Methods for detecting and treating non-small cell lung cancer

Abstract

[0003] Methods for treating a cancer that co-expresses one or more placental alkaline phosphatase (ALPP) proteins and a cancer target antigen in a patient in need thereof are provided, comprising administering to the patient a therapeutic regimen comprising administering one or more standard of care inhibitors or antiproliferative agents that increase cell surface expression of one or more ALPP proteins and one or more ALPP protein targeting agents. Also provided are methods for selecting a patient with a cancer that co-expresses one or more placental alkaline phosphatase (ALPP) proteins and a cancer target antigen, comprising: assaying for baseline cell surface expression levels of one or more ALPP proteins in a biological sample obtained from the patient; and assaying for co-expression of one or more cancer cell surface oncogenic drivers.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 382,234, filed November 3, 2022, which is incorporated herein by reference in its entirety.

[0002] Incorporating a sequence listing The Sequence Listing, contained in the file name "MDA0076-401-PC", measuring 10 kilobytes as measured on a Microsoft Windows operating system and created on October 30, 2022, is filed electronically herewith and is incorporated by reference herein. [Background technology]

[0003] Placental alkaline phosphatase (ALPP) and ALPPL2 are closely related and regulated GPI-anchored proteins expressed on the cell surface in some cancers, while normal tissue expression of ALPP and ALPP2 is largely restricted to the placenta. ALPP and ALPPL2 are currently being investigated as targets for cancer therapy, including immunotherapy trials in China investigating CAR-T cells targeting ALPP in ovarian cancer (Phase 1) and endometrial cancer (Phase 2). Furthermore, the preclinical efficacy of antibody-drug conjugates targeting ALPP / ALPPL2 has been demonstrated in nonhuman primates, serving as the basis for first-in-human Phase 1 clinical trials. Summary of the Invention [Problem to be solved by the invention]

[0004] Although antibody-drug conjugates are potentially effective treatment options for certain types of cancer, this technology may be limited to cancer types that lack high expression of ALPP / ALPP2. There is a need in the art for more effective methods of treating cancers that express ALPP / ALPP2. [Means for solving the problem]

[0005] Evidence is provided herein that surface expression of ALPP / ALPPL2 is enhanced in ALPP-expressing cancer cells after treatment with therapeutic agents that target oncogenic drivers or inhibit cell proliferation. Enhanced expression sensitizes cancer cells to therapies directed against ALPP / ALPPL2, resulting in improved anti-cancer efficacy.

[0006] Thus, provided is a method for treating a cancer that co-expresses one or more placental alkaline phosphatase (ALPP) proteins, e.g., ALPP and / or ALPP2, and a cancer target antigen in a patient in need thereof, comprising administering to the patient a therapeutic regimen that includes administering one or more standard treatment inhibitors or antiproliferative agents that increase cell surface expression of one or more ALPP proteins, and one or more ALPP protein targeting agents.

[0007] Also provided is a method of treating a cancer that co-expresses alkaline phosphatase (ALPP) and / or ALPP2 proteins and endogenously harbors an EGFR-activating mutation in a patient in need thereof, comprising administering to the patient an antibody-drug conjugate that targets ALPP and / or ALPP2 in cells in combination with an EGFR inhibitor.

[0008] Also provided is a method of treating a cancer that co-expresses alkaline phosphatase (ALPP) and / or ALPP2 proteins and endogenously harbors an EGFR-activating mutation in a patient in need thereof, the method comprising: identifying the EGFR-activating mutation in cells from a biological sample obtained from the patient; detecting and / or quantifying placental alkaline phosphatase (ALPP) and / or ALPP2 cell surface expression in the cells; and administering an antibody-drug conjugate therapy that targets ALPP and / or ALPP2 in the cancer cells in conjunction with an EGFR inhibitor.

[0009] Also provided is a method of treating drug-resistant or drug-refractory cancer cells that co-express alkaline phosphatase (ALPP) and / or ALPP2 proteins in a patient in need thereof, comprising: identifying an EGFR-activating mutation in cells from a biological sample obtained from the patient; detecting and / or quantifying ALPP and / or ALPP2 cell surface expression in the cells; and administering an antibody-drug conjugate therapy that targets ALPP and / or ALPP2 in the cancer cells in conjunction with an EGFR inhibitor.

[0010] Also provided is a method for preventing the emergence of resistance to an EGFR inhibitor in cancer cells that co-express alkaline phosphatase (ALPP) and / or ALPP2 proteins, comprising administering an antibody-drug conjugate therapy that targets ALPP and / or ALPP2 in cancer cells in conjunction with an EGFR inhibitor.

[0011] In some embodiments, prior to administering the therapeutic regimen, the method further comprises assaying a biological sample taken from the patient for baseline cell surface expression levels of one or more ALPP proteins and for co-expression of one or more cancer cell surface oncogenic drivers.

[0012] Also provided is a method for selecting a patient having a cancer that co-expresses one or more placental alkaline phosphatase (ALPP) proteins and a cancer target antigen, comprising: assaying for baseline cell surface expression levels of one or more ALPP proteins in a biological sample taken from the patient; and assaying for co-expression of one or more cancer cell surface oncogenic drivers.

[0013] These and other aspects of the present invention will become apparent upon reference to the following detailed description. To this end, various references are set forth herein which describe in more detail certain background information, procedures, compounds, and / or compositions, and are each incorporated by reference in their entirety. [Brief explanation of the drawings]

[0014] [Figures 1A-1C] Figure 1A shows anti-ALPPIHC staining of tissue microarrays of normal tissues and lung tumors. Positive ALPP staining was restricted to normal testis and lung tumors. Figure 1B shows anti-ALPPIHC staining of large tissue sections of placenta, breast, and TNBC. Normal placenta and TNBC showed strong positive ALPP staining. Figure 1C shows cell surface coexpression of EGFR and ALPP / ALPPL2. [Figures 2A-2E] Figure 2A shows the surface co-expression of target proteins and ALPP / ALPPL2 on lung adenocarcinoma cells H1650 (EGFR mutant). Figure 2B shows the change in cell surface ALPP levels following tyrosine kinase inhibitor (TKI) treatment on H1650c cells. Figure 2C shows the surface co-expression of target proteins and ALPP / ALPPL2 on breast cancer cells, SKBR3 cells, and MCF7 cells. Figure 2D shows gefitinib treatment on SKBR3 cells expressing ALPP and MCF7 cells lacking ALPP expression. Figure 2E shows anti-HER2 antibody treatment on SKBR3 cells expressing ALPP. [Figures 3A-3D] Figure 3A shows the procedure for proteomic analysis of biotin-labeled surface proteins in EGFR-mutant H1650 and PC9 lung adenocarcinoma cells. Figure 3B shows surface ALPP expression (spectral abundance) in EGFR-mutant H1650 and PC9 cells 24 and 48 hours after gefitinib treatment. Figure 3C shows immunofluorescence for ALPP expression in EGFR-mutant H1650 cells after gefitinib treatment. Figure 3D shows immunoblots for ALPP in EGFR-mutant H1650 and KRAS-mutant H2291 and H1838 cells 24 and 48 hours after treatment with gefitinib or epidermal growth factor (EGF). [Figures 4A-4D]Figure 4A shows the internalization of anti-ALPP antibody in H1650 cells. Figure 4B shows that combined treatment with gefitinib and ALPP-ADC showed the best efficacy in H1650 cells. Figure 4C shows the synergistic effect of gefitinib and ALPP-ADC in H1650, H1651, H2291, and H1944 cells. Figure 4D shows a scheme of orthotopic lung xenotransplantation in an in vivo study. Figure 4E shows that combined treatment with gefitinib (50 mg / kg) and ALPP-ADC (5 mg / kg) showed the best efficacy in an in vivo study. [Figures 5A-5D] Figure 5A shows the surface expression (spectral abundance) of ALPP and EGFR in breast cancer cell lines. Figure 5B shows immunoblotting of ALPP, EGFR, and phospho-EGFR in BT20 and HCC1937TNBC cells after treatment with gefitinib (1 μM). Figure 6C shows representative light-field images of BT20 and HCC1937TNBC cells after treatment with either gefitinib and ALPP-MMAF-ADC, alone or in combination. Figure 5D shows tumor volume curves of TNBC tumor-bearing mice after treatment with gefitinib (50 mg / kg) and ALPP-MAFF-ADC (5 mg / kg), alone or in combination. *p-value <0.05; ****p-value <0.0001. [Figures 6A-6E] ALPP and ALPG expression in lung cancer. Figure 6A shows the spectral abundance (MS / MS events) of ALPP and ALPPL2 in LUAD and SCLC cell lines. Figure 6B shows the gene expression of ALPP and ALPG (ALPPL2) in lung adenocarcinoma (LUAD), squamous cell carcinoma of the lung (LUSC), and small cell lung cancer (SCLC) cell lines obtained from the Cancer Cell Line Encyclopedia (CCLE). Figure 6C shows the gene expression levels of ALPP and ALPG in LUAD and LUSC in The Cancer Genome Atlas (TCGA) database. Figure 6D shows IHC staining of ALPP in NSCLC tumor tissue. Figure 6E shows immunofluorescence staining of ALPP in HCC827 and PC9 cell lines. [Figure 7A-7J]EGFR TKIs upregulate ALPP expression in LUAD cells. Figure 7A shows ALPP levels in the TCGA LUAD database of cells with wild-type or driver mutations in EGFR, KRAS, NF1, and BRAF. Figures 7B-C show immunoblots for ALPP, pAKT-S473, total AKT, pERK-T202 / Y204, total ERK, pSTAT1-Y701, pSTAT1-S727, total STAT1, and β-actin in LUAD cancer cells H2291 and H1650 after 24 and 48 hours of treatment with or without EGF (10 or 20 ng / mL) or gefitinib (0.5 or 1 μM). Figure 7D shows immunoblots for ALPP, EGFR, phosphorylated EGFR (pEGFR Tyr1068), and β-actin. Figure 7E shows flow cytometry analysis of ALPP expression. Figure 7F shows immunofluorescence staining of ALPP (green) and nuclei (red). Figures 7G-H show ALPP expression in LUAD cell lines treated with gefitinib or osimertinib. Gene expression data were obtained from Gene Expression Omnibus (GSE75602, GSE193258). Figure 7I shows immunoblots of ALPP and β-actin in LUAD cell lines HCC827 and H1650 treated with vehicle control or an EGFR inhibitor. Figure 7J shows immunoblots of ALPP and β-actin in LUAD cell lines H1395, H1944, HCC2279, H2291, H1650, H1838, and H1651 treated with vehicle control or gefitinib (1 μM) for 48 hours. [Figures 8A-8L]EGFR TKIs dephosphorylate FoxO3a and induce ALPP expression. Figure 8A shows a GESA analysis of the cell cycle using the TCGA LUAD dataset. Figure 8B shows a KEGG pathway analysis using the TCGA LUAD dataset. Figure 8C shows a flow cytometry analysis of the cell cycle in HCC827 and PC9 cells treated with vehicle control, gefitinib (30 nM), or osimertinib (30 nM). Figure 8D shows immunoblots of ALPP and β-actin in HCC827 and PC9 cells treated for 48 hours with the PI3K inhibitor NVP-BKM120 (100 nM), the MEK inhibitor AZD8330 (100 nM), and the ERK inhibitor SCH772984 (100 nM). Figure 8E shows an Ingenuity pathway analysis of upstream regulators in cells treated with vehicle control or osimertinib. RNA-seq data were obtained from Gene Expression Omnibus (GSE193258). Figures 8F-G show immunoblots of ALPP and β-actin in HCC827 and PC9 cells treated with the FOXO inhibitor AS1842856, vehicle, or gefitinib for 48 hours. Figure 8H shows immunoblots of ALPP and FoxO3a in FoxO3a-overexpressing HCC827 and H1650 LUAD cells treated with vehicle or gefitinib (30 nM) for 48 hours. Figure 8I shows immunoblots of ALPP, FoxO3a, and phosphorylated FoxO3a (Ser294, Ser425) in HCC827 and H1650 cells treated with 30 nM gefitinib for 6 hours. Figure 8J shows immunoblots of FoxO3a in the cytosol and nuclear compartments from HC827 and H1650 cell lines treated with gefitinib. Figure 8K shows immunoblots of ALPP, EGFR, pEGFR (Tyr1068), FoxO3a, pFoxO3a (Ser294), and β-actin in HCC827 and PC9 cells treated with gefitinib (30 nM) or osimertinib (30 nM). Figure 8L shows ChIP-qPCR assays for the promoter region of the ALPP gene in HCC827 and H1650 LUAD cells treated with either vehicle or gefitinib (1 μM) for 6 hours. [Figures 9A-9N]EGFR TKIs are required to maintain the transient and reversible upregulation of ALPP. Figure 9A shows immunoblots of ALPP, EGFR, pEGFR (Tyr1068), and β-actin in HCC827 and PC9 cell lines treated with or without gefitinib or osimertinib (D0) for 1 (D1), 2 (D2), and 4 (D4) days. Figure 9B shows immunofluorescence staining of ALPP in HCC827 and PC9 cell lines treated with gefitinib or osimertinib for 0 (D0), 1 (D1), 2 (D2), and 4 (D4) days. Figure 9C shows flow cytometry analysis of surface ALPP expression in HCC827 and PC9 cells treated with gefitinib (30 nM) and osimertinib (30 nM) for 1, 2, and 4 days. Figure 9D shows single-cell analysis of NSCLC PC9 cells treated with osimertinib for 0, 3, 7, and 14 days. Data were obtained from Gene Expression Omnibus (GSE150949). Figures 9E-9F show (E) ALPP expression levels and (F) the percentage of ALPP+ cells in (D). Figure 9G shows ALPP mRNA levels in HCC827 and PC9 osimertinib-resistant cells. Data were obtained from Gene Expression Omnibus (GSE193258). Figure 9H shows immunoblots for ALPP, EGFR, pEGFR (Tyr1068), and β-actin in HCC827 and PC9 parental cells and DTPCs. Figure 9I shows immunofluorescence staining of ALPP in HCC827 and PC9 parental cells and DTPCs. Figure 9J shows flow cytometry analysis of surface ALPP expression in HCC827 and PC9 parental cells and DTPCs. Figure 9K shows immunoblots of ALPP, EGFR, pEGFR(Tyr1068), and β-actin in HCC827 and PC9 cells after treatment with gefitinib or osimertinib for 48 hours, followed by a washout and resting in TKI-free medium for another 48 hours. Figure 9L shows immunoblots of ALPP, EGFR, pEGFR(Tyr1068), and β-actin. HCC827 and PC9 cells were treated as shown in Figure 9K, rested, and then treated again with gefitinib or osimertinib for 48 hours.Figure 9M shows immunoblots of ALPP, EGFR, pEGFR(Tyr1068), and β-actin in HCC827 and PC9DPTC cells treated with gefitinib for 48 hours followed by a 48-hour rest period. Figure 9N shows immunoblots of ALPP, EGFR, pEGFR(Tyr1068), and β-actin in HCC827 and PC9DPTC cells treated with gefitinib, rested in TKI-free medium for 48 hours, and then treated again with gefitinib for 48 hours. [Figures 10A-10H] EGFR TKI induces ALPP expression in resistant cancer cells. Figure 10A shows immunoblots of ALPP, EGFR, pEGFR (Tyr1068), and β-actin in HCC827 and PC9 parental (P) cells and gefitinib-resistant (GR) cells treated with vehicle control or gefitinib (100 nM) for 48 hours. Figure 10B shows immunofluorescence staining of ALPP in HCC827 and PC9 parental (P) cells and gefitinib-resistant (GR) cells treated with vehicle control or gefitinib (100 nM) for 48 hours. Figure 10C shows flow cytometry analysis of surface ALPP expression in HCC827 and PC9 parental and gefitinib-resistant cells treated with vehicle control or gefitinib (100 nM). Figure 10D shows ALPP mRNA expression levels in parental cells and erlotinib-resistant LUAD cell lines HCC827 and HCC4006. Gene expression data was obtained from Gene Expression Omnibus (GSE121634). Figure 10E shows LPP mRNA expression levels in parental cells and gefitinib-resistant LUAD cell line PC9. Gene expression data was obtained from Gene Expression Omnibus (GSE75602). Figure 10F shows ALPP mRNA expression levels in PC9 xenograft tumors. Gene expression data was obtained from Gene Expression Omnibus (GSE161584). Figure 10G shows ALPP expression in EGFR TKI-naive and -resistant patients. Data was obtained from OncoSG. Figure 10H shows ALPP expression in NSCLC patients before and after failure to osimertinib. [Figures 11A-11E] The combination of EGFR TKI and ALPP-ADC treatment enhances cancer cell death in vitro. Figure 11A shows the cell viability of HCC827 and PC9 cells treated with gefitinib (50 nM) or osimertinib (50 nM), and αALPP-MMAF (1 μg / ml) or IgG-MMAF (1 μg / ml). Figure 11B shows the cell viability of HCC827 and PC9 cells treated with or without gefitinib (50 nM) and osimertinib (50 nM), IgG-MMAF, or α-ALPP-MMAF (1 μg / ml). Figure 11C shows the cell viability of HCC827 and PC9 cells pretreated with vehicle or gefitinib (50 nM) and osimertinib (50 nM) for 4 days, followed by the addition of IgG-MMAF or α-ALPP-MMAF (1 μg / ml). Figure 11D shows the cell viability (right panel) and crystal violet staining (left panel) of gefitinib-resistant cells treated with IgG-MMAF or α-ALPP-MMAF (5 μg / ml). Figure 11E shows the cell viability of HCC827 and PC9 cells pretreated with EGFR TKI (100 nM) for 2 days, then treated with EGFR TKI (100 nM) in combination with IgG-MMAF or α-ALPP-MMAF (5 μg / ml) for 2 days, followed by extended culture in the presence of EGFR TKI (100 nM) for 20 days. [Figures 12A-12H]Gefitinib treatment enhances tumor ALPP expression and enhances the anti-cancer efficacy of ALPP-MMAF-ADC in vivo. Figure 12A shows confocal imaging of H1650 cells treated with or without Phrodo Red-labeled anti-ALPP antibody. Figure 12B shows the cell viability of H1650 cells treated with sequential gefitinib and / or ALPP-MMAF-ADC. Figure 12C shows the morphology of the LUAD cell line H1650 treated with gefitinib and / or ALPP-MMAF-ADC. Figure 12D shows H&E staining of tumor tissue and IHC staining of ALPP in tumor tissue from a subcutaneous xenograft LUAD mouse model of the H1650 cell line treated with or without gefitinib (50 mg / kg). Figure 12E shows a schematic diagram of treatment of an orthotopic xenograft LUAD mouse model with gefitinib (50 mg / kg) and / or ALPP-MMAF-ADC (5 mg / kg). Figure 12F shows IVIS imaging of tumors before drug treatment on day 17 and after treatment on day 29. Figure 12G shows statistical analysis of tumor burden from (F). *P<0.05, **P<0.01. Figure 12H shows hematoxylin and eosin staining of a representative tumor section. [Figure 13] A proposed schematic diagram of ALPP surface upregulation in cancer cells upon EGFR inhibition is shown. [Figures 14A-14C] Figure 14 shows ALPP expression in normal tissues. Figure 14A shows representative immunohistochemistry (IHC) images of ALPP staining in placental tissues. Figure 14B shows immunoblots of ALPP in protein lysates from placenta, lung, breast, and heart tissues. Figure 14C shows representative IHC sections for ALPP in various normal tissues. [Figures 15A-15C]EGFR inhibitors upregulate ALPP expression. Figure 15A shows ALPP levels in an East Asian LUAD cohort with wild-type or driver mutations in EGFR. Figure 15B shows qPCR for ALPP mRNA levels in HCC827 and PC9 cells treated with vehicle control or gefitinib (30 nM) for 48 hours. Figure 12C shows ALPP expression in LUAD cell lines treated with gefitinib or osimertinib. Gene expression data were obtained from Gene Expression Omnibus (GSE80802, GSE75602, GSE193258). [Figures 16A-16C] FoxO3a is a transcriptional regulator of ALPP. Figure 16A shows the mRNA levels of FOXO1 and FOXO3A in HCC827 and H650 cell lines. Data were obtained from CCLE. Figure 16B shows the mRNA levels of FOXO3A in LUAD cells treated with or without gefitinib. Figure 16C shows IPA upstream analysis of LUAD cell lines treated with osimertinib. RNAseq data were obtained from Gene Expression Omnibus (GSE193258). [Figures 17A-17B] FoxO3a is a transcriptional regulator of ALPP. Figure 17A shows potential FoxO3a binding sites (yellow) in the ALPP promoter region (2 kb upstream of the ALPP gene). Figure 17B shows ChIP-qPCR assays for the promoter region of the ALPP gene in HCC827 and H1650 LUAD cells treated with vehicle or gefitinib (1 μM) for 6 hours. [Figures 18A-18C]EGFR TKIs are required to maintain the transient and reversible upregulation of ALPP. Figure 18A shows the mRNA levels of ALPP in HCC2935 and H1975 osimertinib-resistant cells. Data was obtained from Gene Expression Omnibus (GSE193258). Figure 18B shows the IC50 of gefitinib and osimertinib in HCC827 and PC9 cells. Figure 18C shows the IC50 of gefitinib and osimertinib in gefitinib- and osimertinib-resistant cells, respectively. Figure 18C shows the IC50 of gefitinib in gefitinib-resistant HCC827 and PC9 cells. [Figures 19A-19B] The EGFR TKI + ALPP-ADC format enhances the efficacy of cancer cell death in vitro. Figure 19A shows bright-field photographs and crystal violet staining of cells from Figure 11A. Figure 19B shows bright-field photographs and crystal violet staining of HCC827 and PC9 cells treated with IgG-MMAF or α-ALPP-MMAF (5 μg / ml) in combination with an EGFR inhibitor (100 nM) for 2 days, followed by extended culture in the presence of an EGFR TKI (100 nM) for 18 days. [Figure 20] 1 shows IHC analysis of ALPPN in normal tissues of tumor-bearing mice after gefitinib treatment. DETAILED DESCRIPTION OF THE INVENTION

[0015] Methods for treating a cancer that co-expresses one or more placental alkaline phosphatase (ALPP) proteins and a cancer target antigen in a patient in need thereof are provided, comprising administering to the patient a therapeutic regimen comprising administering one or more standard of care inhibitors or antiproliferative agents and one or more ALPP protein targeting agents that increase cell surface expression of one or more ALPP proteins. Also provided are methods for selecting a patient with a cancer that co-expresses one or more placental alkaline phosphatase (ALPP) proteins and a cancer target antigen, comprising assaying for baseline cell surface expression levels of one or more ALPP proteins in a biological sample collected from the patient and assaying for co-expression of one or more cancer cell surface oncogenic drivers.

[0016] Also provided is a method of treating a cancer that endogenously harbors an EGFR-activating mutation in a patient in need thereof, the method comprising administering to the patient an antibody-drug conjugate that targets ALPP and / or ALPP2 in cells in combination with an EGFR inhibitor. Also provided is a method of treating a cancer that endogenously harbors an EGFR-activating mutation in a patient in need thereof, the method comprising identifying an EGFR-activating mutation in cells from a biological sample obtained from the patient; detecting and / or quantitating placental alkaline phosphatase (ALPP) and / or ALPP2 cell surface expression in the cells; and administering to the patient antibody-drug conjugate therapy that targets ALPP and / or ALPP2 in cancer cells in combination with an EGFR inhibitor. Also provided is a method of treating drug-resistant or drug-refractory cancer cells in a patient in need thereof, comprising: identifying EGFR-activating mutations in cells from a biological sample taken from the patient; detecting and / or quantifying ALPP and / or ALPP2 cell surface expression in the cells; and administering an antibody-drug conjugate therapy targeting ALPP and / or ALPP2 in the cancer cells in conjunction with an EGFR inhibitor. Also provided is a method for preventing the emergence of resistance in cancer cells to EGFR inhibitors, the method comprising administering an antibody-drug conjugate therapy that targets ALPP and / or ALPP2 in cancer cells in conjunction with an EGFR inhibitor.Also provided is a method for treating a cancer that endogenously harbors an EGFR-activating mutation in a patient in need thereof, the method comprising administering an antibody-drug conjugate that targets ALPP and / or ALPP2 on the surface of the cancer cells, wherein the cancer cells exhibit increased expression of ALPP and / or ALPP2 compared to healthy lung cells; the antibody-drug conjugate that targets ALPP and / or ALPP2 comprises an antibody that targets ALPP and / or ALPP2 conjugated to MMAF; and the cancer cells contain an activating mutation in the EGFR gene, resulting in resistance to an EGFR tyrosine kinase inhibitor.

[0017] In some embodiments, the cells comprise increased cell surface expression of ALPP and / or ALPP2 relative to healthy cells.

[0018] In some embodiments, the EGFR mutation comprises an exon 19 deletion, a T790M point mutation, and / or an L858R point mutation.

[0019] In some embodiments, the cancer with an EGFR mutation is resistant to inhibitors that target EGFR.

[0020] In some embodiments, the cancer cells are drug-resistant persister cells (DTPCs) or drug-resistant cells (DRCs).

[0021] In some embodiments, administration of antibody-drug conjugate therapy targeting ALPP and / or ALPP2 in cancer cells and an EGFR inhibitor prevents drug-resistant persister cells (DTPCs) from developing into drug-resistant cells (DRCs).

[0022] In some embodiments, the methods treat drug-resistant persister cells (DTPCs) and drug-resistant cells (DRCs) to prevent the emergence of resistance to EGFR inhibitors.

[0023] In some embodiments, the cancers that co-express alkaline phosphatase (ALPP) and / or ALPP2 proteins are ovarian cancer, breast cancer, cervical cancer, endometrial cancer, pancreatic cancer, gastric cancer, colorectal cancer, lung cancer, urothelial cancer, brain cancer, testicular cancer, seminoma, and mesothelioma.

[0024] In some embodiments, the cancers that co-express alkaline phosphatase (ALPP) and / or ALPP2 proteins are testicular germ cell tumors, endometrial carcinoma of the uterine corpus, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, bladder urothelial carcinoma, triple-negative breast cancer, gastric adenocarcinoma, esophageal carcinoma, uterine carcinosarcoma, rectal adenocarcinoma, head and neck squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, clonal adenocarcinoma, mesothelioma, and acute myeloid leukemia.

[0025] In some embodiments, the lung cancer is non-small cell lung cancer.

[0026] In some embodiments, detecting and / or quantifying placental alkaline phosphatase (ALPP) and / or ALPP2 cell surface expression in cancer cells comprises histological analysis, immunohistochemical (IHC) staining for ALPP protein, blood-based tests, tissue-based tests, or imaging techniques.

[0027] In some embodiments, tissue-based tests include biopsy, flow cytometry, immunohistochemistry (IHC), Western blot (WB), polymerase chain reaction (PCR), or immunofluorescence (IF).

[0028] In some embodiments, the tissue-based test comprises a Mammaprint+Blueprint® test or an Oncotype DX® test.

[0029] In some embodiments, blood-based tests include Galleri®, circulating tumor cell (CTC) tests, complete blood counts (CBC), or tests or assays that measure circulating proteins, autoantibodies, cell-free circulating DNA, or extracellular vesicle-derived proteins.

[0030] In some embodiments, the biopsy is analyzed by hematoxylin and eosin (H&E) staining and / or microscopy.

[0031] In some embodiments, the antibody drug conjugate comprises an antibody targeting an ALPP conjugated to a chemotherapeutic agent.

[0032] In some embodiments, the EGFR inhibitor comprises a tyrosine kinase inhibitor (TKI).

[0033] In some embodiments, an antibody drug conjugate therapy targeting ALPP and / or ALPP2 is administered in conjunction with an EGFR inhibitor.

[0034] In some embodiments, the EGFR inhibitor is selected from gefitinib, osimertinib, mobocertinib, amivantamab, CLN081, and / or DZD9008.

[0035] In some embodiments, antibody-drug conjugates include SGN-ALPV, Adcetris®, Kadcyla®, Besponsa®, Mylotarg®, Polivy®, Padcev®, Enhertu®, Trodelvy®, Blenrep®, Zynlonta™, Akalux®, Aidixi®, and Tivdak®.

[0036] In some embodiments, the antibody drug conjugate therapy targeting ALPP and / or ALPP2 in cancer cells and the EGFR inhibitor are administered simultaneously.

[0037] In some embodiments, the antibody drug conjugate therapy targeting ALPP and / or ALPP2 in cancer cells and the EGFR inhibitor are administered sequentially.

[0038] Placental alkaline phosphatase (ALPP) is a membrane-bound, glycosylated, dimeric enzyme first detected in serum during pregnancy and shown to originate from the placenta. Four distinct isotypes of alkaline phosphatase exist: placental type (ALPP), placental type 2 (ALPP2), intestinal ALPP (ALPI), and tissue-nonspecific ALPP (ALPL). Of these four isotypes, ALPP and ALPP2 have been found to be associated with many human cancers, including testicular seminoma, ovarian cancer, and endometrial cancer, among others. Beyond placental trophoblasts, ALPP / ALPP2 expression is virtually absent on normal tissues, providing excellent opportunities for the development of both antibodies requiring a high degree of tumor specificity. Antibodies against ALPP and / or ALPP2 can be conjugated to other classes of drugs, such as DNA crosslinkers or radionuclides (e.g., alpha particles).

[0039] Many features make ALPP and / or ALPP2 attractive candidates for antigen-targeted immunotherapy: (1) ALPP is a membrane-bound protein and therefore an accessible cell surface target for specific binding molecules such as antibodies. (2) ALPP expression is restricted in healthy tissues but increased in malignant tumors, suggesting that it may serve as a tumor-specific antigen with low off-tumor expression. (3) Alkaline phosphatase activity has been reported to induce tumor progression in various cancers, including prostate cancer, head and neck squamous cell carcinoma, and ovarian cancer. Therefore, targeting ALPP as a target for cancer therapy may also enhance tumor control by reducing tumor-derived alkaline phosphatase activity.

[0040] As described herein, increased or enhanced cell surface expression of ALPP and / or ALPP2 in many cancer types offers novel opportunities for the treatment of these cancers. For cancers in which ALPP and / or ALPP2 expression is elevated, not only can these proteins themselves serve as targets for cancer therapy, but also, expression of ALPP and / or ALPP2 may occur in conjunction with activating mutations, such as EGFR activating mutations, or oncogenic driver mutations, i.e., expression of genes involved in the initiation or maintenance of cancer. Such genes may be EGFR or another gene disclosed herein. Increased or enhanced expression of ALPP and / or ALPP2 may also occur in conjunction with genes involved in the initiation or maintenance of cancer, such as oncogenic driver mutations. Expression of ALPP and / or ALPP2 can be enhanced by detecting and / or quantifying cell surface expression levels of ALPP and / or ALPP2. Baseline expression of ALPP and / or ALPP2 may provide useful information related to disease severity and prognosis for the treatment of cancer in an individual. Assessment of baseline cell surface expression of ALPP and / or ALPP2 determines whether there is elevated ALPP / ALPP2, which can serve as a useful first step in determining treatment for a particular cancer. In some embodiments, increased baseline cell surface expression of ALPP and / or ALPP2 increases the sensitivity of cancer cells to therapies directed against ALPP and / or ALPP2. As described herein, the methods of the present disclosure make it possible to prevent the emergence of resistance to drug therapy in cancer cells, such as NSCLC cells.

[0041] Thus, in some embodiments, the methods described herein for treating cancers that express high levels of ALPP / ALPP2 may first employ a step in which baseline levels of ALPP and / or ALPP2 are determined before initiating treatment for the cancer. For cancers found to express high levels of ALPP / ALPP2, standard of care inhibitor therapy, or anti-proliferative agents that target ALPP and / or ALPP2, may be administered to treat the cancer.

[0042] In some embodiments, a standard of care inhibitor or anti-proliferative agent may be administered to an individual with cancer to increase its expression on the surface of cancer cells. In such cases, administration of the standard of care inhibitor or anti-proliferative agent serves the purpose of increasing ALPP and / or ALPP2 expression on the cell surface prior to administration of an ALPP / ALPP2 protein-targeting agent for the treatment of cancer. This "two-hit" approach increases the expression of the target itself to increase the sensitivity of cancer cells to therapies directed against ALPP and / or ALPP2.

[0043] Therefore, in some embodiments, measuring the baseline level of ALPP and / or ALPP2 protein on the cell surface of cancer cells can enable the determination of an appropriate treatment regimen for the cancer. Methods for assaying for baseline cell surface expression levels are well known in the art and include, but are not limited to, histological analysis, immunohistochemical (IHC) staining for ALPP protein, electron microscopy, mass spectrometry, immunofluorescence, blood-based tests, tissue-based tests, or imaging techniques. In some embodiments, detecting and / or quantifying placental alkaline phosphatase (ALPP) and / or ALPP2 cell surface expression, i.e., baseline values, in cancer cells comprises histological analysis, immunohistochemical (IHC) staining for ALPP protein, blood-based tests, tissue-based tests, or imaging techniques. Any method capable of determining ALPP and / or ALPP2 levels in a biological sample from an individual can be used and is intended to be encompassed within the scope of the present disclosure.

[0044] The blood-based test can be any blood-based test known or available in the art, such as a Galleri® test, a circulating tumor cell (CTC) test, a complete blood count (CBC), or a test or assay that measures circulating proteins, autoantibodies, cell-free circulating DNA, or extracellular vesicle-derived proteins. In some embodiments, a blood-based assay or test for detecting the expression or level of ALPP and / or ALPP2 can include the use of a labeled ligand or antibody.

[0045] The tissue-based test described herein can be any tissue-based test known or available in the art, such as biopsy, flow cytometry, immunohistochemistry (IHC), Western blot (WB), polymerase chain reaction (PCR), or immunofluorescence (IF). Such tests can utilize specific protein markers or reagents (e.g., stains) to detect and quantify the amount of ALPP and / or ALPP2 protein present in a biological sample, for example, using antibodies or any other specific method for determining cell surface expression of ALPP and / or ALPP2. For example, specific tissue-based tests known in the art include, but are not limited to, the Mammaprint+Blueprint® test, the Signatara™ test, the Altera Tumor Genomic Profile test, or the Oncotype DX® test. In some embodiments, the tissue-based test can be the Mammaprint+Blueprint® test or the Oncotype DX® test.

[0046] A biological sample suitable for the methods described herein can be any biological sample, for example, a blood sample, or a biopsy, or a cell culture sample. Depending on the cancer type, certain biological samples may be more advantageous, for example, a biopsy for solid cancers, or a blood-based sample for blood cancers, although any biological sample may be useful with the methods described herein.

[0047] In some embodiments, the biopsy is analyzed by hematoxylin and eosin (H&E) staining and / or microscopy.

[0048] Oncogenic drivers and activating mutations, e.g., EGFR activating mutations, are well known in the art and may include genes responsible for the initiation or maintenance of cancer. As described herein, cell surface oncogenic drivers are useful cancer target antigens. Targeting oncogenic drivers as cancer target antigens can be combined with targeting ALPP and / or ALPP2, or can be separate. In some embodiments, ALPP and / or ALPP2 can be targeted for sequential treatment with activating mutations or oncogenic driver mutations, e.g., EGFR activating mutations, or can be targeted simultaneously in the treatments disclosed herein, meaning that antibody-drug conjugate therapy targeting ALPP and / or ALPP2 described herein and EGFR activating mutations described herein are administered together or simultaneously. In some embodiments, activating mutations or oncogenic driver mutations, e.g., EGFR activating mutations and / or cell surface oncogenic drivers, are directly targeted by inhibitor molecules or by chemotherapeutic treatments described herein. In some embodiments, activating mutations, e.g., EGFR activating mutations or oncogenic driver mutations, can be targeted using any of the cancer therapies described herein, alone or in combination with ALPP and / or ALPP2. In some embodiments, ALPP and / or ALPP2 are targeted using the antibody-drug conjugates described herein, and EGFR activating mutations are targeted using an EGFR inhibitor therapy. These therapeutics can be administered simultaneously or sequentially. Oncogenic drivers useful in treating cancer can be genes and / or encoded proteins associated with specific types of cancer, e.g., BRCA1 / 2 in breast cancer. Some oncogenic driver genes are found to be mutated in many different cancers, while others are specific to one or a few cancer types.

[0049] Some common oncogenic drivers that may be useful as described herein, i.e., may have mutations that cause or contribute to the initiation or maintenance of the cancers described herein, include, but are not limited to, ALK, ARID1A, BRAF, BRCA1 / 2, CD22, CD40, CD46, CD74, CDKN2A, DDR1, EFNB2, EGFR, EML4, ENG, EPHA2, EPHB2, EPHB4, ERBB1, ERBB2, ERBB3, FAS, FGFR1, FGFR2, HER2, HRA These include S, ICAM1, IGF1R, INSR, ITGA4, ITGAV, ITGB1, ITGB3, JAK, KRAS, MAPK, MAP2K1, MEK, MET, MICB, MST1R, MYC, NF1, NGFR, NRAS, NRP1, NTRK1, PIK3CA, PTEN, PTK7, RAF, RAS, RET, ROR1, ROS1, SEMA4D, STAT, TNFRSF10A, TNFRSF10B, TSC1 / 2, TP53, UMD, TYRO3, YAP1, and / or YAP2. TP53 is associated with more than 25 different cancer types, and many other oncogenic driver genes, such as PIK3CA, KRAS, PTEN, and ARID1A, are associated with 15 or more. In some embodiments, one or more of the above oncogenic driver genes may have a mutation that causes or contributes to the initiation or maintenance of the cancers described herein.

[0050] In some embodiments, the cancer target antigen is a cell surface oncogenic driver.

[0051] In some embodiments, the cancer target antigen comprises one or more of CD22, CD40, CD46, CD74, DDR1, EFNB2, ENG, EPHA2, EPHB2, EPHB4, ERBB3, FAS, FGFR2, ICAM1, IGF1R, INSR, ITGA4, ITGAV, ITGB1, ITGB3, MET, MICB, MST1R, NGFR, NRP1, PTK7, RET, ROR1, SEMA4D, TNFRSF10A, TNFRSF10B, TYRO3, YAP1, and / or YAP2.

[0052] In some embodiments, cell surface oncogenic drivers are directly targeted by inhibitor molecules.

[0053] The specific oncogenic drivers to be targeted can be selected by a physician or clinician for personalized treatment of a specific cancer type. The present disclosure is not intended to be limited to the list of oncogenic driver genes / proteins described herein, but rather exemplifies methods of treating cancers that express ALPP and / or ALPP2 as described herein.

[0054] The most common way to classify cancer is by dividing it into four categories based on whether it can be removed by surgery and where it has spread: resectable, borderline resectable, locally advanced, or metastatic. Resectable cancers can be removed by surgery. The cancerous tumor is located only in or has spread beyond a specific area or organ, but has not entered important arteries or veins in that area. There is no evidence that the tumor has spread to areas outside that area. Borderline resectable is described as a tumor that may be difficult or impossible to remove by surgery when it is first diagnosed, but if chemotherapy and / or radiation therapy can initially shrink the tumor, it may later be resected with negative margins. Negative margins mean that no visible cancer cells remain. Locally advanced cancer is still located only in the immediate area around the tumor, but if it has grown into nearby arteries or veins or into nearby organs, it cannot be removed by surgery. However, there is no sign that it has spread to distant parts of the body. Metastatic means that the cancer has spread beyond the area of ​​the tumor and to other organs such as the liver or to distant areas of the abdomen.

[0055] Cancer treatment options are well established in the literature and may include partial or complete surgical removal of cancerous tissue, or may include administration of one or more cancer therapies described herein (e.g., chemotherapeutic agents, immunotherapeutic agents, antibody drug conjugates, therapeutic radiation, etc.).

[0056] There are numerous chemotherapeutic agents approved for cancer treatment and are well known in the art. Many of these are described herein, but it should be noted that the present disclosure is not limited to the cancer types or agents described herein. Those skilled in the art will understand that the present disclosure can extend to any suitable cancer type and any chemotherapeutic agent or treatment. For example, any cancer type that expresses ALPP and / or ALPP2 or expresses increased cell surface levels of ALPP and / or ALPP2 is within the scope of the methods of the present invention.

[0057] For example, techniques useful for determining cell surface expression of ALPP and / or ALPP2, e.g., techniques useful for detecting and / or quantifying ALPP and / or ALPP2 cell surface expression, include staining techniques, immunohistochemistry, cell viability assays, microscopy, phosphatase assays, flow cytometry, cell surface biotinylation, proton or chymotrypsin sensitivity assays, antibody labeling assays, ELISA, radioligand binding, cAMP enzyme-linked immunosorbent assay, bioluminescence resonance energy transfer (BRET), CREB phosphorylation assays, live cell staining, imaging, or any other technique known or available in the art. Staining techniques are known in the art and may include any stain suitable for measuring proteins, for example, fluorescein stains such as fluorescein-5-isothiocyanate (5-FITC) or fluorescein-5-isothiocyanate (6-FITC), Alexa Fluor 488, Alexa Fluor 647, carboxyfluorescein diacetate (CFSE), R-phycoerythrin (PE), PE-Texas Red, propidium iodide (PI), PE-Cy5, PerCP, PerCP-Cy5.5, PE-Cy7, allophycocyanin (APC), green fluorescent protein (GFP), hematoxylin and eosin (H&E), or other cell staining techniques or dyes known or available in the art.

[0058] In some embodiments, cancer types that exhibit expression of ALPP and / or ALPP2 or that exhibit increased expression of ALPP and / or ALPP2 may be treated according to the methods described herein. In some embodiments, ALPP and / or ALPP2 expression may be increased to a statistically significant degree or level, or may be increased by various magnitudes depending on the type of cancer. Non-limiting examples of cancer types suitable for treatment as described herein include ovarian cancer, breast cancer, intrauterine cancer, pancreatic cancer, gastric cancer, colorectal cancer, lung cancer, urothelial cancer, brain cancer, testicular cancer, seminoma, and mesothelioma. Some specific cancers include, but are not limited to, testicular germ cell tumors, endometrial cancer of the uterine corpus, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, bladder urothelial carcinoma, triple-negative breast cancer, gastric adenocarcinoma, esophageal carcinoma, uterine carcinosarcoma, rectal adenocarcinoma, head and neck squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, clonal adenocarcinoma, mesothelioma, and acute myeloid leukemia.

[0059] In some embodiments, cancers that co-express one or more placental alkaline phosphatase (ALPP) proteins and cancer target antigens include breast cancer, intrauterine cancer, pancreatic cancer, gastric cancer, colorectal cancer, lung cancer, urothelial cancer, brain cancer, testicular cancer, seminoma, and mesothelioma.

[0060] In some embodiments, the lung cancer is non-small cell lung cancer.

[0061] Provided herein are methods for treating cancers that normally express high levels of ALPP and / or ALPP2. Also provided are methods for treating cancers that co-express one or more placental alkaline phosphatase (ALPP) proteins and cancer target antigens in a patient in need thereof, comprising administering a therapeutic regimen to the patient, the therapeutic regimen comprising administering one or more standard of care inhibitors or antiproliferative agents that increase cell surface expression of one or more ALPP proteins, and one or more ALPP protein-targeting agents. In some embodiments, prior to administering the therapeutic regimen, the method further comprises assaying a biological sample obtained from the patient for baseline cell surface expression levels of one or more ALPP proteins and for co-expression of one or more cancer cell surface oncogenic drivers. In some embodiments, the method includes administering a therapeutic regimen, e.g., one or more standard of care inhibitors or anti-proliferative agents, to increase cell surface expression of ALPP and / or ALPP2 in the cancer cells and to sensitize the cancer cells to one or more ALPP protein targeting agents.

[0062] In some embodiments, the present disclosure also provides a method for selecting a patient having a cancer that co-expresses one or more placental alkaline phosphatase (ALPP) proteins and a cancer target antigen, the method comprising: assaying for baseline cell surface expression levels of one or more ALPP proteins in a biological sample taken from the patient; and assaying for co-expression of one or more cancer cell surface oncogenic drivers.

[0063] Chemotherapy is widely used as a standard treatment for cancer, destroying cancer cells and preventing them from growing, dividing, and producing more. Some chemotherapy drugs work by damaging DNA and preventing cells from replicating, resulting in the death of cancer cells. Because cancer cells typically grow and divide faster than normal, non-cancerous cells, these chemotherapy drugs are more effective against actively dividing cancer cells.

[0064] As described herein, treatment regimens that can be used to increase cell surface expression of ALPP and / or ALPP2 can be any standard of care inhibitor or anti-proliferative agent, including any chemotherapeutic agent known in the art for cancer treatment, such as, but not limited to, Evista (raloxifene hydrochloride), bleomycin, ifosfamide (Ifex®), raloxifene hydrochloride, Soltamox (tamoxifen citrate), tamoxifen citrate, Abemaciclib, albumin-bound paclitaxel (Abraxane), (paclitaxel albumin-stabilized nanoparticle formulation), Ado-trastuzumab emtansine, Afinitor (everolimus), Afinitor (albumin-stabilized nanoparticle formulation), Ado-trastuzumab emtansine, ... Disperz (everolimus), Alkeran (Melphan), alpelisib, altretamine (Hexalen®), anastrozole, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), atezolizumab, Avastin (bevacizumab), capecitabine (Xeloda®), carboplatin, cisplatin, cyclophosphamide (Cytoxan®), docetaxel (Taxotere®), doxorubicin hydrochloride, Doxil (doxorubicin hydrochloride liposomal), Ellence (epirubicin hydrochloride), Enhertu® (Fa m-trastuzumab deruxtecan-nxki), epirubicin hydrochloride, eribulin mesylate, etoposide (VP-16, Vepesid), everolimus, exemestane, 5-FU (fluorouracil injection), Fam-trastuzumab deruxtecan-nxki, Fareston (toremifene), Faslodex (fulvestrant), Femara (letrozole), fluorouracil injection, fulvestrant, gemcitabine hydrochloride, Gemzar® (gemcitabine hydrochloride), goserelin acetate, Halaven (eribulin mesylate), Herceptin Hylecta (trastuzumab and hyaluronidase-oysk), Herceptin (trastuzumab), Hycamtin (topotecan hydrochloride),Ibrance (palbociclib), Infugem (gemcitabine hydrochloride), irinotecan (CPT-11, Camptosar®), ixabepilone, Ixempra (ixabepilone), Kadcyla® (Ado-trastuzumab emtansine), Keytruda (pembrolizumab), Kisqali (ribociclib), lapatinib ditosylate, letrozole, liposomal doxorubicin (Doxil®), Lynparza (olaparib), Ma Rgenza (margetuximab-cmkb), margetuximab-cmkb, megestrol acetate, melphalan, methotrexate sodium, mitomycin, neratinib maleate, Nerlynx (neratinib maleate), niraparibut tosylate monohydrate, olaparib, doxorubicin (Adriamycin®), oxaliplatin, paclitaxel (Taxol®), paclitaxel albumin-stabilized nanoparticle formulation, palbociclib, pamidronate disodium, pembro Lizab, pemetrexed (Alimta®), Perjeta (pertuzumab), pertuzumab, pertuzumab, Rubraca (rucaparib camsylate), trastuzumab, and hyaluronidase-zzxf, Phesgo (pertuzumab, trastuzumab, and hyaluronidase-zzxf), Piqray (alpelisib), ribociclib, sacituzumab govitecan-hziy, Soltamox (tamoxifen citrate), talazoparibut tosylate, Talzenna ( Talazoparibut tosylate), tamoxifen citrate, Taxol, Taxotere (docetaxel), Tecentriq (atezolizumab), Tepadina (thiotepa), thiotepa, topotecan hydrochloride, toremifene, trastuzumab (Herceptin®), trastuzumab and hyaluronidase-oysk, Trexall (methotrexate sodium), Trodelvy® (sacituzumab govitecan-hziy), tucatinib, Tukysa (tucatinib), Tykerb (lapatinib ditosylate),Examples of chemotherapy-resistant cancers include Venclexta (Venetoclax), Verzenio (Abemaciclib), vinblastine sulfate, Xeloda (Capecitabine), Vinorelbine (Navelbine®), Zejula (Niraparibut tosylate monohydrate), and Zoladex (Goserelin acetate). As will be appreciated by those skilled in the art, the benefit of a particular cancer from a particular chemotherapy drug or combination thereof, and therefore the individual treatment plan for a particular cancer, may vary as deemed appropriate by the clinician or physician.

[0065] In some embodiments, one or more standard of care inhibitors or antiproliferative agents that increase cell surface expression of ALPP and / or ALPP2 are combined with each other in a combination therapy. The combination therapy can include any combination of cancer treatments as determined appropriate by a clinician or physician. For example, cancer treatment options useful in the methods of the present invention include one or more of chemotherapy drugs, radiation therapy, immunotherapy, DNA crosslinking agents, antibody-drug conjugates, hormone therapy, targeted drug therapy, radionuclides, cryoablation, surgical procedures, etc.

[0066] In some embodiments, the one or more standard of care inhibitors or antiproliferative agents are trastuzumab (Herceptin®), cisplatin, etoposide (VP-16), bleomycin, ifosfamide (Ifex®), paclitaxel (Taxol®), carboplatin, vinblastine, doxorubicin (Adriamycin®), liposomal doxorubicin (Doxil®), docetaxel (Taxotere®), albumin-bound paclitaxel (nab-paclitaxel, Abraxane®), altreta, or cyclosporine. The anti-cancer drug is selected from fluticasone (Hexalen®), capecitabine (Xeloda®), cyclophosphamide (Cytoxan®), gemcitabine (Gemzar®), irinotecan (CPT-11, Camptosar®), melphalan, pemetrexed (Alimta®), topotecan, vinorelbine (Navelbine®), 5-fluorouracil (5-FU), irinotecan (Camptosar), bevacizumab (Avastin), mitomycin, epirubicin, and oxaliplatin, and combinations thereof.

[0067] Combination therapies known in the art include, but are not limited to, one or more standard of care inhibitors or antiproliferative agents, such as a combination of bleomycin, etoposide, and cisplatin; or a combination of etoposide and cisplatin; a combination of VP-16 (etoposide) or vinblastine plus ifosfamide and cisplatin; a combination of carboplatin and paclitaxel; a combination of cisplatin and doxorubicin; a combination of carboplatin and docetaxel; a combination of cisplatin, paclitaxel, and doxorubicin; a combination of ifosfamide (Ifex®) and cisplatin. combinations of ifosfamide (Ifex®) and paclitaxel; combinations of trifluridine and tipiracil (Lonsurf); combinations of oxaliplatin and 5-FU / leucovorin (FOLFOX); combinations of oxaliplatin and capecitabine (CAPOX); combinations of 5-FU / leucovorin, oxaliplatin, and docetaxel; combinations of docetaxel or paclitaxel and either 5-FU or capecitabine; combinations of cisplatin and either 5-FU or capecitabine; or combinations of paclitaxel and carboplatin.

[0068] In some embodiments, antibody-drug conjugates can be used to treat cancers that overexpress ALPP and / or ALPP2 as described herein. In some embodiments, the antibody-drug conjugate specifically targets ALPP and / or ALPP2. Antibody-drug conjugates consist of three major components: an antibody specific for a particular cancer protein, a cytotoxic chemotherapeutic drug, and a linker protein connecting the two. In some embodiments, antibody-drug conjugates useful according to the present disclosure comprise an antibody that targets ALPP and / or ALPP2 conjugated to a chemotherapeutic drug. Any chemotherapeutic drug disclosed herein may be useful for use in an antibody-drug conjugate. Generally, when administered intravenously, the antibody portion targets the specific cancer protein and is taken up by the cancer cell, releasing the cytotoxic anti-cancer drug, which kills the cancer cell. Many antibody-drug conjugates are known in the art. One example is SGN-ALPV, a novel investigational antibody-drug conjugate composed of a humanized anti-ALPP / ALPPL2 monoclonal antibody, a protease-cleavable linker, and the microtubule inhibitor monomethyl auristatin E (MMAE). SGN-ALPV is undergoing phase I clinical trials in patients with solid tumors, such as ovarian tumors, endometrial tumors, non-small cell lung carcinoma, cervical tumors, and testicular tumors.Other useful antibody-drug conjugates include Tivdak®, used to treat cervical cancer; brentuximab vedotin (Adcetris®), used to treat relapsed Hodgkin's lymphoma and systemic anaplastic large cell lymphoma, both of which exhibit high expression of CD30; gemtuzumab ozogamicin (Mylotarg®), which targets the CD33 receptor found on certain types of myeloid cells and is approved for relapsed acute myeloid leukemia; and gemtuzumab ozogamicin (Mylotarg®), which targets the CD22 receptor and is approved for relapsed precursor B-cell acute lymphoblastic leukemia. These include inotuzumab ozogamicin (Besponsa®), approved for advanced lymphocytic leukemia; polatuzumab vedotin-piiq (Polivy®), which targets the CD79b receptor and is approved in combination with certain chemotherapy for relapsed diffuse large B-cell lymphoma; and Ado-trastuzumab emtansine (Kadcyla®), which targets the ERBB2 protein on the surface of certain breast cancer cells and is approved to treat advanced breast cancers that express this protein. Other antibody-drug conjugates include, but are not limited to, Mylotarg®, Adcetris®, Kadcyla®, Besponsa®, Lumoxiti®, Polivy®, Padcev®, Enhertu®, Trodelvy®, Blenrep®, Zynlonta™, Akalux®, Aidixi®, and Tivdak®.

[0069] In some embodiments, antibody-drug conjugates include SGN-ALPV, Adcetris®, Kadcyla®, Besponsa®, Mylotarg®, Polivy®, Padcev®, Enhertu®, Trodelvy®, Blenrep®, Zynlonta™, Akalux®, Aidixi®, and Tivdak®. Those of skill in the art will recognize that any suitable antibody-drug conjugate is useful in accordance with the present disclosure, and thus, other antibody-drug conjugates known or available in the art may be useful as described herein.

[0070] As described herein, the methods of the present disclosure are useful for treating non-small cell lung cancer (NSCLC) in patients whose cancer cells exhibit EGFR-activating mutations. Treatments for NSCLC are known in the art, and numerous therapies are available. In the present disclosure, administration of an antibody-drug conjugate targeting ALPP and / or ALPP2 can be used in conjunction with an EGFR inhibitor to treat NSCLC. As described in the Examples herein, such combination treatment can result in an increased or enhanced, i.e., advantageous, response of cancer cells to treatment compared to administration of either the antibody-drug conjugate or the EGFR inhibitor alone.

[0071] Identification of EGFR activating mutations can be achieved by any technique known and available in the art, including but not limited to DNA sequencing, proteomic analysis, staining techniques, immunohistochemistry, and the like.

[0072] Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapy is considered the standard treatment for lung cancers that inherently harbor EGFR activating mutations, but its effectiveness is limited.EGFR inhibitors are known and available in the art, including but not limited to gefitinib, osimertinib, mobocertinib, amivantamab, CLN081, and / or DZD9008.Other EGFR inhibitors can be used if deemed appropriate by a clinician or physician.The EGFR inhibitor can be a tyrosine kinase inhibitor (TKI).

[0073] In some embodiments, specific drugs or combinations of drugs or compounds described herein and known in the art can be used alone or in combination with cancer target antigens to treat specific types of cancers that overexpress ALPP and / or ALPP2. For example, in some embodiments, testicular cancer can be treated with cisplatin, etoposide (VP-16), bleomycin, ifosfamide (Ifex®), paclitaxel (Taxol®), and / or vinblastine. In some embodiments, combinations of bleomycin, etoposide, and cisplatin can also be used to treat testicular cancer, along with combinations of etoposide and cisplatin, or combinations of VP-16 (etoposide) or vinblastine, ifosfamide, and cisplatin.

[0074] In some embodiments, endometrial cancer may be treated with paclitaxel (Taxol®), carboplatin, doxorubicin (Adriamycin®) or liposomal doxorubicin (Docil®), cisplatin, or docetaxel (Taxotere®). Common combination therapies for endometrial cancer include, but are not limited to, carboplatin and paclitaxel, cisplatin and doxorubicin, and carboplatin and docetaxel, with cisplatin, paclitaxel, and doxorubicin being useful.

[0075] For carcinomas, treatment with ifosfamide (Ifex®) is often used, either alone or in combination with either cisplatin or paclitaxel. For carcinomas that are HER2-positive, trastuzumab (Herceptin®) may be added.

[0076] For ovarian cancer, the combination of a platinum agent, usually cisplatin or carboplatin, and a taxane, such as paclitaxel (Taxol®) or docetaxel (Taxotere®). Other drugs useful in the treatment of ovarian cancer include, but are not limited to, albumin-bound paclitaxel (nab-paclitaxel, Abraxane®), altretamine (Hexalen®), capecitabine (Xeloda®), cyclophosphamide (Cytoxan®), etoposide (VP-16), gemcitabine (Gemzar®), ifosfamide (Ifex®), irinotecan (CPT-11, Camptosar®), liposomal doxorubicin (Doxil®), melphalan, pemetrexed (Alimta®), topotecan, and vinorelbine (Navelbine®).

[0077] Cervical cancer can be treated with a combination of chemotherapy and radiation therapy, referred to herein as concurrent chemoradiotherapy. In some embodiments, cisplatin or carboplatin can be administered weekly during radiation therapy, or cisplatin can be combined with 5-fluorouracil (5-FU) and administered every three weeks during radiation therapy. For recurrent cervical cancer, cisplatin, carboplatin, paclitaxel (Taxol®), topotecan, docetaxel (Taxotere), ifosfamide (Ifex), 5-fluorouracil (5-FU), irinotecan (Camptosar), gemcitabine (Gemzar), and mitomycin can be used. In some embodiments, targeted drug therapy such as bevacizumab (Avastin) can be added in addition to chemotherapy treatment.

[0078] Gastric cancer can be treated with a number of chemotherapy drugs, such as 5-FU (fluorouracil), often administered with the combination drugs leucovorin (folinic acid), capecitabine, carboplatin, cisplatin, docetaxel, epirubicin, irinotecan, oxaliplatin, paclitaxel, or trifluridine and tipiracil (Lonsurf) in pill form. For early-stage cancer, some common drug combinations that can be used before and / or after surgery include, but are not limited to, oxaliplatin and 5-FU / leucovorin (FOLFOX), oxaliplatin plus capecitabine (CAPOX), 5-FU / leucovorin, oxaliplatin, and docetaxel, docetaxel or paclitaxel combined with either 5-FU or capecitabine, cisplatin combined with either 5-FU or capecitabine, or paclitaxel and carboplatin. When chemotherapy is administered along with post-surgical radiation therapy, single agents such as 5-FU or capecitabine may be used. In some embodiments, for advanced gastric cancer or gastric cancer, many of the same drug combinations can be used, although a combination of two drugs rather than three drugs may often be useful to reduce side effects. Some of the most commonly used combinations include, but are not limited to, oxaliplatin and 5-FU / leucovorin (FOLFOX), oxaliplatin and capecitabine (CAPOX), cisplatin combined with either 5-FU or capecitabine, irinotecan combined with 5-FU / leucovorin (FOLFIRI), paclitaxel combined with either cisplatin or carboplatin, docetaxel combined with cisplatin, epirubicin, either cisplatin or oxaliplatin, and either 5-FU or capecitabine, and docetaxel, 5-FU, and either cisplatin, carboplatin, or oxaliplatin.

[0079] Radionuclide therapy uses radioactive substances called radiopharmaceuticals to treat cancer. Radionuclides are introduced into the body by various means and, depending on their properties and route of administration, localize to specific locations, organs, or tissues. Radionuclides can be provided in a variety of particle types, such as α, β, γ, or combinations thereof.

[0080] In some embodiments, the radionuclide is an alpha particle such as polonium-210, bismuth-213, or uranium-238. In some embodiments, the radionuclide comprises an alpha particle selected from polonium-210 and uranium-238.

[0081] In some embodiments, the radionuclide is a beta particle, such as strontium-90, thallium-201, carbon-14, and tritium. In some embodiments, the radionuclide is a gamma particle, such as barium-133, cadmium-109, cobalt-57, cobalt-60, europium-152, manganese-54, sodium-22, zinc-65, and technetium-99m. In some embodiments, the radionuclide can be a combination of these particles, such as cesium-137 and americium-241. Any radionuclide known or available in the art can be used in accordance with the methods described herein.

[0082] In some embodiments, a combination of radionuclides may be used, with each radionuclide in the combination emitting radiation at a different wavelength such that each individual radionuclide is separately distinguishable from the others.

[0083] In some embodiments, the drug treatments described herein may be administered in a clinical setting, or in an alternate setting as deemed appropriate by a clinician or physician. In some embodiments, one or more standard of care inhibitors or antiproliferative agents that increase cell surface expression of one or more ALPP proteins and one or more ALPP protein targeting agents are administered simultaneously or sequentially.

[0084] In some embodiments, the one or more ALPP protein targeting agents comprise an antibody drug conjugate, an immunotherapy, a DNA cross-linking agent, or a radionuclide, or a combination thereof.

[0085] Any of the therapies described herein for treating cancers expressing ALPP and / or ALPP2 alone or in combination with a cancer target antigen, e.g., an oncogenic driver described herein, can be administered to a patient in need thereof (e.g., a patient with cancer) alone or in combination (i.e., by simultaneous or sequential administration) with a second drug therapy or other therapeutic treatment or drug (e.g., a chemotherapy or immunotherapy drug or treatment) that treats the cancer. In one embodiment, the additional therapeutic treatment or drug is included in a pharmaceutical composition described herein. In other embodiments, the additional therapeutic treatment or drug is co-administered, administered simultaneously, or administered sequentially in separate or distinct compositions.

[0086] As will be appreciated by those skilled in the art, the treatments described herein may be administered in any form necessary or useful for the subject for the treatment of cancer, including liquids (e.g., injectable and infusible solutions), semisolids, solids, aqueous solutions, suspensions, emulsions, gels, magmas, mixtures, tinctures, powders, capsules, dispersions, tablets, pellets, pills, powders, liposomes, lozenges, troches, liniments, ointments, lotions, pastes, suppositories, sprays, inhalants, and the like. In some embodiments, the drugs described herein for cancer treatment may be administered in liquid or aqueous form for injection into a patient, or in pills or tablets for oral administration. The dosage form of the drugs described herein may vary depending on the intended mode of administration and therapeutic use. Typically, the dosage form of the drug treatments described herein takes the form of an injectable or infusible solution, or a pill for oral administration.

[0087] In some embodiments, the drugs described herein for treating cancer in a patient can be administered by any route or mode of administration, including intraperitoneal, intravenous, oral, sublingual, rectal, vaginal, ophthalmic, otic, nasal, transdermal, enteral, epidural, intraarterial, intravascular, nasal, respiratory, subcutaneous, topical, transdermal, intramuscular, etc. In other embodiments, the second drug regimen, e.g., chemotherapy regimen, can take the form of an aqueous solution for intravenous administration.

[0088] Unless otherwise specified, the methods described herein may be performed according to procedures exemplified herein or may be performed routinely by methods well known in the art. The following sections provide additional guidance for performing the methods of the present disclosure.

[0089] In some embodiments, the cancer therapy administered to an individual for the treatment of cancer may be provided as a composition. In some embodiments, the methods described herein may include compositions administered as a single composition. In some embodiments, each drug may be administered separately (albeit simultaneously), i.e., in separate solutions or drug forms as described herein. Pharmaceutical formulations are well established and well known in the art.

[0090] In some embodiments, the drug or composition used in the methods described herein may be formulated with excipient materials such as sodium citrate, disodium hydrogen phosphate heptahydrate, monosodium phosphate, Tween 80, and / or stabilizers. The drug or composition used in the methods described herein may be provided at an appropriate concentration, e.g., in a buffer solution, and stored at a temperature appropriate to maintain drug efficacy, e.g., 2-8°C. In some other embodiments, the pH of the composition is about 5.5 to about 7.5 (e.g., 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5).

[0091] Pharmaceutical compositions for the described methods may also include an agent that reduces drug aggregation during formulation. Examples of aggregation-reducing agents include one or more amino acids selected from the group consisting of methionine, arginine, lysine, aspartic acid, glycine, and glutamic acid. Pharmaceutical compositions may also include sugars (e.g., sucrose, trehalose, mannitol, sorbitol, or xylitol) and / or osmolality adjusters (e.g., sodium chloride, mannitol, or sorbitol) and / or surfactants (e.g., polysorbate 20 or polysorbate 80).

[0092] As noted above for cancer treatment in the present disclosure, the compositions used in the described methods can be administered parenterally (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration, typically by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraarticular, intraocular, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injection and infusion.

[0093] The compositions used in the described methods can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for stable storage at high concentrations. Sterile injectable solutions can be prepared by incorporating the agents described herein above in the required amount in an appropriate solvent with one or a combination of the ingredients listed above and sterilizing by filtration. Generally, they are prepared by incorporating the agents described herein and any other required ingredients from those listed above into a sterile vehicle containing a basic dispersion medium. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying, which produce a powder of the agents described herein plus any additional desired ingredients from a previously sterile-filtered solution. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as monostearate salts and gelatin.

[0094] In certain embodiments, the compositions can be prepared using carriers that protect the compound against immediate release, such as sustained-release formulations, such as implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Many methods for preparing such formulations have been described or are generally known. See, for example, *Sustained and Controlled Release Drug Delivery Systems*, *J.R. Robinson*, ed., *Marcel Dekker, Inc., New York (1978).

[0095] In some embodiments, the compositions are formulated in sterile distilled water or phosphate buffered saline. The pH of the pharmaceutical formulation can be about 5.5 to about 7.5 (e.g., 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5).

[0096] Cancer therapies for treating cancers expressing ALPP and / or ALPP2 alone or in combination with a cancer target antigen, e.g., an oncogenic driver, can be provided in a kit. In one embodiment, the kit includes (a) a container housing the individual cancer therapies described herein, and optionally (b) instructional material. The instructional material can be descriptive, instructional, marketing, or other material regarding the use of the methods and / or agents described herein to obtain a therapeutic benefit.

[0097] In some embodiments, the kit also includes a second agent (e.g., a chemotherapeutic or immunotherapeutic agent described herein) for treating a cancer described herein. For example, the kit includes a first container containing a standard of care inhibitor or anti-proliferative agent and a second container containing a second dosing regimen, e.g., a chemotherapeutic or immunotherapeutic agent or drug combination.

[0098] The form of the instructional material in the kit is not limited. In one embodiment, the instructional material can include information about the production of the compound, the molecular weight of the compound, concentration, expiration date, batch or place of manufacture, etc. In one embodiment, the instructional material relates to methods of administering a cancer therapy to treat a subject with cancer, including, for example, administering a chemotherapeutic or immunotherapeutic agent in an appropriate dose, dosage form, or administration format (e.g., a dose, dosage form, or administration format described herein). The information can be provided in a variety of formats, including printed text, computer readable material, video recording, or audio recording, or information providing links or addresses to a wealth of resources, for example, on the internet, can be provided.

[0099] In addition to the cancer treatment or agent, the kit may include materials or reagents necessary to determine baseline levels of ALPP and / or ALPP2, along with other components, such as solvents or buffers, stabilizers, or preservatives. The cancer treatment may be provided in any form described herein, for example, in a substantially pure and / or sterilized liquid, dried, or lyophilized form. In some embodiments, when the agent is provided in a liquid solution, the liquid solution is an aqueous solution. When the agent is provided as a lyophilized product, the lyophilized powder is generally reconstituted by adding a suitable solvent. A solvent, such as sterile water or a buffer solution (e.g., PBS), may optionally be provided in the kit.

[0100] The kit may include one or more containers for the drugs or compositions. In some embodiments, the kit contains separate containers, dividers, or compartments for the drugs and instructional material. For example, the cancer treatment may be contained in a bottle, vial, or syringe, and the instructional material may be contained in a plastic sleeve or packet. In other embodiments, the separate components of the kit are contained in a single, undivided container. For example, the cancer treatment or agent may be contained in a bottle, vial, or syringe with the instructional material attached thereto in the form of a label. In some embodiments, the kit includes multiple individual containers (e.g., packs), each containing one or more unit dosage forms of the agent (e.g., dosage forms described herein). The containers may contain combination unit dosages, e.g., units containing both cancer therapies in a desired ratio. For example, the kit may include multiple syringes, ampoules, foil packets, blister packs, or medical devices, each containing a single combination unit dose. The containers of the kits can be airtight, waterproof (eg, impermeable to moisture change or evaporation), and / or light-tight.

[0101] The kit optionally includes a device suitable for administering the cancer treatment, such as a syringe or other suitable delivery device. The device may be provided pre-filled with one or both of the agents, or may be empty but suitable for loading.

[0102] definition Where a range of values ​​is provided, it is understood that, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, each intervening value between the upper and lower limit of that range, and any other stated value or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges are independently included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range.

[0103] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are described. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the disclosure is not entitled to antedate such publication by virtue of prior disclosure. Any conflict between the disclosures of references discussed herein and the present disclosure should be resolved in favor of the present disclosure. Further, the dates of publication may differ from the actual publication dates and may need to be independently confirmed.

[0104] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Some specific terminology relevant to the description of this disclosure is defined below.

[0105] As used herein and in the appended claims, along with similar references used in the context of describing particular embodiments (particularly in the specific context of the claims below), the singular forms "a," "an," and "the" can be interpreted to encompass both the singular and the plural, unless specifically stated otherwise. Thus, for example, "active agent" refers to not only one active agent but also a combination of two or more different active agents; "dosage form" refers to a combination of dosage forms as well as a single dosage form, etc. In some embodiments, the term "or" as used herein, including the claims, is used to mean "and / or" unless expressly specified to mean alternatives or the alternatives are not mutually exclusive.

[0106] In some embodiments, numbers expressing quantities of ingredients, properties, such as molecular weights, reaction conditions, and the like, used to describe and claim particular embodiments of the present disclosure are understood to be modified in some instances by the term "about." In some embodiments, the term "about" is used to indicate that a value includes the standard deviation of the average for the device or method being employed to determine that value. In some embodiments, the numerical parameters set forth in the written specification and attached claims are approximations that may vary depending on the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as practicable. The numerical values ​​set forth in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values ​​set forth herein is merely intended to serve as a shorthand method of referring to each separate value falling within that range. Unless otherwise specified herein, each individual value is incorporated herein as if individually set forth herein. In some embodiments, "about" means + / - 10% of the specified value.

[0107] The terms "comprise," "have," and "including" are open-ended linking verbs. Any form or tense of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes," and "including," is also open-ended. For example, a method that "comprises," "has," or "includes" one or more steps is not limited to possessing only those one or more steps, but may also include other steps not listed. Similarly, a composition or device that "comprises," "has," or "includes" one or more features is not limited to possessing only those one or more features, but may also include other features not listed.

[0108] As used herein, "co-administration" refers to the combined administration of one or more drugs with another drug. In some embodiments, both drugs are administered at the same time. Co-administration can also refer to a specific period of administration of either or both drugs. For example, as described herein, a drug can be administered several hours or days before the administration of another drug and still be considered co-administered. In some embodiments, co-administration can refer to any time of administration of either drug such that both drugs are present in the patient's body at the same time. In some embodiments, either drug can be administered before or after the other drug, as long as both are present in the patient for a sufficient length of time to provide the intended clinical or pharmacological benefit.

[0109] As used herein, "anti-cancer therapy" refers to a therapy for treating or preventing cancer. For example, an anti-cancer therapy described herein can refer to a chemotherapeutic drug or compound, or a combination of drugs or compounds, for treating cancer. In some embodiments, an anti-cancer therapy can refer to a drug or compound, or a combination of drugs or compounds, that induces immunogenicity in cells (e.g., cancer cells or cancer stem cells). In some embodiments, inducing immunogenicity in cancer cells can include any means of inducing cancer cells to progress to a more differentiated state, e.g., associated with a more limited proliferative capacity and a finite lifespan. This can be achieved, for example, by upregulating or enhancing the expression of self- and tumor antigens, which renders such cells more susceptible to immune-based therapies.

[0110] As used herein, "DNA damaging agent" refers to an agent or compound that introduces damage to the DNA of cancer cells through many mechanisms. For example, DNA damage can result from errors in DNA replication or from disruption of DNA repair mechanisms, which may include double-strand break (DSB) repair via the homologous recombination (HR) and non-homologous end joining (NHEJ) pathways, among others. DNA damaging agents are widely used in oncology to treat both hematological and solid cancers. Some commonly used modalities include, but are not limited to, ionizing radiation, platinum-based agents (e.g., cisplatin, oxaliplatin, and carboplatin), cyclophosphamide, chlorambucil, doxorubicin, and temozolomide. Other drugs commonly used in cancer treatment are described in detail herein. As used herein, "DNA damaging agent" may refer to a chemotherapeutic agent or regimen, or any agent known or available in the art that can introduce DNA damage to cancer cells.

[0111] As used herein, "EGFR" refers to the EGFR gene, also known as the "epidermal growth factor" gene and "HER1." Many mutations in EGFR are known to be associated with cancers, such as NSCLC.

[0112] As used herein, "activating EGFR mutation" refers to a mutation in the EGFR gene in the DNA of a patient's cells that results in abnormal expression of the EGFR gene, i.e., a mutation that "activates" the expression of the EGFR gene. Activating EGFR mutations are most commonly found in non-small cell lung cancer (NSCLC), glioblastoma, and colorectal cancer. Activating EGFR mutations are usually found in exons 18-21 of the EGFR gene, which are the portions of the gene that encode the tyrosine kinase domain of the EGFR protein. EGFR mutations according to the present disclosure can be point mutations, missense mutations, substitution mutations, etc. The most common activating mutations identified in NSCLC are exon 19 deletions and the L858R point mutation in exon 21. Other EGFR mutations found in lung cancer include, but are not limited to, the C797S mutation, and EGFR exon 20 insertions, T790M substitutions, and the L858R missense mutation.

[0113] As described herein, the methods of the present invention are useful for treating NSCLC in patients or subjects with EGFR-activating mutations. Regarding EGFR-activating mutations, the genetic mutations cause cancer cells to be resistant to inhibitors. For example, non-small cell lung cancer with EGFR mutations is resistant to inhibitors that target EGFR, such as EGFR inhibitors. Some patients who will benefit from the methods of the present invention will be patients with NSCLC that is resistant to inhibitors that target EGFR. Combination therapy comprising administering an antibody-drug conjugate targeting ALPP and / or ALPP2 and an EGFR inhibitor described herein.

[0114] As used herein, "drug-resistant cells" or "DRCs" refer to cells that have developed resistance to a particular drug. As used herein, non-small cell lung cancer cells can be or can develop into drug-resistant cells.

[0115] As used herein, "drug-resistant persister cells" or "DTPCs" refer to cells that exhibit a reversible phenotype. DTPCs can regain proliferation and drug sensitivity after discontinuation of drug treatment. DTPCs may eventually acquire several types of drug resistance mechanisms under continuous treatment. As described herein, non-small cell lung cancer cells can be or develop into drug-resistant cells. The methods described herein prevent drug-resistant persister cells (DTPCs) from developing into drug-resistant cells (DRCs). The methods described herein, i.e., administration of an antibody-drug conjugate targeting ALPP and / or ALPP2 in conjunction with an EGFR inhibitor, also treat drug-resistant persister cells (DTPCs) and drug-resistant cells (DRCs) by preventing the emergence of resistance to the EGFR inhibitors described herein.

[0116] Pharmaceutical compositions comprising one or more cancer treatments described herein can contain a therapeutically effective amount of a cancer treatment described herein. The terms "therapeutically effective amount," "pharmacologically effective dose," "pharmacologically effective amount," or simply "effective amount" can be used interchangeably and refer to an amount of an agent effective to achieve an intended pharmacological therapeutic or prophylactic result, e.g., a reduction in cancer cells or cancer cell burden (i.e., a reduction in cancer cell number), tumor size, tumor density, lymph node involvement, metastasis, cancer recurrence or relapse, or associated symptoms in a patient. A pharmacologically effective amount improves one or more symptoms of a disease (e.g., a hematological cancer), prevents disease progression, causes disease regression, or prevents disease. Such an effective amount can be determined based on the effect of the administered agent, e.g., the effect of a cancer treatment described herein, and the combined effect of the agents when multiple agents are used, e.g., the effect of a second treatment or agent described herein following a described cancer treatment. A therapeutically effective amount of an agent may vary depending on factors such as the individual's disease stage, condition, age, sex, and weight, as well as the ability of the compound to elicit a desired response in the individual, e.g., an improvement in at least one disease parameter or an improvement in at least one symptom of the disease. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. In some embodiments, an "effective amount" is an amount that treats (including prevents) one or more symptoms and / or underlying causes of cancer. In some embodiments, an effective amount is a therapeutically effective amount. In some embodiments, an effective amount is an amount that prevents the appearance of one or more signs or symptoms of a particular disease or condition.

[0117] As used herein, "gene expression" or "expression" refers to the processes of gene transcription, translation, and post-translational modification.

[0118] As used herein, "immunotherapeutic" refers to a drug or compound that stimulates or suppresses the immune system to help the body fight cancer, infection, and / or other diseases. An immunotherapeutic may be an antibody, e.g., a monoclonal antibody. In some embodiments, an immunotherapeutic may be an immune checkpoint inhibitor, e.g., a drug that targets programmed cell death protein 1 (PD-1) or its ligand, PDL-1, and / or the cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor. For example, useful immunotherapeutics include, but are not limited to, pembrolizumab (Keytruda®), ipilimumab (Yervoy®), pembrolizumab (Keytruda®), nivolumab (Opdivo®), atezolizumab (Tecentriq®), pidilizumab (CT-011), toripalimab (JS-001), avelumab (Bavencio®), tislelizumab (BGB-A317), durvalumab (Imfinzi®), and cemiplimab (Libtayo®). Immunotherapeutics that target PD-1 include, but are not limited to, pembrolizumab (Keytruda®), nivolumab (Opdivo®), and cemiplimab (Libtayo®). Immunotherapeutics that target PD-L1 include, but are not limited to, atezolizumab (Tecentriq®), avelumab (Bavencio®), and durvalumab (Imfinzi®). Immunotherapeutics that target CTLA-4 include, but are not limited to, ipilimumab (Yervoy®). Additional immunotherapeutics that target these checkpoint inhibitors are known and available in the art and are encompassed within the scope of this disclosure.

[0119] "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material can be incorporated into a pharmaceutical composition administered to a patient without producing any untoward biological effects or adversely interacting with any of the other components of the composition in which it is contained. When the term "pharmaceutically acceptable" is used to refer to a pharmaceutical carrier or excipient, it means that the carrier or excipient has met the required standards of toxicology and manufacturing testing or is included in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration or an equivalent foreign regulatory agency. "Pharmacologically active" (or simply "active") in "pharmacologically active" (or "active") derivatives or analogs refers to derivatives or analogs that have the same type of pharmacological activity as the parent compound, and to approximately the same degree. Some pharmacologically active derivatives may have improved pharmacological activity. The term "pharmaceutically acceptable salts" includes acid addition salts formed, for example, with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0120] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. The compositions may include pharmaceutically acceptable salts, for example, acid addition salts or base addition salts.

[0121] As used herein, "reduce" means a decrease or decrease, such as a reduction in cancer cell burden. In some embodiments, administration of a cancer treatment described herein may "reduce" or decrease the cancer cell burden (i.e., a decrease in the number of cancer cells) in a patient compared to a patient not receiving such a drug, resulting in a reduction in tumor size, tumor density, blast cell involvement, proliferation, percentage of quiescent (G0) cells, lymph node involvement, metastasis, or associated symptoms. "Reduction" can also mean a reduction in disease symptoms as a result of a treatment described herein, alone or co-administered with another drug.

[0122] As used herein, the term "cancer" means a malignant tumor characterized by abnormal proliferation of cells, the growth of which exceeds and is out of step with that of the surrounding normal tissue.

[0123] As used herein, "oncogenic driver" refers to a gene involved in the development or maintenance of cancer. Cancer-causing mutations tend to affect three main types of genes: protooncogenes, tumor suppressor genes, and DNA repair genes. These changes are sometimes called the "drivers" of cancer. These mutations are often found in genes that encode signaling proteins important for maintaining normal cell growth and survival.

[0124] As used herein, "subject" or "patient" means a mammal, preferably a human, who has cancer and who may be further treated.

[0125] As used herein, "healthy" means an individual who is cancer-free.

[0126] As used herein, the term "ELISA" refers to enzyme-linked immunosorbent assay. This assay generally involves contacting a fluorescently labeled sample of a protein with an antibody that has specific affinity for that protein. Detection of these proteins can be achieved by various means, including, but not limited to, laser fluorimetry.

[0127] As used herein, "subject" or "individual" or "patient" refers to any patient for whom both are desired, and generally refers to the recipient of therapy. "Subject" or "patient" refers to any animal classified as a mammal, both human and non-human mammals. Examples of non-human animals include dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Unless otherwise specified, "patient" or "subject" are used interchangeably herein. In some embodiments, subjects suitable for therapeutic use may be primates, e.g., humans and non-human primates.

[0128] As used herein, the terms "treating" and "treatment" or "alleviation" refer to a reduction or decrease in the severity and / or frequency of symptoms, elimination of symptoms and / or underlying causes, and reversal or amelioration of damage. In certain embodiments, "treating" and "treatment" as used herein refer to the prevention of recurrence of symptoms. In other embodiments, "treating" and "treatment" as used herein refer to the prevention of the underlying cause of symptoms associated with a disease or condition, such as blood cancer. The phrase "administering to a patient" refers to the process of introducing a composition or drug into a patient via any art-recognized means of introduction. "Treatment" or "alleviation" also includes the administration of a compound or agent to a subject to alleviate symptoms, arrest or inhibit further development of a disease, condition, or disorder, to prevent or delay the onset of symptoms, complications, or physiological signs of a disease (e.g., cancer). Subjects in need of treatment include subjects already suffering from a disease or condition, subjects who have previously suffered from a disease or condition and are at risk of recurrence, and subjects at risk of developing a disease or condition.

[0129] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all example or exemplary language (e.g., "etc.") provided with respect to specific embodiments herein is intended merely to better clarify the disclosure and does not impose limitations on the scope of the disclosure as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0130] Groupings of alternative elements or embodiments of the disclosure disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in combination with other members of the group or other elements described herein. One or more members of a group may be included in, or deleted from, a group for reasons of convenience or patentability.

[0131] The present disclosure has been described in detail, and it will be apparent that modifications, variations, and equivalents are possible without departing from the scope of the present disclosure as defined in the appended claims. Furthermore, it should be understood that all examples in this disclosure are provided as non-limiting examples. [Example]

[0132] The following examples are included to demonstrate embodiments of the disclosure. The following examples are presented merely as illustrations to assist those skilled in the art in using the present disclosure. The examples are not intended to limit the scope of the disclosure in any way. Those skilled in the art will understand, in light of the present disclosure, that many changes can be made to the specific embodiments disclosed and still obtain similar results without departing from the spirit and scope of the present disclosure.

[0133] To illustrate the methods claimed in this invention, treatment of EGFR-expressing lung adenocarcinoma cells with a small molecule EGFR inhibitor to amplify ALPP cell surface expression is demonstrated in vitro and in vivo. Furthermore, treatment with other therapeutic agents is demonstrated to enhance ALPP protein expression. Treatment of lung adenocarcinoma-bearing mice with the EGFR inhibitor gefitinib, together with an anti-ALPP antibody conjugated with monomethyl auristatin F (MMAF), resulted in significantly improved anti-cancer efficacy compared with gefitinib or MMAF treatment alone. Similar combined efficacy of gefitinib + anti-ALPP-MMAF was confirmed in in vitro and in vivo models of triple-negative breast cancer.

[0134] As described herein below, diagnostic tests for combination therapy involving anti-ALPP / ALPPL2 agents include: (1) assessing baseline ALPP / ALPPL2 surface expression in cancer cells, as measured by histological analysis of tissue biopsies or blood-based tests for evidence of tumor dissemination of ALPP / ALPPL2 expression; and (2) assessing co-expression of cancer cell surface oncogenic drivers that are directly targetable with inhibitors.

[0135] Furthermore, a "two-hit" combination treatment strategy is demonstrated, including: (1) treatment with standard of care inhibitors, antiproliferative agents, to enhance cancer cell surface ALPP / ALPPL2 expression; and (2) anti-ALPP / ALPPL2 antibody-drug conjugates or immunotherapy or radionuclide therapy.

[0136] Example 1 Immunohistochemistry (IHC) analysis of ALPP protein in normal tissues showed limited expression in the testis and placenta, with negative results in the heart, kidney, ovary, stomach, brain, liver, spleen, lung, colon, and mammary gland (Figure 1A, B). Conversely, representative IHC staining of ALPP in lung and breast tumors showed strong positivity (Figure 1A, B). Representative target EGFR and ALPP / ALPPL2 expression by mass spectrometry of biotin-labeled surface proteins frequently coexpressed in seven cancer types is shown in Figure 1C.

[0137] As proof of concept, H1650 lung adenocarcinoma cells, which co-express EGFR and ALPP, were treated with several EGFR-targeting TKIs (Figure 2B), demonstrating upregulation of ALPP (Figures 2A and 2B). Induction of ALPP by EGFR-TKI stimulation was also extendable to breast cancer cells using SKBR3 cells (Figure 2D). When basal ALPP expression was negative in MCF7 breast cancer cells, receptor inhibition did not induce ALPP (Figures 2C and 2D). Stimulation with an anti-HER2 (ERBB2) monoclonal antibody against HER2 expressed by SKBR3 cells similarly induced ALPP compared with EGFR inhibition with small molecule TKIs, confirming the broad applicability of the concept of detecting and targeting cell surface receptors (Figure 2E).

[0138] Targeting EGFR with inhibitors, such as gefitinib in the EGFR-overexpressing mutant lung adenocarcinoma (LUAD) cell lines H1650 and PC9, significantly upregulated surface ALPP expression, as confirmed by mass spectrometry and immunofluorescence (Figure 3A-D). Immunoblotting further confirmed ALPP upregulation after gefitinib treatment, and stimulation of EGFR with its natural ligand, EGF, reduced ALPP expression (Figure 3B). Treatment of the EGFR wild-type, KRAS-mutant LUAD cell lines H2291 and H1838 with gefitinib also increased ALPP expression, albeit to a lesser extent (Figure 3B).

[0139] Targeting cancer cell surface ALPP with an anti-ALPP antibody demonstrated internalization in EGFR-mutant H1650LUAD cells (Figure 4A). Next, an anti-ALPP antibody conjugated with monomethyl auristatin F (MMAF) was synthesized. MMAF is a microtubule inhibitor that induces cell cycle arrest and apoptosis. Briefly, the drug-linker conjugate was Mc-vc-PAB-MMAF. The payload drug in this conjugate, MMAF, is an auristatin derivative that induces cell death by disrupting microtubule dynamics. The linker consists of a valine-citrulline (Val-Cit) dipeptide module that is cleaved in the lysosome after internalization of the antibody-drug conjugate (ADC) and a self-demolishing PBA module that releases MMAF in its native form. Mc-vc-PAB-MMAF was prepared by CBL, and the purity of the compound is over 95%. Mc-vc-PAB-MMAF is conjugated to Cys residues on antibodies via maleimide functional groups. Typical conjugation procedures include partial reduction of antibodies using either dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to generate free interchain cysteine ​​residues. Mc-vc-PAB-MMAF is then conjugated to antibodies via these free cysteine ​​residues via a maleimide-thiol reaction. After conjugation, the remaining free Mc-vc-PAB-MMAF is quenched, and all small molecule species are removed by ultrafiltration to yield the ADC.

[0140] The efficacy of gefitinib and ALPP-MMAF-ADC was tested in vitro using H1650LUAD cells, both alone and in combination, for anticancer efficacy. The combination treatment demonstrated synergistic effects and the highest anticancer efficacy (Figure 4B-C). This strategy was further investigated using an orthotopic xenograft model in H1650 tumor-bearing mice, confirming that the combination of gefitinib and ALPP-MMAF-ADC enhanced anticancer activity compared with control or either treatment alone (Figure 4E).

[0141] To demonstrate broader clinical relevance, additional studies were performed using triple-negative breast cancer (TNBC) cells, which similarly demonstrated high surface ALPP expression correlated with surface EGFR expression. Consistent with the findings in lung cancer, EGFR inhibition by gefitinib increased ALPP expression in the TNBC cell lines BT20 and HCC1937 (Figure 5B). Combination treatment with gefitinib and ALPP-MMAF-ADC resulted in robust anticancer effects in vitro and in an orthotopic xenograft model of TNBC (Figure 5C-D).

[0142] Table 1 shows a list of cell surface targets, in addition to EGFR and HER2, that are co-expressed with ALPP / ALPPL2 on lung adenocarcinoma and breast tumors that are known to be treated with drugs. These surface target candidates have good overlap and extendibility across various cancer types.

[0143] A diagnostic test that determines cell surface target and ALPP / ALPPL2 co-expression by IHC or other means will allow for the assessment of potential benefit from combining inhibitors with ALPP-targeted therapy across a wide range of cancer types.

[0144] [Table 1]

[0145] [Table 2]

[0146] [Table 3]

[0147] Example 2 Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapy is considered the standard of care for lung cancers harboring endogenous EGFR-activating mutations, but its efficacy is limited. Placental alkaline phosphatase (ALPP) and the related ALPPL2 are expressed in various solid tumors, including lung cancer. While tumors with EGFR-activating mutations suppress ALPP expression, EGFR TKIs have been shown to upregulate and maintain ALPP surface expression in lung adenocarcinoma (LUAD). EGFR inhibition results in the dephosphorylation and activation of FoxO3a, a transcriptional regulator of ALPP, leading to its overexpression. In vitro evaluation of the combination of an EGFR TKI and an ALPP antibody conjugated with monomethyl auristatin F (ALPP-MMAF) significantly increased cell death in the EGFR-mutated LUAD cell lines HCC827 and PC9 compared with either treatment alone. EGFR TKIs also upregulated ALPP expression and enhanced targeted cell death of drug-resistant LUAD cells. Gefitinib treatment in a gefitinib-resistant LUAD xenograft model upregulated ALPP expression in tumor cells, but not in normal tissues. Gefitinib plus ALPP-MMAF induced greater tumor regression than gefitinib or ALPP-MMAF alone. These findings support the use of combination therapy containing an EGFR inhibitor together with an ALPP antibody-drug conjugate for LUAD.

[0148] The epidermal growth factor receptor (EGFR), a type 1 receptor tyrosine kinase, is activated by EGF and other ligands and transmits important cell proliferation signals. It is expressed in over 60% of lung adenocarcinomas (LUAD) and is mutated in 40–60% of tumors from Southeast Asian patients and 10–15% of Caucasians. EGFR TKI therapy is used as the standard of care for lung cancer patients who inherently harbor activating EGFR mutations. Despite prolonged overall survival and high initial responses to KI therapy, the disease eventually progresses, primarily due to insufficient tumor cell killing and subsequent acquisition of drug resistance. TKI-refractory tumors also become resistant to most available drugs, as well as immune checkpoint inhibitors. There remains an unmet need for novel treatment modalities for patients with activating EGFR mutations.

[0149] Placental alkaline phosphatase (ALPP, also known as PLAP) and ALPPL2 (also known as ALPG) are members of the alkaline phosphatase family that are exclusively expressed in the placenta and share 98% similarity in their amino acid sequences. Several studies have described the expression of ALPP and ALPPL2 in various cancer types. To this end, evaluation of ALPP protein levels in 12,381 tumors by immunohistochemistry demonstrated strong ALPP expression in seminomas, primarily embryonal carcinomas of the testis, and yolk sac tumors, with reduced expression in other cancer types. In lung cancer, ALPP expression is relatively reduced and restricted to adenocarcinomas.

[0150] The restricted expression of ALPP and ALPPL2 in normal tissues and their accessibility on the surface of cancer cells support their potential as therapeutic targets. To this end, chemical library screening yielded a selective and potent ALPP inhibitor that specifically bound to ALPP-positive tumors in vitro and targeted cervical cancer in mouse models of the disease. A fluorescent derivative of the ALPP inhibitor served as a bispecific engager to target chimeric antigen receptor (CAR) T cells to fluorescein on LPP-positive tumor cells for cancer cell killing mediated by CAR T cells. Targeting ALPP with α-ALPP and α-CD3 bispecific antibodies resulted in the death of ALPP-positive colorectal cancer cell lines. The expression of ALPP in colorectal cancer has led to the investigation of CAR T cell therapy. ALPP-CAR T cells mediate potent cytotoxicity against cancer cells, while the combination of ALPP-CAR T cells with anti-PD-1, PD-L1, or LAG-3 checkpoint inhibitors further enhanced the therapeutic efficacy of CAR T cells. Second-generation CART cells carrying a fully humanized scFv against ALPP effectively killed ALPP-expressing HeLa cells. Clinical trials using α-ALPPCART cells have been initiated for ovarian and endometrial cancer, and another clinical trial is underway to evaluate the efficacy of an ALPP / ALPPL2 antibody-drug conjugate in advanced solid tumors.

[0151] In the current study, we investigated the factors involved in regulating ALPP expression in LUAD. We demonstrated that ALPP expression in LUAD with EGFR-activating mutations was significantly lower than in EGFR wild-type (WT) tumors, and that EGFR TKIs substantially upregulate ALPP expression. FoxO3a was identified as an upstream transcriptional regulator of ALPP in the setting of EGFR inhibition. Combination therapy with EGFR TKIs and ALPP-MMAF resulted in enhanced anticancer activity and prevented the formation of drug-resistant cell clones from the EGFR TKI-sensitive LUAD cell lines HCC827 and PC9 in vitro. The combination therapy also enhanced anticancer responses compared with gefitinib or ALPP-MMAF alone in a xenograft mouse model of LUAD.

[0152] cell culture Detailed information about the human cancer cell lines used in this study is shown in Table 4. Cells were cultured in RPMI 1640 (Cat. No. 10-040-CV, Corning) supplemented with 10% inactivated fetal bovine serum (FBS, Cat. No. 16140-071, Gibco) and maintained at 37°C in a humidified atmosphere containing 5% CO2.

[0153] Antibodies, chemicals, and virus strains Detailed information on the antibodies, chemicals, and virus strains used in this study is shown in Table 5. ALPP antibody (catalog no. NB110-3638, Novus biologicals) was conjugated with monomethyl auristatin F (MMAF) by Creative Biolabs.

[0154] Proteomic analysis ALPP and ALPPL2 expression in whole cell extracts and surfaceome compartments was analyzed by mass spectrometry as described above. 19~23 Each data set was normalized to the total number of spectral counts in each compartment. 24 .

[0155] FoxO3a overexpression The FoxO3a open reading frame was inserted into the pLOC-RFP vector and packaged into lentivirus. Stable FoxO3a overexpression in the LUAD cell lines HCC827 and H1650 was achieved by lentiviral infection, followed by selection of blasticidin-resistant cells and verification of FoxO3a overexpression by immunoblotting.

[0156] Immunofluorescence LUAD cells fixed with 1% paraformaldehyde were stained with ALPP antibody (catalog no. NB110-3638, Novus biologicals) and Alexa488-conjugated secondary antibody. Images were acquired in the z-series on a spinning disk confocal system.

[0157] Flow cytometry LUAD cells were stained with ALPP antibody (catalog no. NB110-3638, Novus Biologicals) and Alexa488-conjugated secondary antibody (catalog no. A11029, Invitrogen), followed by flow cytometry analysis using an LSRII flow cytometer (BD).

[0158] For flow cytometry-based cell cycle analysis, ethanol-fixed cells were stained in Hoechst 33342 (1 μg / ml, Cat. No. 62249, Invitrogen), followed by cell cycle analysis using an LSRII flow cytometer (BD).

[0159] Immunohistochemistry (IHC) analysis The tissue microarrays for IHC staining of ALPP in this study included 204 surgically removed lung cancer tumor specimens collected under institutional review board protocols and stored as formalin-fixed, paraffin-embedded specimens at The University of Texas Specialized Program of Research Excellence thoracic tissue bank at The University of Texas MD Anderson Cancer Center. Patient characteristics of the analyzed cohort are shown in Table 3. Human normal tissue microarrays for IHC staining of ALPP were obtained from Novus Biologicals (catalog number NB110-3638). IHC staining was performed as described above. 25 .

[0160] Cell viability assay The cell viability of LUAD cells was determined using the CellTiter96 Aqueous One Solution Cell Proliferation Assay (MTS) kit (catalog no. G3580, Promega).

[0161] Chromatin immunoprecipitation (ChIP) ChIP was performed in HCC827 and H1650 cells using the Pierce Magnetic ChIP Kit (catalog no. 26157, Thermo Scientific) according to the manufacturer's instructions. Nuclear lysates were incubated with antibodies (positive antibody: 10 μL; negative antibody: 2 μL; FoxO3a antibody: 8 μL (catalog no. 720128, Invitrogen)) overnight at 4°C. DNA-protein complexes were then enriched with Protein A / G magnetic beads, and DNA was recovered using a DNA clean-up column. The relative abundance of genomic DNA fragments was determined using Sso Advanced Universal SYBR Green Supermix (catalog no. 1725271, Bio-Rad) with the primers listed in SEQ ID NOs: 1–10 in Table 2 below.

[0162] [Table 4]

[0163] Total RNA extraction and quantitative PCR (qPCR) Total RNA was extracted using the RNeasy Mini Kit (Cat. No. 74104, QIAGEN). Reverse transcription of total RNA was performed using the High-Capacity Reverse Transcriptase Kit (Cat. No. 4368813, Applied Biosystems). TaqMan Universal Master Mix II (Cat. No. 4440040, Applied Biosystems) was used for ALPP expression analysis. The ALPP probe was purchased from Thermo Fisher Scientific (Hs03046558_s1).

[0164] Immunoblotting Total protein was extracted in RIPA buffer (catalog no. 89901, Pierce) containing complete protease inhibitor cocktail (catalog no. 04693116001, Roche) and phosSTOP (catalog no. 04906837001, Roche). Antibody information is shown in Table 4.

[0165] Antibody internalization assay Antibody internalization was assessed using the pHrodo™ iFL Red Microscale Protein Labeling Kit (Cat. No. P36014, Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, 100 μg of anti-ALPP antibody (Cat. No. MAB59051, R&D Systems) in 100 μl of PBS was mixed with 10 μl of 1 M sodium bicarbonate and 5 μl of 2 mM pHrodo, followed by incubation at room temperature for 30 minutes. pHrodo-conjugated anti-ALPP antibody was then purified using gel resin. Cells were washed twice with PBS and incubated with pHrodo-conjugated anti-ALPP antibody (10 μg / ml) for 24 hours, followed by fixation with 2% paraformaldehyde, washing twice with PBS, and mounting with DAPI. Images were captured using a fluorescence microscope.

[0166] In-vivo studies The animal experimental protocol was approved by the University of Texas MD Anderson Cancer Center IRB and in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the NIH (Bethesda, MD). BALB / c nude mice (catalog no. 194, Charles River) were housed in a specific pathogen-free facility. For the orthotopic xenograft model of LUAD, a total of 1 × 10 firefly luciferase-expressing BALB / c nude mice were cultured in a specific pathogen-free facility. 6H1650 cell line cells were suspended in 50 μl of 50% Matrigel Matrix (Corning) / Opti-MEM medium and injected into the lungs of 8-10 week-old nude mice. For gefitinib treatment, mice were administered gefitinib (50 mg / kg) by oral gavage for 10 days, starting on day 17 after cancer cell inoculation. For gefitinib / ALPP-ADC combination treatment, mice were administered an additional ALPP antibody conjugated with MMAF (ALPP-MMAF) (5 mg / kg, intravenously) on days 19 and 26. Tumor growth was monitored twice weekly for 5 weeks using a Xenogeny In Vivo Imaging System (IVIS, Alameda, CA). Mice were euthanized on day 30. Tumors were collected and processed for routine histological and immunohistochemical analysis.

[0167] Gene Set Enrichment Analysis (GESA) To perform GESA, we calculated signal enrichment scores using the “ReactomePA” and “clusterProfiler” packages in R and the GSEA dataset (gsea-msigdb.org / gsea / index.jsp) with a significance threshold of FDR q-value <0.05. 26,27 The 'enrichplot' package was applied to visualize the enrichment results.

[0168] Single-cell RNA-sequencing (scRNA-seq) Single-cell RNA-sequencing GEO dataset (GSE150949) 28The data were retrieved from [https: / / www.r-project.org / ]. The Seurat package (version 4.3.0) implemented in R statistical software (version 4.3) (https: / / www.r-project.org / ) was used for preprocessing, principal component analysis (PCA)-based dimensionality reduction, and t-SNE to visualize cell clusters from different treatment groups. The expression and percentage of ALPP were calculated accordingly. Furthermore, cells were classified into S / G2M or G1 phase, and cell cycle scores were calculated by the CellCycleScoring function under the Seurat package. 29 .

[0169] statistical analysis For continuous variables, statistical significance was determined by a two-tailed Student's t-test unless otherwise specified. For categorical variables, statistical significance was determined by Fisher's exact test for categorical comparisons of two classes or by a chi-square test for trend of multiple categorical variables. Figures were created with GraphPad Prism Version 9.0.0.

[0170] result ALPP and ALPPL2 expression in LUAD To evaluate the expression of ALPP and ALPPL2 in lung cancer, we assessed ALPP and ALPPL2 protein levels in whole-cell extracts (WCEs) and surface compartments of 49 lung adenocarcinoma (LUAD) and 22 small cell lung cancer (SCLC) cell lines. This revealed that ALPP, and to a lesser extent ALPPL2, are predominantly expressed in LUAD compared with SCLC (Figure 6A and Table 4). Evaluation of their mRNA expression levels in 48 LUAD, 24 lung squamous cell carcinoma (LUSC), and 50 SCLC cell lines from the Cancer Cell Line Encyclopedia (CCLE), similarly revealed that ALPP is predominantly expressed in LUAD (Figure 6B), consistent with the proteomics results. Analysis of ALPP and ALPPL2 mRNA expression in LUAD and LUSC in The Cancer Genome Atlas (TCGA) also revealed higher expression in LUAD compared with LUSC (Figure 6C).

[0171] Because the expression level of ALPP is higher than that of ALPPL2 in LUAD (Figures 6A-C), we focused on ALPP as a potential therapeutic target. ALPP expression was assessed by immunohistochemistry (IHC) using tissue microarrays (TMAs) consisting of 140 LUAD and 64 LUSC tumors. Of 204 tumors, 37 (18.1%) stained positive for cytosolic ALPP, and 23 (11.3%) stained positive for membrane ALPP (Figure 6D). ALPP surface expression was also confirmed by immunofluorescence in EGFR-mutant HCC827 and PC9 LUAD cells (Figure 6E). Positive membrane staining for ALPP was more frequent in LUAD tumors (21 of 140) than in LUSC tumors (2 of 64; Fisher's exact test p-value: 0.015), consistent with previous reports. Positive ALPP membrane staining was associated with smoking status (chi-squared test for trend p-value: 0.0001) (Table 3). There was no statistical evidence of an association between positive tumor ALPP membrane staining and gender or age (Table 3). ALPP expression in normal human tissues was evaluated. Except for placenta and testis tissue, ALPP protein expression was negative in other tissues, including lung (Figures 14A-C).

[0172] [Table 5]

[0173] EGFR TKIs upregulate ALPP expression in LUAD cells The relatively low expression of ALPP in LUAD may limit its usefulness as a therapeutic target. Although oncogene activation typically leads to unchecked cell proliferation, previous studies have reported an inverse association between ALPP expression and cell proliferation. To investigate whether oncogenes contribute to the suppression of ALPP expression, ALPP gene expression in LUAD was compared with wild-type and driver mutations in EGFR, KRAS, NF1, and BRAF. LUAD with expressed EGFR driver mutations had reduced ALPP compared with EGFR wild-type (WT) tumors in the TCGA dataset (Figure 7A), consistent with findings from an independent cohort of 181 East Asian patients with LUAD (Figure 15A). To test whether the effect of EGFR signaling on ALPP expression depends on its activating mutation status, gefitinib-resistant EGFR-mutant H1650 and EGFR wild-type H2291 LUAD cells were cultured in growth medium with or without epidermal growth factor (EGF), the endogenous ligand for EGFR. ALPP expression was dramatically reduced in both cell lines cultured in EGF-containing growth medium (Figure 7B). EGF induced AKT phosphorylation at serine 473 (Ser473) and ERK phosphorylation at threonine 202 (T202) and tyrosine 204 (Y204) residues, whereas treatment with the EGFR TKI gefitinib suppressed AKT and ERK phosphorylation (Figure 7C). Furthermore, EGFR TKI-sensitive EGFR-mutant HCC827 and PC9 cells treated with gefitinib or osimertinib showed approximately 60-fold and 110-fold increases in ALPP expression (Figures 7D and 7B). Immunofluorescence staining and flow cytometry analysis demonstrated enrichment of cell surface expression of ALPP upon EGFR inhibition (Figures 7E and 7F). Expression levels of ALPP from independent datasets confirmed a consistent increase with EGFR TKI treatment (Figures 7G-H and 15C).

[0174] To determine whether other EGFR inhibitors induce ALPP expression, we treated EGFR-mutant H1650 and HCC827 LUAD cells with inhibitors such as lapatinib, afatinib, and osimertinib, and similar results were obtained (Figure 7I). EGFR inhibitor-induced ALPP upregulation was also confirmed in the EGFR wild-type LUAD cell lines H2291, H1838, and H1651 (Figure 7J). However, the ALPP-nonexpressing cell lines H1395, H1944, and HCC2279 did not upregulate ALPP after gefitinib treatment, whereas the ALPP-positive LUAD cell lines H2291, H1650, H1838, and H1651 showed increased ALPP levels (Figure 7J).

[0175] FoxO3a is a transcriptional regulator of ALPP Given the inverse association between ALPP expression and cell proliferation, GESA analysis of the TCGA LUAD dataset confirmed the inverse correlation between ALPP expression and cell cycle (Figure 8A). KEGG pathway analysis also revealed cell cycle, with one of the top pathways inversely correlated with ALPP expression (Figure 8B). Treatment of HCC827 and PC9 cells with gefitinib or osimertinib revealed a statistically significant increase in cell cycle arrest at G0 / G1 (Figure 8C).

[0176] We next examined whether signaling pathways immediately downstream of EGFR are involved in ALPP upregulation. Small molecule inhibition of PI3K / AKT (NVP-BKM120) or MEK / ERK (AZD8330, SCH772984) signaling in HCC827 and H1650LUAD cells resulted in a dramatic induction of ALPP expression (Figure 8D). FoxO family transcription factors are involved in cell cycle control and are regulated by PI3K / AKT and MEK / ERK signaling. At the mRNA level, EGFR-mutant H1650 and HCC827 LUAD cells expressed low levels of FoxO1 but significant levels of FoxO3 (Figure 16A). Transcription of FOXO3A was not affected by gefitinib treatment (Figure 16B). Ingenuity pathway analysis of upstream regulators revealed consistent activation of FoxO3a, but not FoxO1, across four LUAD cell lines treated with osimertinib (Figures 8E and 16C). Phosphorylation of FoxO3a at Ser294 and Ser425 leads to nuclear export and cytosolic retention of FoxO3a, resulting in reduced transcriptional activity and degradation. Inhibition of FoxO3a using the small molecule inhibitor AS1842856 reduced ALPP protein expression and attenuated gefitinib-mediated ALPP upregulation in LUAD cells (Figures 8F–G). Conversely, overexpression of FoxO3a in both H1650 and HCC827 cell lines resulted in upregulation of basal ALPP expression, which was further enhanced when the respective cancer cells were treated with gefitinib (Figure 8H). Treatment of EGFR-mutant H1650 and HCC827 LUAD cells with gefitinib inhibited FoxO3a phosphorylation at Ser294 and Ser425 residues, reduced cytosolic FoxO3a, and promoted its nuclear translocation, suggesting transcriptional activation (Figures 8I-8J). Osimertinib also inhibited EGFR and FoxO3a phosphorylation and increased ALPP expression (Figure 8K).

[0177] To clarify whether FoxO3a is a transcriptional regulator of ALPP, we first used the online tool PROMO for in silico prediction of putative transcription factors for ALPP. Seven potential binding sites for FoxO3a were identified within a 2-kb promoter region upstream of the ALPP gene (Figure 17A). Next, ChIP-qPCR was performed and confirmed that gefitinib treatment induced FoxO3a binding to the ALPP promoter sequence, which concomitantly increased ALPP mRNA in both H1650 and HCC827 LUAD cell lines (Figure 8L and Figure 17B). Collectively, these data demonstrated that FoxO3a is a transcriptional regulator of ALPP.

[0178] EGFR TKIs maintain ALPP upregulation Treatment of HCC827 and PC9 cells with gefitinib or osimertinib for 1 to 4 days resulted in a gradual and sustained increase in the percentage of ALPP and ALPP+ cells (Figures 9A-C). Previous single-cell transcriptomics studies included PC9 cells treated with osimertinib. We queried this dataset for ALPP expression and found a progressive increase in ALPP gene expression after osimertinib treatment (Figures 9D-E). Interestingly, the proportion of ALPP-positive cells dramatically increased after osimertinib treatment, with less than 1% of LUAD cells expressing basal ALPP on day 0 and nearly 50% of LUAD cells expressing ALPP by day 14 (Figure 9F). Drug-resistant persister cells (DTPCs) are a cell population that tolerate and survive drug treatment and contribute to subsequent therapy resistance and disease relapse. ALPP expression was determined to be maintained in DTPC, and sustained expression in osimertinib-DTPC was confirmed (Figures 9G and 18A). DTPC were further produced from HCC9827 and PC9 LUAD cell lines (Figures 18B-18C), and ALPP expression was assessed in gefitinib-DTPC and osimertinib-DTPC, both of which showed sustained ALPP expression (Figures 9H-9I), suggesting the maintenance of ALPP expression by EGFR TKIs in drug-resistant cells. ALPP+ Gefitinib-DTPC and osimertinib-DTPC also showed a significant increase (Figure 9J).

[0179] To determine whether continuous treatment with EGFR inhibitors is required for sustained ALPP expression, HCC827 and PC9 cells were treated with gefitinib or osimertinib for 48 hours, followed by an additional 48 hours in the absence of the inhibitor. ALPP expression was significantly reduced in the absence of the EGFR inhibitor and restored with the addition of the inhibitor (Figures 9K-9L). Similar results were observed with gefitinib-DTPC (Figures 9M-9N).

[0180] To further investigate whether ALPP expression is upregulated in EGFR inhibitor-resistant cells, gefitinib-resistant (GR) HCC827 and PC9 cells were generated (Figure 18D). ALPP expression was higher in GR cells compared with parental cells, and gefitinib treatment further enhanced ALPP upregulation (Figures 10A-B). Flow cytometry analysis of ALPP demonstrated that the majority of gefitinib-resistant cells expressed high levels of ALPP upon gefitinib challenge (Figure 10C). These results are consistent with ALPP upregulation in erlotinib-resistant HCC827 and HCC4006 and gefitinib-resistant PC9 LUAD cells (Figures 10D-E). Transcriptomic analysis of erlotinib-treated PC9 xenograft tumors also revealed increased ALPP mRNA expression in erlotinib-responsive tumors compared with treatment-naive tumors, and high ALPP gene expression persisted in erlotinib-resistant tumors (Figure 10F). Using publicly available transcriptomic data, we also examined the transcription of ALPP in treatment-naive and EGFR TKI-resistant tumors. Increased levels of ALPP occurred in NSCLC resistant to first- and second-generation EGFR TKIs compared with treatment-naive tumors (Figure 10G). Similarly, paired comparison of ALPP expression in tumors before and after developing resistance to osimertinib revealed a general upregulation of ALPP (Figure 10H).

[0181] Combining EGFR TKI and ALPP-ADC therapy enhances cancer cell killing The in vitro efficacy of a combination regimen consisting of an EGFR TKI and an anti-ALPP antibody conjugated with monomethyl auristatin F (MMAF), a microtubule inhibitor that induces apoptosis, was evaluated. The combination resulted in increased cell death compared with either treatment alone (Figures 11A and 19A). Titration of the ADC revealed dose-dependent cytotoxicity of ALPP-MMAF (Figure 11B). Following treatment with gefitinib or osimertinib, ALPP-MMAF treatment also resulted in strong cytotoxicity (Figure 11C). Similarly, ALPP-MMAF demonstrated efficacy in gefitinib-resistant HCC827 and PC9 cells (Figure 11D). Sequential treatment of HCC827 and PC9 cells with gefitinib or osimertinib led to the emergence of drug-resistant clones, and the addition of ALPP-MMAF to gefitinib- or osimertinib-treated HCC827 and PC9 cells effectively suppressed the formation of EGFR TKI-resistant clones (Figures 11E and 19B).

[0182] We then tested this dual regimen in the gefitinib-resistant cell line H1650. Surface ALPP was targeted in H1650 LUAD cells using a Phosphoro-Red-conjugated ALPP antibody, demonstrating internalization (Figure 12A). The combined treatment demonstrated synergistic effects with potent tumor-killing activity (Figures 12B-C).

[0183] Furthermore, firefly luciferase-expressing H1650 (H1650 LucLUAD cells were orthotopically implanted into BALB / c nude mice, and tumor-bearing mice were treated daily with gefitinib at 50 mg / kg or saline control for 10 days. Tumor and other organ tissues were subsequently collected and ALPP expression was assessed by IHC. Consistent with the in vitro observations, tumor-bearing mice treated with gefitinib showed a significant increase in tumoral ALPP surface expression (Figure 12D). ALPP protein staining in other tissues was negative or showed no appreciable difference compared to vehicle controls (Figure 20). H1650 Luc Using an independent cohort of tumor-bearing mice, the anti-cancer efficacy of gefitinib at 50 mg / kg and ALPP-MMAF at 5 mg / kg, alone and in combination, was evaluated (Figure 12E). Treatment with gefitinib or ALPP-MMAF alone resulted in a significant reduction in H1650 tumors compared to controls. Luc There was a statistically significant reduction in tumor relative fluorescence units (RFU). Notably, tumor-bearing mice treated with gefitinib + ALPP-MMAF showed a complete response and were tumor-free based on IVIS imaging (Figures 12F-G).

[0184] Consideration EGFR TKI monotherapy is the frontline treatment for lung cancer patients who harbor endogenous EGFR-activating mutations. Despite high initial responses to these drug therapies, resistance to EGFR TKIs inevitably emerges, resulting in cancer progression within 1–2 years of therapy initiation. Combination therapy can maximize antitumor efficacy and virtually prevent the development of drug resistance. Herein, we demonstrate that ALPP upregulation driven by EGFR inhibitors confers improved efficacy against TKI-sensitive and refractory cancer cells to a viable "two-hit" combination regimen containing an EGFR TKI and ALPP-MMAF (Figure 13).

[0185] ALPP is a GPI-anchored protein expressed on the cell surface in some cancers, whereas normal tissue expression is primarily restricted to the placenta. It is currently being explored as a target for cancer therapy. However, ALPP expression levels are relatively low in most cancer types. In lung cancer, ALPP is primarily expressed in adenocarcinoma. Consistent with previous studies, evaluation of ALPP mRNA and surface protein expression in human lung tumors and cancer cell lines demonstrated variable expression, with frequent outliers identified in LUAD. In lung tumor TMAs, ALPP surface staining was found in 11.3% of all lung tumors analyzed, with the highest frequency of ALPP surface staining identified in LUAD (17.6%). Notably, positive ALPP staining was associated with smoking status. Previous studies have reported that serum levels of ALPP are up to 10-fold increased in smokers compared with nonsmokers. Furthermore, an epigenome-wide association study (EWA) comparing current, former, and never-smoker individuals from 1,793 participants revealed that CpG island methylation at the ALPP locus was lower in smokers than in nonsmokers. More recently, a study identified ALPP as one of the risk biomarkers for smoking-related lung cancer. Data suggest that basal expression of ALPP is required for EGFR TKI-mediated upregulation, suggesting that EGFR inhibition may more potently enhance ALPP expression in patients with a smoking history.

[0186] Previous studies have documented an inverse correlation between ALPP expression and cell proliferation state. Consistent with these observations, we found that significant ALPP expression was upregulated in cancer cells exposed to EGFR inhibitors. Importantly, the enhanced ALPP expression in cancer cells was transient and reversible, suggesting that ALPP expression may be associated with cell dormancy. In support of this, FoxO3a, a key regulator of cell dormancy, was identified as an upstream transcriptional regulator of ALPP. FoxO3a is one of four related FoxO transcription factors that protect cells against a wide range of physiological stresses and plays a central role in DNA repair, growth arrest, and apoptosis in response to DNA damage and oxidative stress. Inactivation of FoxO3a, commonly reported in various malignant diseases, is attributed to overactivation of the PI3K-AKT or MEK-ERK signaling pathway, leading to nuclear exclusion and proteasomal degradation, and negatively regulating FoxO3a transcriptional activity via its sustained phosphorylation at Thr32, Ser253, and Ser294. Previous studies have well documented that EGFR signaling suppresses FoxO3a activation. Inhibition of EGFR by erlotinib results in the nuclear accumulation and transcriptional activation of FoxO3a. EGF induces FoxO3a phosphorylation, and inactivation is prevented by gefitinib treatment in NSCLC cell lines. In the present study, we found that EGFR TKI treatment suppressed FoxO3a phosphorylation in LUAD cells, resulting in FoxO3a nuclear translocation with concomitant increases in ALPP mRNA and protein levels. Inhibition of FoxO3a with the small molecule inhibitor AS1842856 reduced ALPP protein expression and prevented EGFR TKI-mediated ALPP upregulation, and ChIP-qPCR assays demonstrated direct binding of FoxO3a to the promoter of ALPP. Collectively, our data identify FoxO3a as a transcriptional regulator of ALPP underlying EGFR TKI-driven upregulation of ALPP.

[0187] These findings prompted us to evaluate whether enhanced surface ALPP expression in cancer cells mediated by EGFR TKIs could enhance the efficacy of ALPP-targeted therapy. To this end, we demonstrated that combination therapy consisting of gefitinib / osimertinib and ALPP-MMAF significantly improved cancer killing compared with either treatment alone. Treatment of mice bearing EGFR-mutant LUAD tumors with gefitinib specifically increased ALPP surface expression in tumors, but not in normal tissues. Combinatorial treatment of mice bearing EGFR-mutant LUAD tumors with gefitinib plus ALPP-MMAF resulted in significant anticancer activity, exceeding the efficacy of gefitinib or ALPP-MMAF treatment alone. This synergistic combination approach demonstrated the potential of targeting both EGFR signaling and ALPP expression to achieve improved therapeutic outcomes in EGFR-mutant lung cancer.

[0188] Acquired drug resistance to EGFR TKIs, which usually occurs within two years of initiating EGFR inhibitor treatment, remains a significant clinical challenge. EGFR-dependent resistance, often due to the acquisition of additional EGFR genetic mutations (e.g., EGFRT790M, exon 19 deletion, L858R, or C797S), occurs in approximately 50% of cases with early-generation EGFR TKIs or in approximately 10-20% of patients treated with osimertinib, and may be addressed by using different generation EGFR inhibitors or combination regimens. EGFR-independent resistance involves the expression of other receptor tyrosine kinases, EMT, and small cell lung cancer transformation. Therapies for the vast majority of EGFR-independent resistance remain inadequate. Our findings demonstrate that ALPP is upregulated in cancer cells closely linked to EGFR signaling. Importantly, EGFR TKIs sustain ALPP expression even in drug-refractory cancer cells. In vitro evaluation of the efficacy of ALPP-MMAF in DTPC- and gefitinib-resistant cells revealed effective killing of cancer cells refractory to gefitinib or osimertinib. Furthermore, ALPP-MMAF in combination with gefitinib or osimertinib also prevented the formation of drug-resistant clones. A recent study aimed to identify surface therapeutic targets in EMT-associated EGFR TKI-resistant NSCLC and listed ALPP as one of the highly upregulated proteins. These findings support the feasible application of ALPP-targeting regimens in addressing EGFR-independent resistance.

[0189] In conclusion, our study provides mechanistic insight into ALPP upregulation in cancer cells and identifies FoxO3a as a transcriptional regulator of ALPP. Importantly, our study demonstrates a novel combination therapeutic strategy that exploits enhanced ALPP expression in cancer cells, potentially enhancing the efficacy of targeted therapy in LUAD patients with EGFR-activating mutations.

[0190] [Table 6]

[0191] [Table 7]

[0192] [Table 8]

[0193] [Table 9]

[0194] [Table 10]

[0195] [Table 11]

[0196] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or set forth in the Application Data Sheets are incorporated herein by reference in their entirety. Where necessary, concepts from the various patents, applications, and publications can be used to modify aspects of the embodiments to provide further embodiments.

[0197] These and other changes can be made to the embodiments in light of the above-detailed specification. Generally, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but rather, such claims should be construed to encompass all possible embodiments, along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the present disclosure.

Claims

1. 1. A method of treating a cancer that co-expresses alkaline phosphatase (ALPP) and / or ALPP2 proteins and endogenously harbors an activating EGFR mutation in a patient in need thereof, comprising administering to the patient an antibody-drug conjugate that targets ALPP and / or ALPP2 in cells in combination with an EGFR inhibitor.

2. 1. A method of treating a cancer that co-expresses alkaline phosphatase (ALPP) and / or ALPP2 proteins and endogenously harbors an activating EGFR mutation in a patient in need thereof, comprising: identifying said EGFR activating mutation in cells from a biological sample obtained from said patient; detecting and / or quantifying ALPP and / or ALPP2 cell surface expression in said cells; administering an antibody drug conjugate therapy that targets ALPP and / or ALPP2 in cancer cells in conjunction with an EGFR inhibitor.

3. 1. A method of treating drug-resistant or drug-refractory cancer cells that co-express alkaline phosphatase (ALPP) and / or ALPP2 proteins in a patient in need thereof, comprising: identifying an EGFR activating mutation in cells from a biological sample obtained from said patient; detecting and / or quantifying ALPP and / or ALPP2 cell surface expression in said cells; administering an antibody drug conjugate therapy targeting ALPP and / or ALPP2 in said cancer cells in conjunction with an EGFR inhibitor.

4. 1. A method for preventing the emergence of resistance to an EGFR inhibitor in cancer cells that co-express alkaline phosphatase (ALPP) and / or ALPP2 proteins, the method comprising administering an antibody-drug conjugate therapy that targets ALPP and / or ALPP2 in the cancer cells in conjunction with the EGFR inhibitor.

5. The method of any one of claims 1 to 4, wherein the cancer cells comprise increased cell surface expression of ALPP and / or ALPP2 relative to healthy cells.

6. The method of any one of claims 1 to 5, wherein the EGFR mutation comprises an exon 19 deletion, a T790M point mutation, and / or a L858R point mutation.

7. The method of any one of claims 1 to 6, wherein the cancer harboring an EGFR mutation is resistant to an inhibitor that targets EGFR.

8. The method of any one of claims 1 to 7, wherein the cancer cells are drug-resistant persister cells (DTPCs) or drug-resistant cells (DRCs).

9. 9. The method of any one of claims 1 to 8, wherein the drug-resistant persister cells (DTPCs) are prevented from developing into drug-resistant cells (DRCs) by administering the antibody-drug conjugate therapy targeting ALPP and / or ALPP2 in the cancer cells and the EGFR inhibitor.

10. The method of any one of claims 1 to 9, wherein drug-resistant persister cells (DTPCs) and drug-resistant cells (DRCs) are treated to prevent the emergence of resistance to EGFR inhibitors.

11. 11. The method of any one of claims 1 to 10, wherein the cancers co-expressing alkaline phosphatase (ALPP) and / or ALPP2 proteins are ovarian cancer, breast cancer, cervical cancer, endometrial cancer, pancreatic cancer, gastric cancer, colorectal cancer, lung cancer, urothelial cancer, brain cancer, testicular cancer, seminoma, and mesothelioma.

12. 12. The method of any one of claims 1 to 11, wherein the cancer is testicular germ cell tumor, endometrial cancer of the uterine corpus, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, bladder urothelial carcinoma, triple-negative breast cancer, gastric adenocarcinoma, esophageal carcinoma, uterine carcinosarcoma, rectal adenocarcinoma, head and neck squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, clonal adenocarcinoma, mesothelioma, and acute myeloid leukemia.

13. 12. The method of claim 11, wherein the lung cancer is non-small cell lung cancer.

11. 11. The method of any one of claims 1 to 10, wherein detecting and / or quantifying placental alkaline phosphatase (ALPP) and / or ALPP2 cell surface expression in the cancer cells comprises histological analysis, immunohistochemistry (IHC) staining for ALPP protein, blood-based tests, tissue-based tests, or imaging techniques.

12. 12. The method of claim 11, wherein the tissue-based test comprises tissue biopsy, flow cytometry, immunohistochemistry (IHC), Western blot (WB), polymerase chain reaction (PCR), or immunofluorescence (IF).

13. 12. The method of claim 11, wherein the tissue-based test comprises a Mammaprint+Blueprint® test or an Oncotype DX® test.

14. 12. The method of claim 11, wherein the blood-based test comprises a Galleri® test, a circulating tumor cell (CTC) test, a complete blood count (CBC), or a test or assay that measures circulating proteins, autoantibodies, cell-free circulating DNA, or extracellular vesicle-derived proteins.

15. 13. The method of claim 12, wherein the tissue biopsy is analyzed by hematoxylin and eosin (H&E) staining and / or microscopy.

16. The method of any one of claims 1 to 15, wherein the antibody-drug conjugate comprises an antibody targeting ALPP conjugated to a chemotherapeutic drug.

17. The method of any one of claims 1 to 16, wherein the EGFR inhibitor comprises a tyrosine kinase inhibitor (TKI).

18. 18. The method of any one of claims 1 to 17, wherein the antibody drug conjugate therapy targeting ALPP and / or ALPP2 is administered in conjunction with the EGFR inhibitor.

19. 19. The method of any one of claims 1 to 18, wherein the EGFR inhibitor is selected from gefitinib, osimertinib, mobocertinib, amivantamab, CLN081, and / or DZD9008.

20. 17. The method of claim 16, wherein the antibody-drug conjugate comprises SGN-ALPV, Adcetris®, Kadcyla®, Besponsa®, Mylotarg®, Polivy®, Padcev®, Enhertu®, Trodelvy®, Blenrep®, Zynlonta™, Akalux®, Aidixi®, and Tivdak®.

21. 21. The method of any one of claims 1 to 20, wherein the antibody drug conjugate therapy targeting ALPP and / or ALPP2 in the cancer cells and the EGFR inhibitor are administered simultaneously.

22. 22. The method of any one of claims 1 to 21, wherein the antibody drug conjugate therapy targeting ALPP and / or ALPP2 in the cancer cells and the EGFR inhibitor are administered sequentially.

23. 1. A method of treating a cancer that endogenously harbors an activating EGFR mutation in a patient in need thereof, comprising: administering an antibody-drug conjugate that targets ALPP and / or ALPP2 on the surface of cancer cells; the cancer cells exhibit increased expression of ALPP and / or ALPP2 relative to healthy cells; the antibody-drug conjugate targeting ALPP and / or ALPP2 comprises an antibody targeting ALPP and / or ALPP2 conjugated to MMAF; The method, wherein the cancer cells contain an activating mutation in the EGFR gene, resulting in resistance to EGFR tyrosine kinase inhibitors.

Images