tumor regression similar to that of osimertinib monotherapy (FIG. 4F). In both models, there were significant extensions of progression-free survival (PFS) ranging from 114 to 220 days (median 128 days) for TM00219, and 98 to 112 days (median 103 days) for TM00199. The difference in the probability of PFS in the two treatment arms is significant (p = 0.0004, FIG. 4G and FIG. 4H).

[0114] Example 4: H3 Alone has no Effect on the Growth of Osimertinib-Naive PDX Tumors.

[0115] Mice bearing an established TM00219 PDX tumor (49 to 106 mm3) were randomized to receive PBS or anti-HDGF antibody H3 (13 mg / kg, intraperitoneally, b.i.w.). Tumor volumes were measured twice a week and plotted as a spider plot of tumor volume changes over the treatment course. FIG. 5A and FIG. 5B show the results for the PBS arm and the H3 arm, respectively. H3 alone had no effect on the growth of osimertinib naive PDX tumors or tumor progressed on osimertinib monotherapy.

[0116] Example 5: Effect of Anti-HDGF Antibody H3 on Post-progression Tumor.

[0117] Mice bearing an established TM00219 PDX tumor (300 to 350 mm3, n = 4) were treated with osimertinib (10 mg / kg per os). After progression, anti-HDGF antibody H3 (13 mg / kg, intraperitoneally, b.i.w.) was added to the treatment regimen in combination with osimertinib. See FIG. 6. Arrows indicate the approximate time of antibodyAttorney Docket No. 15024-395PC0 administration. The tumor volumes (measured twice a week) were plotted in FIG. 6A. The effect of osimertinib monotherapy was shown in FIG. 6B for comparison (reproduced from FIG. 4).

[0118] Example 6: Reactivation of mTOR and MAPK Pathways in PDX Tumors Receiving Osimertinib Monotherapy.

[0119] To identify the potential mechanism leading to the limited response in osimertinib monotherapy, the early changes in the major pro-proliferative signaling pathways downstream of EGFR were investigated in serially collected TM00219 tumors after initiation of osimertinib administration by IHC and western blotting. After oral osimertinib administration, significant suppression of EGFR Y1068 phosphorylation was observed by 6 hours in IHC stained tumor tissue (see FIG. 7A).

[0120] In this study, the dynamics of EGFR signaling was examined during early stage of osimertinib treatment in TM00219 PDX tumor. Mice bearing TM00219 tumors were treated with osimertinib (10 mg / kg p.o., every 24 hours) for up to 72 hours. Duplicate tumor samples were serially collected at indicated time interval. Each tumor was split far formalin-fixed paraffin-embedding (FFPE) and snap freezing in liquid nitrogen. Duplicate, formalin-fixed FFPE sections were stained with P-EGFR (#1058) (see FIG. 7A), P-Erk 1 / 2 (T202 / Y204) (FIG. 7B), P-Aktl (S473) (FIG. 7C), P-PRAS40 (T245) (FIG. 7D). Stained slides were scanned with OlympusTMVS120 or AperioTMCS2 Pathology Slice Scanner AT 40x resolution. Representative images were extracted from OlyVIA at 4x view (see FIG. 7A, FIG. 7B, and FIG. 7D) or from lmagescopeTMat 8x view see FIG. 7B and FIG. 7E). Tumor proteins were extracted from snap frozen tissue (10 ug / lane), then separated by PAGE, blotted on PVDF membrane, and probed with the indicated antibodies (see FIG. 7E).

[0121] In the downstream Akt / mTOR and MAPK pathways, phosphorylation of Erk 1 / 2, Akt, and PRAS40, also showed significant suppression by 16 hours in IHC-stained tumor samples. However, 24 to 30 hours after initiation of osimertinib treatment, there was an apparent increase in phosphorylation of these proteins in IHC stained tumor samples (see FIG. 7B, FIG. 7C and FIG. 7D). In several tumors, positive phosphoprotein staining was most prominent in small loci. At the same time,Attorney Docket No. 15024-395PC0 phosphorylation of EGFR Y1068 remained at low level. See FIG. 7A.

[0122] Western blot examination of tumor proteins extracted from the corresponding serially collected samples confirmed suppression of EGFR Y1068 phosphorylation starting from 6 hours after osimertinib dosing and was undetectable by 72 hours (FIG. 7E). The phosphorylation of MEK 1 / 2, Erk 1 / 2 on the Ras / MAPK pathway and Akt, PRAS40, and p70s6k of the Akt / mTOR pathway began declining at 6-hour post osimertinib dosing, remaining low at 16 hours. However, there was a significant increase of phosphorylation of these proteins by western blot beginning at 24 hours, in line with the increase in phosphorylation observed in IHC-stained tumor tissues (FIG. 7E). During this period, the total level of these proteins likely remains unchanged as exemplified by EGFR, Akt, and 4EBP1. FIG. 7F.

[0123] Example 7: Anti-HDGF Antibody Attenuates Reactivation of Akt / mTOR and MAPK Pathways in PDX Tumors Treated with Osimertinib.

[0124] We next examined the effect of anti-HDGF antibody on these signaling pathways in tumor treated with osimertinib for 10 to 13 days. At this point of treatment, there was approximately 30% tumor size reduction.

[0125] Mice with established TM00219 PDX tumor were randomized into two arms (n = 6 each) to receive osimertinib (10 mg / kg p.o.) or osimertinib plus anti-HDGF antibody H3 (13 mg / kg intraperitoneally, twice per week). After 10 days of treatment, tumors were collected from euthanized animals, divided for FFPE and snap freeze in liquid nitrogen. See FIG. 8A through FIG. 8D. IHC staining of tumor FFPE sections using P- Erkl / 2 (T202 / Y204) (FIG. 8A), P-Aktl (5473) (FIG. 8B), P-PRAS40 (T246) (FIG. 8C), and P-4EBP1 (T37 / 46) (FIG. 8D). Showing representative field of stained tumor at 4x view on OlyVIATM(P-ERK and P-PRAS40), or 8x view on lmagescopeTM(P-Akt and P- 4EBP1). The staining intensity of tumors in the two treatment arms were scored and plotted as Cleveland dot graph. Naive tumor staining is also shown as comparison in the figures. Asterisks mark the data points where there is a significant difference between the treatment arms.

[0126] In IHC staining of phosphorylated Erk1 / 2, Akt, and PRAS40, there were regions of strong positive staining in tumors from osimertinib monotherapy arm. InAttorney Docket No. 15024-395PC0 contrast, the staining intensities of these phosphorylated proteins in tumors from osimertinib plus anti-HDGF antibody combination treatment arm were generally much weaker. See FIG. 8A through FIG. 8D. The difference in the staining intensity of P- Erk1 / 2, P-Akt, and P-PRAS40 between the two arms was significant as shown in the Cleveland dot graph.

[0127] We further examined the level of phosphorylation in the Akt / TOR and MAPK pathways in proteins extracted from these tumors by western blotting. See FIG. 8E. Protein extract (10 ug / lane) from a matching set of tumors were separated by PAGE, blotted on PVDF membrane and probed with indicated antibodies. The Western blot images were scanned and quantitated using lmageJTM. Mean density for each treatment group were plotted as bar graph ± S.D., with the Y-axis showing image density relative to the naive group. N, naive group; 0, osimertinib; OH, osimertinib plus anti-HDGF antibody. Asterisks mark the data points where there is a significant difference between the treatment arms.

[0128] Stronger staining of phosphorylated MEK 1 / 2, Erk 1 / 2, p70S6K and 4EBP1 were observed in osimertinib monotherapy arm comparing to osimertinib plus anti- HDGF antibody combination arm, while changes in Akt and PRAS40 were not significant. It is interesting to note that the level of EGFR phosphorylation in both treatment arms were increased compared to P-EGFR level at 72 hours (see FIG. 8E). However, this level of EGFR phosphorylation in a particular tumor did not always correlate well with the level of phosphorylation in the downstream pathways. Inaddition, there was a noticeable reduction of total 4EBP1 level in both treatment armscompared to naive samples.

[0129] Example 8: Hypothetical Mechanisms of HDGF Mediated Tolerance to Osimertinib in NSCLC Cells with EGFR Mutation.

[0130] To investigate hypothetical mechanisms of HDGF mediated tolerance to osimertinib in NSCLC cells with EGFR mutation, a study was performed in the HDGF- mediated transition model of acquiring resistance to TKI. In this model, sensitive cells acquire transitory tolerance to TKI prior to emergence of new genetic or epigenetic driver alterations. The progression-free period is likely dominated by cells tolerant to TKI. SeeAttorney Docket No. 15024-395PC0 FIG. 2A, where, in the direct mode, HDGF released from dying or dead tumor cells could bind to receptors on surviving tumor cells, to enhance their tolerance to osimertinib. In the indirect mode (see FIG. 2B), HDGF first activates tumor stroma cells to secrete stroma cell factors, such as cytokines, growth factors, extracellular vesicle, or enhanced stroma cell surface ligand expression. These factors in turn interact with tumor cells to promote their tolerance to osimertinib. These two modes of actions are not mutually exclusive.

[0131] Example 9: Osimertinib Tolerance did not Evoke MET Activation or EGFR C797X Secondary Mutation.

[0132] To evaluate the contribution of MET activation in the incomplete response to osimertinib of the PDX tumors, we examined the level of MET and P-MET expression in TM00219 in comparison with Hcc827 cells. For this study, we investigated the status of P-MET expression and C797X secondary mutation in naïve and treated TM00219 PDX tumor. The expression of MET and P-MET in TM00219 tumors and Hcc827 cells were examined by western blot. See FIG. 9A (lane a, Hcc827 cell cultured in 10% FBS; lane b, Hcc827 cells treated with 250 µM crizotinib for 60 minutes). Naive, TM00219 tumor from untreated mice, Osi, TM00219 tumor from mice treated with osimertinib; Osi + H3, TM000219 tumor from mice treated with osimertinib plus H3 for 10 days. The samples collected for the data shown in FIG. 8 were used here. Beta actin (ACTB) western staining was developed with 3,3′-Diaminobenzidine (DAB) substrate. Total protein were visualized by Ponceaus S staining of the membrane.

[0133] C797X secondary mutation status was examined by sequencing genomic DNA in TM00219 tumors. In medium containing 10% FBS, Hcc827 cells showed a high level of MET and P-MET expression that can be abolished by treatment with crizotinib. However, the TM00219 PDX tumors displayed significantly lower levels of MET expression and negligible levels of P-MET expression in naive tumor and in arms treated with osimertinib or osimertinib plus H3 for 13 days. More importantly, the level of MET expression in the PDX tumor did not correlate with the drug treatment regimen (see FIG. 9A).Attorney Docket No. 15024-395PC0

[0134] We next examined if the C797X secondary mutation is involved in the incomplete response to osimertinib of these PDX tumors. A DNA fragment spanning EGFR intron 19-20 to intron 20-21 (genomic DNA fragments flanking EGFR gene exon 20) were amplified from osimertinib naive, osimertinib and osimertinib plus H3 treatment tumors, then sequenced with a primer on exon 20. Four tumors from each arm were sequenced. Representative chromatogram (trace) from the treated arms were shown. The amino acid sequence flanking 797 is shown in single letter designation. The sequencing chromatograms of the two treated arms were similar to naive tumors, and no indication of C797X mutation was found. See FIG. 9B.

[0135] Example 10: Elevated Expression of HDGF in TKI Tolerant Tumor Cells.

[0136] To examine the relationship between HDGF expression and TKI tolerance, we examined the expression of HDGF in tumors from different treatment arms. For this study, the FFPE sections of TM00219 PDX tumors from the different treatment arms used in FIG. 8 were stained with a mouse anti-HDGF antibody T221. See FIG. 10A, which shows representative fields (10x) of staining in naive, osimertinib or osimertinib plus anti-HDGF antibody H3 treated arms. FIG. 10B and FIG. 10C show representative slides from naive (FIG. 10B) and osimertinib-treated (FIG. 10C) tumor viewed under different power: from left to right are low power (1.5x) wide field views, medium power (10x) and high power (40x) view. The images were extracted from lmagescopeTMview.

[0137] In IHC of naive tumors, HDGF staining was seen in the nucleus of most of the tumor cells with intensity ranging from weak to medium to strong. Weak cytoplasmic staining of HDGF was also observed in naive tumor cells. See FIG. 10A and FIG. 10B).

[0138] Interestingly, in both osimertinib monotherapy and combination therapy arms, all surviving tumor cells displayed nearly uniform medium strong to strong nuclear staining and negligible cytoplasmic staining (see FIG. 10A and FIG. 10C). However, we did not observe significant difference in the mean intensity between osimertinib monotherapy and osimertinib plus anti-HDGF antibody combination arms despite some variations within the treatment arms. In addition, we did not observe significant changes of HDGF staining intensity in western blot analysis of tumor protein extracted from different treatment arms (see FIG. 8E).Attorney Docket No. 15024-395PC0 References.

[0139] All publications listed below and throughout the specification are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. 1. Bray F, Ferlay J, Soerjomataram I et al. (2018) Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians 68:394- 424. doi.org / 10.3322 / caac.21492 2. Midha A, Dearden S, McCormack R (2015) EGFR mutation incidence in non-small- cell lung cancer of adenocarcinoma histology: A systematic review and global map by ethnicity (mutMapll). Am J Cancer Res 5:2892-2911. 3. Lemmon MA, Schlessinger J (2010) Cell signaling by receptor tyrosine kinases. Cell 141:1117-1134. doi.org / 10.1016 / j.cell.2010.06.011 4. 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Its homology with high mobility group-1 protein. J Biol Chem 269:25143-25149. 33. Chen S-C, Hu T-H, Huang C-C et al. (2015) Hepatoma-derived growth factor / nucleolin axis as a novel oncogenic pathway in liver carcinogenesis. Oncotarget 6:16253-16270. doi.org / 10.18632 / oncotarget.3608 34. Lin Y-W, Huang S-T, Wu J-C et al. (2019) Novel HDGF / HIF-la / VEGF axis in oral cancer impacts disease prognosis. BMC Cancer 19:165. doi.org / 10.1186 / s12885-019- 6229-5 35. Guo Y, Xu H, Huang M et al. (2023) BLM promotes malignancy in PCa by inducing KRAS expression and RhoA suppression via its interaction with HDGF and activation of MAPK / ERK pathway. J Cell Commun Signal 17:757-772. doi.org / 10.1007 / s12079-022- 00717-8 36. Bao C, Wang J, Ma W et al. (2014) HDGF: A novel jack-of-all-trades in cancer. Future Oncol 10:2675- 2685. doi.org / 10.2217 / fon.14.194 37. Ren H, Tang X, Lee JJ et al. 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Claims

Attorney Docket No. 15024-395PC0 CLAIMS 1. A method of treating lung cancer in a subject in need, comprising: administering to the subject a therapeutically effective amount of HDGF antibody and a therapeutically effective amount of an EGFR inhibitor chemotherapeutic agent.

2. The method of claim 1 wherein the EGFR inhibitor chemotherapeutic agent is selected from the group consisting of erlotinib, gefitinib, afatinib, dacomitinib, lazertinib, icotinib, and osimertinib.

3. The method of claim 2 wherein the EGFR inhibitor chemotherapeutic agent is osimertinib.

4. The method of claim 1 wherein the HDGF antibody is H3.

5. The method of claim 1 wherein the lung cancer is primary lung cancer, recurrent lung cancer, or metastatic lung cancer.

6. The method of claim 1 wherein the lung cancer is non-small cell lung cancer.

7. A method for overcoming acquired resistance to EGFR-targeted therapy of lung cancer in a subject in need, comprising: administering to the subject a therapeutically effective amount of HDGF antibody and a therapeutically effective amount of an EGFR inhibitor chemotherapeutic agent.

8. The method of claim 7 wherein the EGFR inhibitor chemotherapeutic agent is selected from the group consisting of erlotinib, gefitinib, afatinib, dacomitinib, Lazertinib, icotinib, and osimertinib.

9. The method of claim 8 wherein the EGFR inhibitor chemotherapeutic agent is osimertinib.Attorney Docket No. 15024-395PC0 10. The method of claim 7 wherein the HDGF antibody is H3.

11. The method of claim 7 wherein the lung cancer is primary lung cancer, recurrent lung cancer, or metastatic lung cancer.

12. The method of claim 7 wherein the lung cancer is non-small cell lung cancer.

13. A pharmaceutical composition comprising an anti-HDGF antibody for the treatment of lung cancer in combination with an EGFR inhibitor chemotherapeutic agent.

14. The composition of claim 13 wherein the EGFR inhibitor chemotherapeutic agent is osimeritinib.

15. The composition of claim 13 wherein the anti-HDGF antibody is H3.

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