Methods for the detection and treatment of resistant cancers co-expressing alpp and / or alpg / alppl2

Abstract

Provided are methods for treating treatment-resistant non-small-cell lung cancer (NSCLC) in a patient in need thereof comprising administering a chimeric antigen receptor (CAR) cell therapy, antibody drug conjugate (ADC), or bispecific T cell engager (BiTE) targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, an AKT mutation, an ERK mutation, a CDK4 / 6 mutation, a BRAF mutation, and an FGFR mutation.

Description

Attorney Docket No. MDA0083-401-PC METHODS FOR THE DETECTION AND TREATMENT OF RESISTANT CANCERS CO-EXPRESSING ALPP AND / OR ALPG / ALPPL2

[0001] This application claims the benefit of priority of United States provisional application no.63 / 596,026, filed November 03, 2023, and United States provisional application no.63 / 556,191, February 21, 2024, the contents of which are incorporated by reference as if written herein in their entirety. GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under CA070907 awarded by the National Institutes of Health. The government has certain rights in the invention. INCORPORATION OF SEQUENCE LISTING

[0003] The sequence listing that is contained in the file named “MDA0083-401-PC,” which is 30,692 bytes as measured in the Microsoft Windows operating system and was created on November 1, 2024, is filed electronically herewith and incorporated herein by reference.

[0004] While cancer patients with activating mutations in oncogenes such as epidermal growth factor receptor (EGFR), Kirsten rat sarcoma virus (KRAS), rearranged during transfection (RET), anaplastic lymphoma kinase (ALK), fibroblast growth factor receptor (FGFR), and human epidermal growth factor 2 (HER2) are initially responsive to agents targeting these specific molecules, resistant disease inevitably emerges. Cancers with acquired resistance to particular targeted treatments exhibit increased expression of alkaline phosphatase placental type (ALPP) and alkaline phosphatase, germ cell (ALPG, also known as ALPPL2), which are closely related and regulated GPI-anchored proteins that are expressed on the cell surface in numerous cancers, while normal tissue expression of ALPP and ALPPL2 is largely limited to the placenta. Novel treatments are needed to combat acquired resistance to cancer therapies and effectively treat resistant cancers, for example by targeting drug-tolerant persister cells (DTPCs) and / or drug-resistant cells (DRCs). The present disclosure addresses these needs by disclosing methods for treating treatment- resistant non-small-cell lung cancer (NSCLC) with acquired resistance to targeted therapeutic agents by targeting cell surface ALPP and / or ALPPL2, or targeting ALPG / ALPPL2, either alone, or in combination with a therapy targeting the specific genetic mutation that resulted in resistance of the cancer cells to therapy. SUMMARYAttorney Docket No. MDA0083-401-PC

[0005] Provided herein is a method for treating treatment-resistant non-small-cell lung cancer (NSCLC) in a patient in need thereof, comprising administering a chimeric antigen receptor (CAR) cell therapy or antibody drug conjugate (ADC) targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from the group consisting of an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, and a MET mutation.

[0006] Also provided is a method for treating treatment-resistant non-small-cell lung cancer (NSCLC) in a patient in need thereof, comprising: administering a chimeric antigen receptor (CAR) cell therapy or antibody drug conjugate (ADC) targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, an AKT mutation, an ERK mutation, a CDK4 / 6 mutation, a BRAF mutation, and an FGFR mutation.

[0007] Also provided is a method for treating treatment-resistant non-small-cell lung cancer (NSCLC) in a patient in need thereof comprising: identifying a genetic mutation in a NSCLC cell from a biological sample obtained from the patient; detecting and / or quantifying placental alkaline phosphatase (ALPP) and / or ALPPL2 cell surface expression in the NSCLC cell; administering a chimeric antigen receptor (CAR) cell therapy or antibody drug conjugate (ADC) targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting the genetic mutation; wherein the genetic mutation is a mutation selected from the group consisting of an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, and a MET mutation.

[0008] Also provided is a method for treating treatment-resistant non-small-cell lung cancer (NSCLC) in a patient in need thereof comprising: identifying a genetic mutation in a NSCLC cell from a biological sample obtained from the patient; detecting and / or quantifying placental alkaline phosphatase (ALPP) and / or ALPPL2 cell surface expression in the NSCLC cell; administering a chimeric antigen receptor (CAR) cell therapy targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting the genetic mutation; wherein the genetic mutation is a mutation selected from an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, an AKT mutation, an ERK mutation, a CDK4 / 6 mutation, a BRAF mutation, and an FGFR mutation.Attorney Docket No. MDA0083-401-PC

[0009] Also provided is a method for treating drug-tolerant persister non-small-cell lung cancer (NSCLC) cells or drug-resistant NSCLC cells in a patient in need thereof comprising: identifying a genetic mutation in a NSCLC cell from a biological sample obtained from the patient; detecting and / or quantifying ALPP and / or ALPPL2 cell surface expression in the NSCLC cell; administering a chimeric antigen receptor (CAR) cell therapy or antibody drug conjugate (ADC) targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting the genetic mutation; wherein the genetic mutation is a mutation selected from the group consisting of an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, and a MET mutation.

[0010] Also provided is a method for treating drug-tolerant persister non-small-cell lung cancer (NSCLC) cells or drug-resistant NSCLC cells in a patient in need thereof comprising: identifying a genetic mutation in a NSCLC cell from a biological sample obtained from the patient; detecting and / or quantifying ALPP and / or ALPPL2 cell surface expression in the NSCLC cell; administering a chimeric antigen receptor (CAR) cell therapy targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting the genetic mutation; wherein the genetic mutation is a mutation selected from an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, an AKT mutation, an ERK mutation, a CDK4 / 6 mutation, a BRAF mutation, and an FGFR mutation.

[0011] Also provided is a method of preventing emergence of resistance of non-small- cell lung cancer (NSCLC) to a drug therapy comprising administering a chimeric antigen receptor (CAR) cell therapy or antibody drug conjugate (ADC) targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from the group consisting of an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, and a MET mutation.

[0012] Also provided is a method of preventing emergence of resistance of non-small- cell lung cancer (NSCLC) to a drug therapy comprising administering a chimeric antigen receptor (CAR) cell therapy targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, anAttorney Docket No. MDA0083-401-PC AKT mutation, an ERK mutation, a CDK4 / 6 mutation, a BRAF mutation, and an FGFR mutation.

[0013] Also provided is a method for treating non-small-cell lung cancer (NSCLC) that is resistant to a targeted inhibitor in a patient in need thereof comprising: administering a CAR- T cell or CAR NK cell or an antibody drug conjugate (ADC) targeting ALPP and / or ALPPL2 on the surface of the NSCLC cells; wherein the NSCLC cells exhibit increased expression of ALPP and / or ALPPL2 relative to a healthy lung cell; wherein the CAR-T cell targeting ALPP and / or ALPPL2 comprises a single-chain variable-fragment (scFv) derived from an ALPP- specific antibody; wherein the NSCLC cells comprise: a mutation in the EGFR gene resulting in resistance to EGFR tyrosine kinase inhibitor osimertinib; or a mutation in the KRAS gene resulting in resistance to KRAS inhibitors adagrasib and / or sotorasib; or a mutation in the RET gene resulting in resistance to RET inhibitor selpercatinib and / or pralsetinib; or a mutation in the ALK gene resulting in resistance to ALK inhibitors alectinib and / or lorlatinib; or a mutation in the HER2 gene resulting in resistance to HER2 inhibitor lapatinib; or a mutation in the MET gene resulting in resistance to MET inhibitors tepotinib and capmatinib; wherein the CAR-T cell specifically binds to and eliminates the NSCLC cells having increased ALPP and / or ALPPL2; and wherein the CAR-T cell comprises a sequence set forth in SEQ ID NOs:1-18.

[0014] Also provided is a method for treating a non-small cell lung cancer (NSCLC) expressing elevated cell surface levels of alkaline phosphatase, germ cell (ALPG / ALPPL2) protein, comprising administering to the patient: (i) a treatment therapy targeting ALPG / ALPPL2; and / or (ii) a second treatment therapy; wherein the NSCLC has acquired resistance to a targeted inhibitor of an oncogene gene chosen from EGFR, KRAS, RET, ALK, HER2, and FGFR.

[0015] Also provided is a method for treating a non-small cell lung cancer (NSCLC) expressing elevated cell surface levels of alkaline phosphatase, germ cell (ALPG / ALPPL2) protein, comprising: identifying a genetic mutation in a NSCLC cell from a biological sample obtained from the patient; detecting and / or quantifying ALPG / ALPPL2 cell surface expression in the NSCLC cell; administering a chimeric antigen receptor (CAR) cell therapy targeting ALPG / ALPPL2 in the NSCLC cell, and / or a therapy targeting the genetic mutation; wherein the genetic mutation is a mutation selected from the group consisting of an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, and an FGFR mutation.Attorney Docket No. MDA0083-401-PC

[0016] Also provided is a method for treating drug-tolerant persister non-small-cell lung cancer (NSCLC) cells or drug-resistant NSCLC cells in a patient in need thereof comprising: identifying a genetic mutation in a NSCLC cell from a biological sample obtained from the patient; detecting and / or quantifying alkaline phosphatase, germ cell (ALPG / ALPPL2) cell surface expression in the NSCLC cell; administering a chimeric antigen receptor (CAR) cell therapy targeting ALPG / ALPPL2 in the NSCLC cell, and / or a therapy targeting the genetic mutation; wherein the genetic mutation is a mutation selected from the group consisting of an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, and an FGFR mutation.

[0017] Also provided is a method of preventing emergence of resistance of non-small- cell lung cancer (NSCLC) to a drug therapy comprising administering a chimeric antigen receptor (CAR) cell therapy targeting alkaline phosphatase, germ cell (ALPG / ALPPL2) in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from the group consisting of an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, and an FGFR mutation.

[0018] Also provided is a method of selecting a patient having a non-small cell lung cancer (NSCLC) for treatment with a cancer therapy targeting alkaline phosphatase, germ cell (ALPG / ALPPL2) comprising: identifying a genetic mutation in a NSCLC cell from a biological sample obtained from the patient; detecting and / or quantifying ALPG / ALPPL2 cell surface expression in the NSCLC cell; administering a chimeric antigen receptor (CAR) cell therapy targeting ALPG / ALPPL2 in the NSCLC cell, and / or a therapy targeting the genetic mutation; wherein the genetic mutation is a mutation selected from the group consisting of an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, and an FGFR mutation.

[0019] Also provided is a method for treating a non-small cell lung cancer (NSCLC) expressing elevated cell surface levels of alkaline phosphatase, germ cell (ALPG / ALPPL2) protein, comprising: administering a CAR-T cell targeting ALPG / ALPPL2 on the surface of the NSCLC cells; wherein the CAR-T cell targeting ALPG / ALPPL2 comprises a single-chain variable-fragment (scFv) derived from an ALPPL2-specific antibody; wherein the NSCLC cells comprise: a mutation in the EGFR gene resulting in resistance to EGFR tyrosine kinase inhibitor osimertinib; or a mutation in the KRAS gene resulting in resistance to KRAS inhibitors adagrasib, sotorasib, ARS1323, and / or AZD4785; or a mutation in the RET gene resulting in resistance to RET inhibitor selpercatinib; or a mutation in the ALK gene resulting in resistance to ALK inhibitors crizotinib, alectinib, brigatinib, and / or lorlatinib; or aAttorney Docket No. MDA0083-401-PC mutation in the HER2 gene resulting in resistance to HER2 inhibitors lapatinib, BI1810631, and / or trastuzumab-deruxtecan; or a mutation in the FGFR gene resulting in resistance to FGFR inhibitors Balversa, anlotinib, erdafitinib, infigratinib, pemazyre, and / or pemigatinib; wherein the CAR-T cell specifically binds to and eliminates the NSCLC cells having increased ALPG / ALPPL2; and wherein the CAR-T cell comprises a sequence set forth in SEQ ID NOs:1-18.

[0020] Also provided is a method for treating treatment-resistant non-small-cell lung cancer (NSCLC) in a patient in need thereof comprising: administering a bispecific T-cell engager (BiTE) targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from the group consisting of an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, and an FGFR mutation.

[0021] Also provided is a method for treating treatment-resistant non-small-cell lung cancer (NSCLC) in a patient in need thereof comprising administering a bispecific T-cell engager (BiTE) targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, an AKT mutation, an ERK mutation, a CDK4 / 6 mutation, a BRAF mutation, and an FGFR mutation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG.1A – FIG.1C – Show that placental alkaline phosphatase (ALPP) is upregulated in epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)- resistant and drug-tolerant persister cells (DTPCs). FIG.1A shows an unbiased strategy to identify surface targets in EGFR TKI drug-resistant cells (DRCs) and DTPCs using public datasets from GEO database. FIG.1B shows identification of top candidate genes commonly upregulated in EGFR TKI DRCs and DTPCs of different cell lines in multiple datasets. ALPP is commonly upregulated in EGFR TKI DRCs and DTPCs. FIG.1C shows expression of the top candidate genes in normal tissues. The figure was generated using the Genotype- Tissue Expression (GTEx) dataset.

[0023] FIG.2A – FIG.2F – Show that ALPP is upregulated on EGFR TKI (osimertinib) resistant cells. FIG.2A shows that mRNA levels of ALPP are upregulated in H1975 cells with acquired resistance to the EGFR tyrosine kinase inhibitor osimertinib (OSI) (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. FIG.2B shows flow cytometry data demonstrating upregulated ALPP expression on HCC827 osimertinib-resistant (OR) cells compared toAttorney Docket No. MDA0083-401-PC HCC827 parental cells. FIG.2C shows representative flow cytometry data demonstrating upregulated ALPP expression on H1975 OR cells compared to H1975 parental cells. FIG.2D shows summarized data demonstrating upregulated ALPP expression on H1975 OR cells. **p < 0.01, ***p < 0.001. FIG.2E shows representative flow cytometry data demonstrating upregulated ALPP expression on HCC4006 OR cells compared to HCC4006 parental cells. FIG.2F shows summarized data demonstrating upregulated ALPP expression on H1975 OR cells.

[0024] FIG.3A – FIG.3D – Show that ALPP is upregulated in osimertinib-refractory NSCLC patient tissues. FIG.3A shows that mRNA levels of ALPP are upregulated in tissues collected at disease progression compared to pre-osimertinib treatment. FIG.3B shows that mRNA levels of ALPG (ALPPL2) are upregulated in tissues collected at disease progression compared to pre-osimertinib treatment. FIG.3C shows immunohistochemistry (IHC) staining of ALPP in EGFR-TKI naïve tissues. FIG.3D shows IHC staining of ALPP in osimertinib- refractory tissues.

[0025] FIG.4A – FIG.4G – Show that ALPP expression can be targeted by CAR-T cells in EGFR TKI-resistant NSCLC cells. FIG.4A shows a diagram of an ALPP chimeric antigen receptor (CAR) construct. FIG.4B shows activity of ALPP-targeting CAR-T cells against ALPP-expressing NSCLC cells. ALPP CAR-T cells killed ALPP-expressing cells (H1650). FIG.4C shows that ALPP CAR-T cells killed ALPP overexpressing (H1975- ALPP) and ALPG overexpressing (H1975-ALPG) cells but not parental (ALPP negative) cells. FIG.4D shows that ALPP CAR-T cells (M25-M228BB3Z) killed HCC4006 OR cells but not HCC4006 parental cells. FIG.4E shows that ALPP CAR-T cells (M25-M228BB3Z) killed H1975 OR cells but not H1975 parental cells. FIG.4F shows that ALPP CAR-T cells (H2-M228BB3Z) killed HCC4006 OR cells but not HCC4006 parental cells. FIG.4G shows that ALPP CAR-T cells (H2-M228BB3Z) killed H1975 OR cells but not H1975 parental cells.

[0026] FIG.5A – FIG.5B – Show that ALPP expression can be targeted by CAR-NK cells in EGFR TKI-resistant NSCLC cells. FIG.5A shows that ALPP CAR-NK cells killed HCC4006 OR cells more effectively as compared to HCC4006 parental cells. FIG.5B shows that ALPP CAR-NK cells specifically killed HCC4006 OR cells by blocking natural cytotoxicity pathways.

[0027] FIG.6A – FIG.6E – Show that ALPP is upregulated on EGFR TKI (osimertinib) drug tolerant persister cells (DTPCs). FIG.6A shows that mRNA levels of ALPP are upregulated in NSCLC cell lines (H1975, HCC827, and HCC4006) after treatment with theAttorney Docket No. MDA0083-401-PC EGFR inhibitor OSI for 14 days (n = 3). ***p < 0.001. FIG.6B shows representative flow cytometry data demonstrating upregulated ALPP expression on H1975 DTPCs, HCC827 DTPCs, and HCC4006 DTPCs. FIG.6C shows summarized data showing upregulated ALPP expression on DTPCs. ***p < 0.001. FIG.6D shows that mRNA levels of ALPP are upregulated in HCC827 cells treated with osimertinib for 2, 4, 8, 16, and 24 hours. ***p < 0.001. FIG.6E shows that protein levels of ALPP are upregulated in H1975 xenograft tumors treated with osimertinib for two weeks in vivo.

[0028] FIG.7A – FIG.7C – Show that ALPP expression can be targeted in EGFR TKI DTPCs. FIG.7A shows that ALPP CAR-T cells killed H1975 OSI DTPCs but not H1975 parental cells. FIG.7B shows that ALPP CAR-T cells killed HCC827 OSI DTPCs but not HCC827 parental cells. FIG.7C shows that ALPP CAR-T cells killed HCC4006 OSI DTPCs but not HCC4006 parental cells.

[0029] FIG.8A – FIG.8B – Show that ALPP expression can be targeted in EGFR TKI DTPCs in vivo. FIG.8A shows that ALPP CAR-NK cells inhibited H1975 xenograft growth when administrated with osimertinib at the same period. FIG.8B shows that ALPP CAR-NK cells inhibited H1975 xenograft growth when administrated after osimertinib treatment for 2 weeks.

[0030] FIG.9A – FIG.9C – Show that ALPP is upregulated on KRAS G12C inhibitor DTPCs. FIG.9A shows that mRNA levels of ALPP are upregulated in KRAS mutant NSCLC cells (H358, H1373, and Calu-1) after treatment with the KRAS G12C inhibitor (adagrasib) for 14 days (n = 3). ***p < 0.001. FIG.9B shows representative flow cytometry data demonstrating upregulated ALPP expression on H358 sotorasib DTPCs, H1373 adagrasib DTPCs, and Calu-1 adagrasib DTPCs. FIG.9C shows summarized data demonstrating upregulated ALPP expression on KRAS inhibitor DTPCs. ***p < 0.001.

[0031] FIG.10A – FIG.10F – Show that ALPP expression can be targeted in KRAS G12C DTPCs. FIG.10A shows ALPP-targeting CAR-T cells (M25-M228BB3z CAR) killed H358 sotorasib DTPCs but not H358 parental cells. FIG.10B shows ALPP-targeting CAR-T cells (H2-M228BB3z CAR) killed H358 sotorasib DTPCs but not H358 parental cells. FIG. 10C shows ALPP-targeting CAR-T cells (M25-M228BB3z CAR) killed H1373 adagrasib DTPCs but not H1373 parental cells. FIG.10D shows ALPP-targeting CAR-T cells (H2- M228BB3z CAR) killed H1373 adagrasib DTPCs but not H1373 parental cells. FIG.10E shows ALPP-targeting CAR-T cells (M25-M228BB3z CAR) killed Calu-1 adagrasib DTPCs but not Calu-1 parental cells. FIG.10F shows ALPP-targeting CAR-T cells (H2-M228BB3z CAR) killed Calu-1 adagrasib DTPCs but not Calu-1 parental cells.Attorney Docket No. MDA0083-401-PC

[0032] FIG.11A – FIG.11F – Show that ALPP is upregulated in NSCLC cells with acquired resistance to KRAS G12C inhibitors. FIG.11A shows that mRNA levels of ALPP are upregulated in H23 and H358 cells treated with KRAS G12C inhibitor sotorasib and H23 and H358 cells with acquired resistance to KRAS G12C inhibitor sotorasib. The figure was generated from GEO dataset GSE229070. FIG.11B shows that mRNA levels of ALPP are upregulated in clones of H358 cells with acquired resistance to KRAS G12C inhibitor sotorasib. FIG.11C shows that mRNA levels of ALPP are upregulated in clones of H358 cells with acquired resistance to KRAS G12C inhibitor adagrasib. FIG.11D shows summarized western blot data demonstrating upregulated ALPP expression on H358 sotorasib-resistant clones. FIG.11E shows summarized flow cytometry data demonstrating upregulated ALPP expression on sotorasib-resistant H358 clones. FIG.11F shows summarized flow cytometry data demonstrating upregulated ALPP expression on adagrasib- resistant H358 clones. ***p < 0.001.

[0033] FIG.12 – Shows that ALPP expression can be targeted in NSCLC cells with acquired resistance to KRAS G12C inhibitors. FIG.12A shows ALPP-targeting CAR-T cells killed H358 sotorasib-resistant clones but not H358 parental cells.

[0034] FIG.13A – FIG.13F – Show that ALPP is upregulated in RET fusion positive NSCLC cells that have been treated with a RET inhibitor (RET inhibitor DTPCs) and RET inhibitor resistant NSCLC cells. FIG.13A shows that mRNA levels of ALPP are upregulated in TH1101 cells (KIF5B-RET fusion) after treatment with the RET inhibitor pralsetinib for 1, 2, or 14 days (n = 3). ***p < 0.001. FIG.13B shows that mRNA levels of ALPP are upregulated in TH1101 cells (KIF5B-RET fusion) after treatment with the RET inhibitor selpercatinib for 1, 2, or 14 days (n = 3). ***p < 0.001. FIG.13C shows that mRNA levels of ALPP are upregulated in LC2 / AD cells (CCDC6-RET fusion) after treatment with the RET inhibitor pralsetinib for 1, 2, or 14 days (DTPC) and LC2 / AD cells with acquired resistance to the RET inhibitor pralsetinib (n = 3). ***p < 0.001. FIG.13D shows that mRNA levels of ALPP are upregulated in LC2 / AD cells (CCDC6-RET fusion) after treatment with the RET inhibitor selpercatinib for 1, 2, or 14 days (DTPC) and LC2 / AD cells with acquired resistance to the RET inhibitor selpercatinib (n = 3). ***p < 0.001. FIG.13E shows representative flow cytometry data demonstrating upregulated ALPP expression on LC2 / AD cells with acquired resistance to the RET inhibitor selpercatinib compared to LC2 / AD parental cells. FIG.13F shows summarized data demonstrating upregulated ALPP expression on LC2 / AD selpercatinib-resistant cells. ***p < 0.001.Attorney Docket No. MDA0083-401-PC

[0035] FIG.14A – FIG.14D –Show that ALPP expression can be targeted in NSCLC cells with acquired resistance to RET inhibitors and RET inhibitor DTPCs. FIG.14A shows ALPP-targeting CAR-T cells killed LC2 / AD selpercatinib-resistant cells and LC2 / AD selpercatinib DTPCs, but not LC2 / AD parental cells. FIG.14B shows ALPP-targeting CAR- NK cells killed more LC2 / AD selpercatinib-resistant cells compared to LC2 / AD parental cells. FIG.14C shows that ALPP CAR-NK cells specifically killed LC2 / AD selpercatinib- resistant cells by blocking natural cytotoxicity pathways. FIG.14D shows ALPP-targeting Antibody Drug Conjugate (ADC) killed more LC2 / AD selpercatinib-resistant cells compared to control ADC.

[0036] FIG.15A – FIG.15D – Show that ALPP is upregulated in tumor cells bearing ALK fusions after treatment with an ALK inhibitor (DTPCs) and ALK inhibitor resistant NSCLC cells. FIG.15A shows that mRNA levels of ALPP were upregulated in H3122 cells (EML4-ALK fusion) after treatment with the ALK inhibitors crizotinib for 14 days (DTPC). ***p < 0.01. FIG.15B shows that mRNA levels of ALPP were upregulated in H2228 cells (EML4-ALK fusion) after treatment with the ALK inhibitors (alectinib and lorlatinib). The figure was generated from the GEO dataset GSE188406. FIG.15C shows that mRNA levels of ALPP are upregulated in A925L cells (EML4-ALK fusion) after treatment with the ALK inhibitor alectinib. The figure was generated from GEO dataset GSE188406. FIG.15D shows that mRNA levels of ALPP are upregulated in different clones of H2228 cells with acquired resistance to the ALK inhibitor alectinib. The figure was generated from GEO dataset GSE73167.

[0037] FIG.16A – 16B – Show that ALPP is upregulated in breast cancer and stomach cancer cells after treatment with a HER2 inhibitor to generate DTPCs. FIG.16A shows that mRNA levels of ALPP are upregulated in breast cancer cells (BT474, EFM192, HCC1419, and SKBR3) after treatment with the HER2 inhibitor lapatinib. The figure was generated from GEO dataset GSE155341. FIG.16B shows that mRNA levels of ALPP are upregulated in stomach cancer cell line N87 after treatment with the HER2 inhibitor lapatinib. The figure was generated from GEO dataset GSE89127.

[0038] FIG.17 – Shows that ALPP is upregulated in bladder cancer cell line RT112 treated with the FGFR inhibitor infigratinib (BGJ398). The figure was generated from GEO dataset GSE89127.

[0039] FIG.18 – Shows that ALPP is upregulated in H358 treated with KRAS inhibitors ARS1323 and AZD4785 and the MEK inhibitor selumetinib. The figure was generated from GEO dataset GSE206867.Attorney Docket No. MDA0083-401-PC

[0040] FIG.19A – 19C – Show that ALPP is upregulated in NSCLC treated with osimertinib and MEK inhibitor trametinib. FIG.19A shows that mRNA levels of ALPP are upregulated in NSCLC (HCC827 and PC9) after treatment with a combination of the EGFR inhibitor osimertinib and the MEK inhibitor trametinib. The figure was generated from GEO dataset GSE131594. FIG.19B shows that mRNA levels of ALPP are upregulated in PC9 cells after treatment with osimertinib and trametinib in vitro at single cell level. The figure was generated from GEO single-cell RNA-seq dataset GSE138693. FIG.19C shows that mRNA levels of ALPP are upregulated in PC9 cells after treatment with osimertinib and combination of osimertinib and trametinib in vivo at single cell level. The figure was generated from GEO single-cell RNA-seq dataset GSE138693.

[0041] FIG.20A – 20C – Show that ALPP is upregulated in NSCLC treated with MET inhibitors. FIG.20A shows that MET is upregulated in HCC827 osimertinib-resistant (OR) clones (OR7, OR6, OR4, OR3, OR2). FIG.20B shows that mRNA levels of ALPP are upregulated in HCC827 OR cells after treatment with the MET inhibitors tepotinib and capmatinib. ***p < 0.001. FIG.20C shows that protein levels of ALPP are upregulated in HCC827 OR cells after treatment with the MET inhibitors tepotinib and capmatinib.

[0042] FIG.21A – FIG.21C – Show that alkaline phosphatase, germ cell (ALPG) is upregulated in epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)- resistant and drug-tolerant persister cells (DTPCs). FIG.21A shows an unbiased strategy to identify surface targets in EGFR TKI drug-resistant cells (DRCs) and DTPCs using public datasets from GEO database. FIG.21B shows identification of top candidate genes commonly upregulated in EGFR TKI DRCs and DTPCs of different cell lines in multiple datasets. ALPG (ALPPL2) is commonly upregulated in EGFR TKI DRCs and DTPCs. FIG. 21C shows expression of the top candidate genes in normal tissues. The figure was generated using the Genotype-Tissue Expression (GTEx) dataset.

[0043] FIG.22A – FIG.22I – Show that ALPG (ALPPL2) is upregulated on EGFR TKI (osimertinib) resistant cells. FIG.22A shows that mRNA levels of ALPG (ALPPL2) are upregulated in H1975 cells with acquired resistance to the EGFR tyrosine kinase inhibitor osimertinib (OR5) (n = 3). ***p < 0.001. FIG.22B shows that mRNA levels of ALPG (ALPPL2) are upregulated in HCC4006 cells with acquired resistance to the EGFR tyrosine kinase inhibitor osimertinib (OR2 and OR7) (n = 3). ***p < 0.001. FIG.22C shows that mRNA levels of ALPG (ALPPL2) are upregulated in HCC827 cells with acquired resistance to the EGFR tyrosine kinase inhibitor erlotinib (ER1 and ER3) (n = 3). ***p < 0.001. FIG. 22D shows representative flow cytometry data demonstrating that monoclonal antibody 8B6Attorney Docket No. MDA0083-401-PC recognizes both ALPP and ALPG (ALPPL2). FIG.22E shows representative flow cytometry data demonstrating that monoclonal antibody H17E2 recognizes both ALPP and ALPG (ALPPL2). FIG.22F shows representative flow cytometry data demonstrating upregulated ALPP / ALPPL2 expression on H1975 osimertinib-resistant (OR) cells compared to H1975 parental cells. FIG.22G shows summarized data demonstrating upregulated ALPP / ALPPL2 expression on H1975 OR cells. **p < 0.01, ***p < 0.001. FIG.22H shows representative flow cytometry data demonstrating upregulated ALPP / ALPPL2 expression on HCC4006 OR cells compared to HCC4006 parental cells. FIG.22I shows summarized data demonstrating upregulated ALPP / ALPPL2 expression on HCC4006 OR cells.

[0044] FIG.23A – FIG.23E – Show that ALPG (ALPPL2) is upregulated in osimertinib-refractory NSCLC patient tissues. FIG.23A shows that mRNA levels of ALPG (ALPPL2) are upregulated in tissues collected at disease progression compared to pre- osimertinib treatment. FIG.23B shows that protein levels of ALPP / ALPPL2 in tissues collected after progression on osimertinib (OSI refractory; n = 41) are upregulated compared to that at baseline (TKI naïve; n = 57) by immunohistochemistry (IHC) staining. FIG.23C shows that staining positivity of ALPP / ALPPL2 in tissues collected after progression on osimertinib (OSI refractory; n = 41) is increased compared to that at baseline (TKI naïve; n = 57) by IHC staining. FIG.23D shows that staining intensity of ALPP / ALPPL2 in tissues collected after progression on osimertinib (OSI refractory; n = 41) is increased compared to that at baseline (TKI naïve; n = 57) by IHC staining. FIG.23E shows representative images of ALPP / ALPPL2 IHC staining in EGFR-TKI naïve and OSI refractory tissues. Original magnification, 400×; scale bars, 20 µm.

[0045] FIG.24A – FIG.24G – Show that ALPG (ALPPL2) expression can be targeted by CAR-T cells in EGFR TKI-resistant NSCLC cells. FIG.24A shows a diagram of an ALPP / ALPPL2 chimeric antigen receptor (CAR) construct. FIG.24B shows activity of ALPP / ALPPL2-targeting CAR-T cells against ALPP / ALPPL2-expressing NSCLC cells. ALPP / ALPPL2 CAR-T cells killed ALPP / ALPPL2-expressing cells (H1650). FIG.24C shows that ALPP / ALPPL2 CAR-T cells killed ALPP-overexpressing (H1975-ALPP) and ALPG-overexpressing (H1975-ALPG) cells but not parental (ALPP-negative) cells. FIG. 24D shows that ALPP / ALPPL2 CAR-T cells (M25-M228BB3Z) killed HCC4006 OR cells but not HCC4006 parental cells. FIG.24E shows that ALPP / ALPPL2 CAR-T cells (M25- M228BB3Z) killed H1975 OR cells but not H1975 parental cells. FIG.24F shows that ALPP / ALPPL2 CAR-T cells (H2-M228BB3Z) killed HCC4006 OR cells but not HCC4006Attorney Docket No. MDA0083-401-PC parental cells. FIG.24G shows that ALPP / ALPPL2 CAR-T cells (H2-M228BB3Z) killed H1975 OR cells but not H1975 parental cells.

[0046] FIG.25A – FIG.25B – Show that ALPG (ALPPL2) expression can be targeted by CAR-NK cells in EGFR TKI-resistant NSCLC cells. FIG.25A shows that ALPP / ALPPL2 CAR-NK cells killed HCC4006 OR cells more effectively as compared to HCC4006 parental cells. FIG.25B shows that ALPP / ALPPL2 CAR-NK cells specifically killed HCC4006 OR cells by blocking natural cytotoxicity pathways.

[0047] FIG.26A – FIG.26E – Show that ALPG (ALPPL2) is upregulated on EGFR TKI (osimertinib) drug tolerant persister cells (DTPCs). FIG.26A shows that mRNA levels of ALPG (ALPPL2) are upregulated in NSCLC cell lines (H1975 and HCC827) after treatment with the EGFR inhibitor OSI for 14 days (n = 3). **p < 0.01, ***p < 0.001. FIG.26B shows representative flow cytometry data demonstrating upregulated ALPP / ALPPL2 expression on H1975 DTPCs, HCC827 DTPCs, and HCC4006 DTPCs. FIG.26C shows summarized data showing upregulated ALPP / ALPPL2 expression on DTPCs. ***p < 0.001. FIG.26D shows that mRNA levels of ALPG (ALPPL2) are upregulated in PC9 xenograft tumors treated with osimertinib for two weeks in vivo. ***p < 0.001. FIG.26E shows that protein levels of ALPP / ALPPL2 are upregulated in H1975 xenograft tumors treated with osimertinib for two weeks in vivo.

[0048] FIG.27A – FIG.27C – Show that ALPG (ALPPL2) expression can be targeted in EGFR TKI DTPCs. FIG.27A shows that ALPP / ALPPL2 CAR-T cells killed H1975 OSI DTPCs but not H1975 parental cells. FIG.27B shows that ALPP / ALPPL2 CAR-T cells killed HCC827 OSI DTPCs but not HCC827 parental cells. FIG.27C shows that ALPP / ALPPL2 CAR-T cells killed HCC4006 OSI DTPCs but not HCC4006 parental cells.

[0049] FIG.28A – FIG.28D – Show that ALPG (ALPPL2) expression can be targeted in EGFR TKI DRCs and DTPCs in vivo. FIG.28A shows that ALPP / ALPPL2 CAR-T cells inhibited H1975-OR17 xenograft growth. FIG.28B shows that ALPP / ALPPL2 CAR-NK cells inhibited H1975-OR17 xenograft growth. FIG.28C shows that ALPP / ALPPL2 CAR- NK cells inhibited H1975 xenograft growth when administrated with osimertinib at the same period. FIG.28D shows that ALPP / ALPPL2 CAR-NK cells inhibited H1975 xenograft growth when administrated after osimertinib treatment for 2 weeks.

[0050] FIG.29A – FIG.29D – Show that ALPG (ALPPL2) is upregulated on KRAS G12C inhibitor DTPCs. FIG.29A shows that mRNA levels of ALPG (ALPPL2) are upregulated in KRAS mutant NSCLC cells (H358, H1373, and Calu-1) after treatment with the KRAS G12C inhibitor adagrasib for 14 days. **p < 0.01, ***p < 0.001. FIG.29B showsAttorney Docket No. MDA0083-401-PC that mRNA levels of ALPG (ALPPL2) are upregulated in KRAS mutant NSCLC cells (H358) after treatment with the KRAS G12C inhibitor sotorasib. ***p < 0.001. FIG.29C shows representative flow cytometry data demonstrating upregulated ALPP expression on H358 sotorasib DTPCs, H1373 adagrasib DTPCs, and Calu-1 adagrasib DTPCs. FIG.29D shows summarized data demonstrating upregulated ALPP expression on KRAS inhibitor DTPCs. ***p < 0.001.

[0051] FIG.30A – FIG.30F – Show that ALPG (ALPPL2) expression can be targeted in KRAS G12C DTPCs. FIG.30A shows ALPP / ALPPL2-targeting CAR-T cells (M25- M228BB3z CAR) killed H358 sotorasib DTPCs but not H358 parental cells. FIG.30B shows ALPP / ALPPL2-targeting CAR-T cells (H2-M228BB3z CAR) killed H358 sotorasib DTPCs but not H358 parental cells. FIG.30C shows ALPP / ALPPL2-targeting CAR-T cells (M25- M228BB3z CAR) killed H1373 adagrasib DTPCs but not H1373 parental cells. FIG.30D shows ALPP / ALPPL2-targeting CAR-T cells (H2-M228BB3z CAR) killed H1373 adagrasib DTPCs but not H1373 parental cells. FIG.30E shows ALPP / ALPPL2-targeting CAR-T cells (M25-M228BB3z CAR) killed Calu-1 adagrasib DTPCs but not Calu-1 parental cells. FIG. 30F shows ALPP / ALPPL2-targeting CAR-T cells (H2-M228BB3z CAR) killed Calu-1 adagrasib DTPCs but not Calu-1 parental cells.

[0052] FIG.31A – FIG.31D – Show that ALPG (ALPPL2) is upregulated in NSCLC cells with acquired resistance to KRAS G12C inhibitors. FIG.31A shows that mRNA levels of ALPG (ALPPL2) are upregulated in H358 cells with acquired resistance to KRAS G12C inhibitor sotorasib (SR). ns p >= 0.05, **p < 0.01, ***p < 0.001. FIG.31B shows summarized western blot data demonstrating upregulated ALPP / ALPPL2 expression on H358 sotorasib-resistant (SR) clones. FIG.31C shows summarized flow cytometry data demonstrating upregulated ALPP / ALPPL2 expression on sotorasib-resistant H358 clones. FIG.31D shows summarized flow cytometry data demonstrating upregulated ALPP / ALPPL2 expression on adagrasib-resistant H358 clones. ***p < 0.001.

[0053] FIG.32 – Shows that ALPG (ALPPL2) expression can be targeted in NSCLC cells with acquired resistance to KRAS G12C inhibitors. The figure shows ALPP / ALPPL2- targeting CAR-T cells killed H358 sotorasib-resistant clones but not H358 parental cells.

[0054] FIG.33A – FIG.33G – Show that ALPG (ALPPL2) is upregulated in RET fusion-positive NSCLC cells that have been treated with a RET inhibitor (RET inhibitor DTPCs) and RET inhibitor-resistant NSCLC cells. FIG.33A shows that mRNA levels of ALPG (ALPPL2) are upregulated in RET-fusion NSCLC cells (TH1101) after treatment with the RET inhibitors pralsetinib and selpercatinib for 14 days (Pral-DTPC and Selp-DTPC,Attorney Docket No. MDA0083-401-PC respectively). FIG.33B shows that mRNA levels of ALPG (ALPPL2) are upregulated in RET-fusion NSCLC cells (LC2 / ad) after treatment with the RET inhibitors pralsetinib and selpercatinib for 14 days (Pral-DTPC and Selp-DTPC, respectively), as well as in pralsetinib- resistant (Pral-R) and selpercatinib-resistant (Sel-R) LC2 / ad cells. FIG.33C shows representative WB data demonstrating upregulated ALPP / ALPPL2 expression in TH1101 and LC2 / ad DTPCs, as well as LC2 / ad cells with acquired resistance to the RET inhibitors. FIG.33D shows summarized data demonstrating upregulated ALPP / ALPPL2 expression on LC2 / ad after treatment with the RET inhibitors selpercatinib for 14 days (Sel-DTPC). ***p < 0.001. FIG.33E shows representative western blot data demonstrating upregulated ALPP / ALPPL2 expression on LC2 / ad cells with acquired resistance to the RET inhibitor selpercatinib compared to LC2 / as parental cells. FIG.33F shows representative flow cytometry data demonstrating upregulated ALPP / ALPPL2 expression on LC2 / ad cells with acquired resistance to the RET inhibitor selpercatinib compared to LC2 / as parental cells. FIG. 33G shows summarized data demonstrating upregulated ALPP / ALPPL2 expression on LC2 / ad selpercatinib-resistant cells. ***p < 0.001.

[0055] FIG.34A – FIG.34D –Show that ALPG (ALPPL2) expression can be targeted in NSCLC cells with acquired resistance to RET inhibitors and RET inhibitor DTPCs. FIG.34A shows ALPP / ALPPL2-targeting CAR-T cells killed LC2 / AD selpercatinib-resistant cells and LC2 / AD selpercatinib DTPCs, but not LC2 / AD parental cells. FIG.34B shows ALPP / ALPPL2-targeting CAR-NK cells killed more LC2 / AD selpercatinib-resistant cells compared to LC2 / AD parental cells. FIG.34C shows that ALPP / ALPPL2 CAR-NK cells specifically killed LC2 / AD selpercatinib-resistant cells by blocking natural cytotoxicity pathways. FIG.34D shows ALPP / ALPPL2-targeting Antibody Drug Conjugate (ADC) killed more LC2 / AD selpercatinib-resistant cells compared to control ADC.

[0056] FIG.35A – FIG.35F – Show that ALPG (ALPPL2) is upregulated in tumor cells bearing ALK fusions after treatment with an ALK inhibitor (DTPCs) and ALK inhibitor- resistant NSCLC cells. FIG.35A shows that mRNA levels of ALPG (ALPPL2) were upregulated in H3122 cells (EML4-ALK fusion) after treatment with the ALK inhibitors (alectinib, brigatinib, and crizotinib) for 14 days. FIG.35B shows western blot data of ALPP / ALPPL2 upregulation in H3122 cells (EML4-ALK fusion) after treatment with the ALK inhibitors (alectinib and brigatinib) for 14 days. FIG.35C shows that mRNA levels of ALPG (ALPPL2) were upregulated in H2228 cells (EML4-ALK fusion) after treatment with the ALK inhibitors (alectinib and lorlatinib). The figure was generated from the GEO dataset GSE188406. FIG.35D shows that mRNA levels of ALPG (ALPPL2) are upregulated inAttorney Docket No. MDA0083-401-PC A925L cells (EML4-ALK fusion) after treatment with the ALK inhibitor alectinib. The figure was generated from GEO dataset GSE188406. FIG.35E shows that mRNA levels of ALPG (ALPPL2) were upregulated in H3122 cells (EML4-ALK fusion) after treatment with the ALK inhibitors crizotinib for 7 days (DTPC). FIG.35F shows that mRNA levels of ALPG (ALPPL2) are upregulated in different clones of H2228 cells with acquired resistance to the ALK inhibitor alectinib. The figure was generated from GEO dataset GSE73167.

[0057] FIG.36A – FIG.36B – Show that ALPG (ALPPL2) is upregulated in breast cancer and stomach cancer cells after treatment with a HER2 inhibitor to generate DTPCs. FIG.36A shows that mRNA levels of ALPG (ALPPL2) are upregulated in breast cancer cells (BT474, EFM192, and SKBR3) after treatment with the HER2 inhibitor lapatinib. The figure was generated from GEO dataset GSE155341. FIG.36B shows that mRNA levels of ALPG (ALPPL2) are upregulated in stomach cancer cell line N87 after treatment with the HER2 inhibitor lapatinib. The figure was generated from GEO dataset GSE89127.

[0058] FIG.37 – Shows that ALPG (ALPPL2) is upregulated in bladder cancer cell line RT112 treated with the FGFR inhibitor infigratinib (BGJ398). The figure was generated from GEO dataset GSE89127.

[0059] FIG.38 – Shows that ALPG (ALPPL2) is upregulated in H358 treated with KRAS inhibitors ARS1323 and AZD4785 and the MEK inhibitor selumetinib. The figure was generated from GEO dataset GSE206867.

[0060] FIG.39 – Shows that ALPG (ALPPL2) is upregulated in NSCLC treated with osimertinib and MEK inhibitor trametinib. The figure shows that mRNA levels of ALPG (ALPPL2) are upregulated in NSCLC (HCC827 and PC9) after treatment with a combination of the EGFR inhibitor osimertinib and the MEK inhibitor trametinib. The figure was generated from GEO dataset GSE131594.

[0061] FIG.40A – FIG.40B – Show that ALPG (ALPPL2) is upregulated in NSCLC treated with MET inhibitors. FIG.40A shows that MET is upregulated in HCC827 osimertinib-resistant (OR) clone OR2. FIG.40B shows that protein levels of ALPP / ALPPL2 are upregulated in HCC827 OR cells after treatment with the MET inhibitors tepotinib and capmatinib.

[0062] FIG.41A – FIG.41E – Show that ALPP / ALPG expression can be targeted in NSCLC cells with acquired resistance to EGFR TKIs by bispecific T cell engager (BiTE). FIG.41A shows structure of ALPP / ALPG targeting bispecific T cell engager (BiTE) and a proposed mechanism of action. FIG.41B shows that ALPP / ALPG-targeting M25-BiTE specifically killed H1650 cell when co-cultured with human peripheral blood mononuclearAttorney Docket No. MDA0083-401-PC cells (PBMCs) at an effector:target (E:T) ratio of 10:1 for 72 hours. FIG.41C shows that ALPP / ALPG-targeting M25-BiTE specifically killed H1975 OR5 and OR16 cells when co- cultured with human PBMCs at an E:T ratio of 10:1 for 72 hours. FIG.41D shows that ALPP / ALPG-targeting M25-BiTE specifically killed HCC4006 OR2 and OR7 cells when co- cultured with human PBMCs at an E:T ratio of 10:1 for 72 hours. FIG.41E shows that ALPP / ALPG-targeting M25-BiTE specifically killed LC2 / AD selpercatinib-resistant cells when co-cultured with human PBMCs at an E:T ratio of 10:1 for 72 hours.

[0063] FIG.42A – 42B – Show that ALPP expression can be targeted in EGFR TKI DRCs in vivo. FIG.42A shows that ALPP / ALPPL2 CAR-T cells inhibited H1975-OR17 xenograft growth. FIG.42B shows that ALPP / ALPPL2 CAR-NK cells inhibited H1975- OR17 xenograft growth.

[0064] FIG.43A – 43F – Show that ALPP is upregulated in RET fusion-positive NSCLC cells that have been treated with a RET inhibitor (RET inhibitor DTPCs) and RET inhibitor-resistant NSCLC cells. FIG.43A shows that mRNA levels of ALPP are upregulated in RET-fusion NSCLC cells (TH1101) after treatment with the RET inhibitors pralsetinib and selpercatinib for 14 days (Pral-DTPC and Selp-DTPC, respectively). FIG.43B shows that mRNA levels of ALPP are upregulated in RET-fusion NSCLC cells (LC2 / ad) after treatment with the RET inhibitors pralsetinib and selpercatinib for 14 days (Pral-DTPC and Selp- DTPC, respectively), as well as in pralsetinib-resistant (Pral-R) and selpercatinib-resistant (Sel-R) LC2 / ad cells. FIG.43C shows that mRNA levels of ALPP are upregulated in RET- fusion NSCLC cells (MDA-L-30) after treatment with the RET inhibitors pralsetinib and selpercatinib for 14 days (Pral-DTPC and Selp-DTPC, respectively). FIG 43D shows representative WB data demonstrating upregulated ALPP / ALPPL2 expression in TH1101, LC2 / ad, and MDA-L-30 DTPCs, as well as LC2 / ad cells with acquired resistance to the RET inhibitors. FIG.43E shows representative flow cytometry data demonstrating upregulated ALPP / ALPPL2 expression on LC2 / ad cells with acquired resistance to the RET inhibitor selpercatinib compared to LC2 / as parental cells. FIG.22F shows summarized data demonstrating upregulated ALPP / ALPPL2 expression on LC2 / ad after treatment with the RET inhibitors selpercatinib for 14 days (Sel-DTPC), as well as on LC2 / ad selpercatinib- resistant cells (Sel-R). ***p < 0.001.

[0065] FIG.44A – 44F – Show that ALPP is upregulated in RET fusion-positive NSCLC cells that been treated with a RET inhibitor. FIG.44A shows that mRNA levels of ALPP are upregulated in RET-fusion NSCLC cells (TH1101) after treatment with the RET inhibitors pralsetinib and selpercatinib for 24 or 48 hours. FIG.44B shows representative WBAttorney Docket No. MDA0083-401-PC data demonstrating upregulated ALPP / ALPPL2 expression in RET-fusion NSCLC cells (TH1101) after treatment with the RET inhibitors pralsetinib and selpercatinib for 24 or 48 hours. FIG.44C shows that mRNA levels of ALPP are upregulated in RET-fusion NSCLC cells (LC2 / ad) after treatment with the RET inhibitors pralsetinib and selpercatinib for 24 or 48 hours. FIG.44D shows representative WB data demonstrating upregulated ALPP / ALPPL2 expression in RET-fusion NSCLC cells (LC2 / ad) after treatment with the RET inhibitors pralsetinib and selpercatinib for 24 or 48 hours. FIG.44E shows that mRNA levels of ALPP are upregulated in RET-fusion NSCLC cells (MDA-L-30) after treatment with the RET inhibitors pralsetinib and selpercatinib for 24 hours. FIG.44F shows representative WB data demonstrating upregulated ALPP / ALPPL2 expression in RET-fusion NSCLC cells (MDA-L-30) after treatment with the RET inhibitors pralsetinib and selpercatinib for 48 or 72 hours.

[0066] FIG.45A – 45D – Show that ALPP is upregulated in tumor cells bearing ALK fusions after treatment with an ALK inhibitor (DTPCs) and ALK inhibitor-resistant NSCLC cells. FIG.45A shows that mRNA levels of ALPP were upregulated in H3122 cells (EML4- ALK fusion) after treatment with the ALK inhibitors (alectinib, brigatinib, and crizotinib) for 14 days. FIG.45B shows western blot data of ALPP / ALPPL2 upregulation in H3122 cells (EML4-ALK fusion) after treatment with the ALK inhibitors (alectinib and brigatinib) for 14 days. FIG.45C shows that mRNA levels of ALPP were upregulated in LG0812 PDX (ALK fusion positive) after treatment with the ALK inhibitors (alectinib). The figure was generated from the GEO dataset GSE255958. FIG.45D shows that mRNA levels of ALPP are upregulated in FA34 and TS485T PDOs (ALK fusion) with acquired resistance to ALK inhibitor alectinib (OA) or crizotinib (OC) compared to TKI sensitive cells (OS). The figure was generated from GEO dataset GSE223779.

[0067] FIG.46A – 46B – Show the generation of ALPP / ALPG-targeting bispecific T cell engager (BiTE). FIG.46A shows structure of ALPP / ALPG targeting bispecific T cell engager (BiTE) and a proposed mechanism of action. FIG.46B shows that ALPP / ALPG- targeting M25-BiTE specifically killed H1650 cell when co-cultured with human peripheral blood mononuclear cells (PBMCs) at an effector:target (E:T) ratio of 10:1 for 72 hours.

[0068] FIG.47A – 47F – Show that ALPP expression on drug-resistant cells and drug- tolerant persister cells of NSCLC cells by ALPP / ALPG-targeting bispecific T cell engager (TCE). FIG.47A shows that ALPP / ALPG-targeting M25-BiTE specifically killed HCC4006 osimertinib-resistant (OR2 and OR7) cells when co-cultured with human PBMCs at an E:T ratio of 10:1 for 72 hours. FIG.47B shows that ALPP / ALPG-targeting M25-BiTEAttorney Docket No. MDA0083-401-PC specifically killed H1975 osimertinib-resistant (OR5 and OR16) cells when co-cultured with human PBMCs at an E:T ratio of 10:1 for 72 hours. FIG.47C shows that ALPP / ALPG- targeting M25-BiTE specifically killed HCC827 osimertinib DTPCs when co-cultured with human PBMCs at an E:T ratio of 10:1 for 72 hours. FIG.47D shows that ALPP / ALPG- targeting M25-BiTE specifically killed H1975 osimertinib DTPCs when co-cultured with human PBMCs at an E:T ratio of 10:1 for 72 hours. FIG.47E shows that ALPP / ALPG- targeting M25-BiTE specifically killed H358 sotorasib-resistant (SOTO3, SOTO7, and SOTO10) cells when co-cultured with human PBMCs at an E:T ratio of 10:1 for 72 hours. FIG.47F shows that ALPP / ALPG-targeting M25-BiTE specifically killed LC2 / AD selpercatinib-resistant cells when co-cultured with human PBMCs at an E:T ratio of 10:1 for 72 hours.

[0069] FIG.48A – 48E – Show that ALPP is upregulated in tumor cells bearing KRAS mutation after KRAS inhibition. FIG.48A shows that mRNA levels of ALPP were upregulated in tumor cells (H358, Mia Paca-2, and SW837 cells) bearing KRAS G12C mutation after treatment with the KRAS G12C inhibitors MRTX1257 for 24 hours. The figure was generated with data from NCBI SRA PRJNA1054293. FIG.48B shows that mRNA levels of ALPP were upregulated in pancreatic cancer cells (HPAC, Mia Paca-2, Pa01C, Pa02C, Pa04C, Pa14C, Pa16C, and PANC-1 cells) bearing KRAS mutation after treatment with the KRAS siRNA for 24 hours. The figure was generated with data from NCBI SRA PRJNA980201. FIG.48C shows that mRNA levels of ALPP were upregulated in pancreatic cancer cells (HPAC, Mia Paca-2, and AsPC1 cells) bearing KRAS mutation after treatment with the KRAS G12C inhibitor MRTX849 or KRAS G12D inhibitor MRTX1133 for 24 hours. The figure was generated with data from NCBI SRA PRJNA578935 and PRJNA831648. FIG.48D shows that mRNA levels of ALPP were upregulated in 11 lung (H358, H2122, H2030, H1373, and A427 cells), pancreatic (HPAC, Mia Paca-2, and AsPC1), colon (SNU1033 and LS180), and stomach cancer (AGS) cells bearing KRAS mutation after treatment with the KRAS G12C inhibitor MRTX849 or KRAS G12D inhibitor MRTX1133 for 24 hours. The figure was generated with data from NCBI SRA PRJNA578935 and PRJNA831648. FIG.48E shows that mRNA levels of ALPP were upregulated in tumor cells (Mia Paca-2) bearing KRAS G12C mutation after treatment with the KRAS G12C inhibitors adagrasib or sotorasib for 48 hours.

[0070] FIG.49A – 49C – Show that ALPP is upregulated in tumor cells after MEK-ERK pathway inhibition. FIG.49A shows that mRNA levels of ALPP were upregulated in pancreatic cancer cells (Pa16C) bearing KRAS G12D mutation after treatment with theAttorney Docket No. MDA0083-401-PC KRAS G12D inhibitor MRTX1133, MEK inhibitor trametinib, or ERK inhibitor SCH772984 for 24 hours. The figure was generated with data from NCBI SRA PRJNA1063706. FIG.49B shows that mRNA levels of ALPP were upregulated in NSCLC cells (HCC827) bearing EGFR mutation after treatment with the EGFR inhibitor osimertinib, MEK inhibitor trametinib, or ERK inhibitors ulixertinib and SCH772984 for 24 hours. FIG.49C shows summarized flow cytometry data of ALPP upregulation in NSCLC cells (HCC827) bearing EGFR mutation after treatment with the EGFR inhibitor osimertinib, MEK inhibitor trametinib, or ERK inhibitors ulixertinib and SCH772984 for 24 hours.

[0071] FIG.50A – 50H – Show that ALPP is upregulated in tumor cells after MEK inhibition. FIG.50A shows that mRNA levels of ALPP were upregulated in pancreatic cancer cells (Mia PaCa2) bearing KRAS G12C mutation after treatment with the MEK inhibitor trametinib. The figure was generated with data from GSE206267. FIG.50B shows that mRNA levels of ALPP were upregulated in pancreatic cancer cells (BXPC3) bearing BRAF V600E mutation after treatment with the MEK inhibitor trametinib. The figure was generated with data from GSE112282. FIG.50C shows that mRNA levels of ALPP were upregulated in PATU8902 (pancreatic cancer, KRAS G12V), A375 (skin cancer, BRAF V600E), Calu1 (NSCLC, KRAS G12C), and HCT116 (colon cancer, KRAS G13D) after treatment with the MEK inhibitor trametinib. The figure was generated with data from GSE78519. FIG.50D shows that mRNA levels of ALPP were upregulated in HCT116 (colon cancer, KRAS G13D) after treatment with the MEK inhibitor cobimetinib. The figure was generated with data from GSE186618. FIG.50E shows that mRNA levels of ALPP were upregulated in SW480 xenograft (colon cancer, KRAS G12V) after treatment with the MEK inhibitor selumetinib (AZD). The figure was generated with data from GSE98922. FIG.50F shows that mRNA levels of ALPP were upregulated in SK-MEL-113 cells (melanoma, NF1null) after treatment with the MEK inhibitor trametinib (Tram). The figure was generated with data from GSE213588. FIG.50G shows that mRNA levels of ALPP were upregulated in NF cells (malignant peripheral nerve sheath tumor, NF1null) after treatment with the MEK inhibitor selumetinib. The figure was generated with data from GSE212964. FIG.50H shows that mRNA levels of ALPP were upregulated in S462TY cells (malignant peripheral nerve sheath tumor, NF1null) after treatment with the MEK inhibitor selumetinib. The figure was generated with data from GSE262030.

[0072] FIG.51A – 51C – Show that ALPP is upregulated in tumor cells after ERK inhibition. FIG.51A shows that mRNA levels of ALPP were upregulated in pancreatic cancer cells (HPAC, HPAF-II, Pa01C, Pa04C, Pa14C, PANC-1, and SW1990 cells) bearingAttorney Docket No. MDA0083-401-PC KRAS mutation (KRAS G12C or G12D) after treatment with the ERK inhibitor SCH772984 for different time (1h, 4h, 12h, and 24h). The figure was generated with data from PRJEB25806. FIG.51B shows that protein levels of ALPP were upregulated in pancreatic cancer cells (ASPC-1, HPAC, Mia Paca-2, PANC-1, Pa16C, and SW1990 cells) bearing KRAS mutation (KRAS G12C or G12D) after treatment with the ERK inhibitor SCH772984 for 24h. The figure was generated with data from PXD048531 (10.6019 / PXD048531). FIG. 51C shows that mRNA levels of ALPP were upregulated in pancreatic cancer patients after treatment with the ERK inhibitor ulixertinib. The figure was generated with data from GSE213797.

[0073] FIG.52 – Shows that ALPP is upregulated in breast cancer cells (CAL-120) after treatment with FGFR inhibitor infigratinib for 4 hours, 48 hours, or 12 days. The figure was generated with data from GSE168023.

[0074] FIG.53A – 53B – Show that ALPP is upregulated in triple-negative breast cancer (TNBC) cells after AKT inhibition. FIG.53A shows that mRNA levels of ALPP were upregulated in TNBC cell MDA-MB-468 after treatment with the AKT inhibitor ipatasertib. The figure was generated with data from GSE205729. FIG.53B shows that mRNA levels of ALPP were upregulated in TNBC cell SUM149PT after treatment with the AKT inhibitor ipatasertib. The figure was generated with data from GSE205730.

[0075] FIG.54A – 54B – Show that ALPP is upregulated in tumor cells with CDK4 / 6 inhibition. FIG.54A shows that mRNA levels of ALPP were upregulated in NSCLC cell H358 after treatment with the CDK4 / 6 inhibitor palbociclib. The figure was generated with data from GSE253204. FIG.54B shows that ALPP is upregulated in CDK4 / 6 inhibitor (palbociclib) resistant breast cancer cells (MCF-7). The figure was generated with data from GSE234514.

[0076] FIG.55A – 55C – Show that ALPP is upregulated in tumor cells after BRAF inhibition. FIG.55A shows that mRNA levels of ALPP were upregulated in thyroid cancer cells (CUTC5 and CUTC16) after treatment with the BRAF inhibitor dabrafenib, MEK inhibitor ulixertinib, or their combination. The figure was generated with data from GSE221329. FIG.55B shows that mRNA levels of ALPP were upregulated in thyroid cancer cells (8505C and WRO) after treatment with the BRAF inhibitor vemurafenib. The figure was generated with data from GSE178267. FIG.55C shows that mRNA levels of ALPP were upregulated in melanoma cells (WM1366, NRAS mutation) with acquired resistance against BRAF inhibitor encorafenib and MEK inhibitor binimetinib. The figure was generated with data from GSE245262.Attorney Docket No. MDA0083-401-PC

[0077] FIG.56 – Shows that ALPP is upregulated in drug-tolerant persister cells of colorectal adenocarcinoma (HT29 cells, with BRAF V600E mutation) after treatment with DNA topoisomerase I inhibitor irinotecan. The figure was generated with data from GSE189625.

[0078] FIG.57A – 57C – Shows that ALPP is upregulated in chemo drug-tolerant persister cells. FIG.57A shows that mRNA levels of ALPP were upregulated in breast cancer drug-tolerant persister cells after treatment with chemotherapy drug (docetaxel). The figure was generated with data from GSE162285. FIG.57B shows that mRNA levels of ALPP were upregulated in prostate cancer drug-tolerant persister cells after treatment with chemotherapy drug (docetaxel or vinblastine). The figure was generated with data from GSE162285. FIG. 57C shows that mRNA levels of ALPP were upregulated in pancreatic cancer cell Mia Paca-2 after treatment with chemotherapy drug gemcitabine. The figure was generated with data from GSE189764.

[0079] FIG.58 – Shows that ALPP is upregulated in head and neck squamous cell carcinoma (HNSCC) after treatment with EGFR blocking antibody cetuximab. The figure was generated with data from GSE137524. DETAILED DESCRIPTION

[0080] Provided are methods for treating treatment-resistant non-small-cell lung cancer (NSCLC) in a patient in need thereof comprising: administering a chimeric antigen receptor (CAR) cell therapy targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from the group consisting of an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, and an FGFR mutation.

[0081] Also provided are methods for treating treatment-resistant non-small-cell lung cancer (NSCLC) in a patient in need thereof comprising: identifying a genetic mutation in a NSCLC cell from a biological sample obtained from the patient; detecting and / or quantifying placental alkaline phosphatase (ALPP) and / or ALPPL2 cell surface expression in the NSCLC cell; administering a chimeric antigen receptor (CAR) cell therapy targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting the genetic mutation; wherein the genetic mutation is a mutation selected from the group consisting of an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, and an FGFR mutation.

[0082] Also provided are methods for treating drug-tolerant persister non-small-cell lung cancer (NSCLC) cells or drug-resistant NSCLC cells in a patient in need thereof comprising:Attorney Docket No. MDA0083-401-PC identifying a genetic mutation in a NSCLC cell from a biological sample obtained from the patient; detecting and / or quantifying ALPP and / or ALPPL2 cell surface expression in the NSCLC cell; administering a chimeric antigen receptor (CAR) cell therapy or antibody drug conjugate (ADC) targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting the genetic mutation; wherein the genetic mutation is a mutation selected from the group consisting of an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, and an FGFR mutation.

[0083] Also provided are methods of preventing emergence of resistance of non-small- cell lung cancer (NSCLC) to a drug therapy comprising administering a chimeric antigen receptor (CAR) cell therapy or antibody drug conjugate (ADC) targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from the group consisting of an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, and an FGFR mutation.

[0084] Also provided is a method for treating non-small-cell lung cancer (NSCLC) that is resistant to a targeted inhibitor in a patient in need thereof comprising: administering a CAR- T cell targeting ALPP and / or ALPPL2 on the surface of the NSCLC cells; wherein the NSCLC cells exhibit increased expression of ALPP and / or ALPPL2 relative to a healthy lung cell; wherein the CAR-T cell targeting ALPP and / or ALPPL2 comprises a single-chain variable-fragment (scFv) derived from an ALPP-specific antibody; wherein the NSCLC cells comprise: a mutation in the EGFR gene resulting in resistance to EGFR tyrosine kinase inhibitor osimertinib; or a mutation in the KRAS gene resulting in resistance to KRAS inhibitors adagrasib and / or sotorasib; or a mutation in the RET gene resulting in resistance to RET inhibitor selpercatinib and / or pralsetinib; or a mutation in the ALK gene resulting in resistance to ALK inhibitors alectinib and / or lorlatinib and / or crizotinib; or a mutation in the HER2 gene resulting in resistance to HER2 inhibitor lapatinib; or a mutation in the MET gene resulting in resistance to a MET inhibitor, such as tepotinib and capmatinib; or a mutation in the MEK gene resulting in resistance to a MEK inhibitor, such as binimetinib (MEK162), Cotellic, Koselugo, Mekinist, Mektovi, cobimetinib or XL518, selumetinib, trametinib (GSK1120212), PD-325901, CI-1040, PD035901, and / or TAK-733; or a mutation in the FGFR gene resulting in resistance to an FGFR inhibitor, such as Balversa, anlotinib, erdafitinib, infigratinib, pemazyre, and / or pemigatinib, wherein the CAR- T cell specifically binds to and eliminates the NSCLC cells having increased ALPP and / or ALPPL2; and wherein the CAR-T cell comprises a sequence set forth in SEQ ID NOs:1-18. Also provided are methods for treating treatment-resistant non-small-cell lung cancerAttorney Docket No. MDA0083-401-PC (NSCLC) in a patient in need thereof comprising: administering an antibody-drug conjugate (ADC) targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from the group consisting of an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, and an FGFR mutation. ADC targeting of ALPP and / or ALPG / ALPPL2 may be used interchangeably with CAR-T / CAR-NK targeting of ALPP / ALPPL2. In some embodiments, the methods described herein may be used to target ALPP in cells with EGFR tyrosine kinase inhibitor (TKI) resistance that is mediated by MET upregulation.

[0085] Also provided are methods for treating a non-small cell lung cancer (NSCLC) expressing elevated cell surface levels of alkaline phosphatase, germ cell (ALPG / ALPPL2) protein, comprising administering to the patient: (i) a treatment therapy targeting ALPG / ALPPL2; and / or (ii) a second treatment therapy; wherein the NSCLC has acquired resistance to a targeted inhibitor of an oncogene gene chosen from EGFR, KRAS, RET, ALK, HER2, MET, MEK, and FGFR. Also provided are methods for treating a non-small cell lung cancer (NSCLC) expressing elevated cell surface levels of alkaline phosphatase, germ cell (ALPG / ALPPL2) protein, comprising: identifying a genetic mutation in a NSCLC cell from a biological sample obtained from the patient; detecting and / or quantifying ALPG / ALPPL2 cell surface expression in the NSCLC cell; administering a chimeric antigen receptor (CAR) cell therapy targeting ALPG / ALPPL2 in the NSCLC cell, and / or a therapy targeting the genetic mutation; wherein the genetic mutation is a mutation selected from the group consisting of an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, and an FGFR mutation. Also provided are methods for treating drug-tolerant persister non-small-cell lung cancer (NSCLC) cells or drug-resistant NSCLC cells in a patient in need thereof comprising: identifying a genetic mutation in a NSCLC cell from a biological sample obtained from the patient; detecting and / or quantifying alkaline phosphatase, germ cell (ALPG / ALPPL2) cell surface expression in the NSCLC cell; administering a chimeric antigen receptor (CAR) cell therapy targeting ALPG / ALPPL2 in the NSCLC cell, and / or a therapy targeting the genetic mutation; wherein the genetic mutation is a mutation selected from the group consisting of an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, and an FGFR mutation. Also provided are methods of preventing emergence of resistance of non-small-cell lung cancer (NSCLC) to a drug therapy comprising administering a chimeric antigen receptor (CAR) cell therapy targeting alkaline phosphatase, germ cell (ALPG / ALPPL2) in the NSCLC cell, and / or a therapy targeting a genetic mutationAttorney Docket No. MDA0083-401-PC selected from the group consisting of an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, and an FGFR mutation. Also provided are methods of selecting a patient having a non-small cell lung cancer (NSCLC) for treatment with a cancer therapy targeting alkaline phosphatase, germ cell (ALPG / ALPPL2) comprising: identifying a genetic mutation in a NSCLC cell from a biological sample obtained from the patient; detecting and / or quantifying ALPG / ALPPL2 cell surface expression in the NSCLC cell; administering a chimeric antigen receptor (CAR) cell therapy targeting ALPG / ALPPL2 in the NSCLC cell, and / or a therapy targeting the genetic mutation; wherein the genetic mutation is a mutation selected from the group consisting of an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, and an FGFR mutation.

[0086] Also provided is a method for treating a non-small cell lung cancer (NSCLC) expressing elevated cell surface levels of alkaline phosphatase, germ cell (ALPG / ALPPL2) protein, comprising: administering a CAR-T cell targeting ALPG / ALPPL2 on the surface of the NSCLC cells; wherein the CAR-T cell targeting ALPG / ALPPL2 comprises a single-chain variable-fragment (scFv) derived from an ALPPL2-specific antibody; wherein the NSCLC cells comprise: a mutation in the EGFR gene resulting in resistance to EGFR inhibitor, such as a tyrosine kinase inhibitor, e.g., osimertinib, mobocertinib, amivantamab, CLN081, and / or DZD9008; or a mutation in the KRAS gene resulting in resistance to KRAS inhibitors adagrasib and / or sotorasib; or a mutation in the RET gene resulting in resistance to RET inhibitors selpercatinib and / or pralsetinib;; or a mutation in the ALK gene resulting in resistance to ALK inhibitors crizotinib, alectinib, brigatinib, and / or lorlatinib; or a mutation in the HER2 gene resulting in resistance to HER2 inhibitors lapatinib, BI1810631 (zongertinib), and / or trastuzumab-deruxtecan; or a mutation in the MET gene resulting in resistance to MET inhibitors tepotinib and capmatinib; or a mutation in the MEK gene resulting in resistance to MEK inhibitors binimetinib (MEK162), Cotellic, Koselugo, Mekinist, Mektovi, cobimetinib or XL518, selumetinib, trametinib (GSK1120212), PD- 325901, CI-1040, PD035901, and / or TAK-733; or a mutation in the FGFR gene resulting in resistance to FGFR inhibitors Balversa, anlotinib, erdafitinib, infigratinib, pemazyre, and / or pemigatinib; wherein the CAR-T cell specifically binds to and eliminates the NSCLC cells having increased ALPG / ALPPL2; and wherein the CAR-T cell comprises a sequence set forth in SEQ ID NOs:1-18.

[0087] Also provided is a method for treating a non-small cell lung cancer (NSCLC) expressing elevated cell surface levels of alkaline phosphatase, germ cell (ALPG / ALPPL2)Attorney Docket No. MDA0083-401-PC protein, comprising: administering a CAR-T cell targeting ALPG / ALPPL2 on the surface of the NSCLC cells; wherein the CAR-T cell targeting ALPG / ALPPL2 comprises a single-chain variable-fragment (scFv) derived from an ALPPL2-specific antibody; wherein the NSCLC cells comprise: a mutation in the EGFR gene resulting in resistance to EGFR inhibitor, such as a tyrosine kinase inhibitor, e.g., osimertinib, mobocertinib, amivantamab, CLN081, and / or DZD9008; or a mutation in the KRAS gene resulting in resistance to KRAS inhibitors adagrasib and / or sotorasib; or a mutation in the RET gene resulting in resistance to RET inhibitors selpercatinib and / or pralsetinib;; or a mutation in the ALK gene resulting in resistance to ALK inhibitors crizotinib, alectinib, brigatinib, and / or lorlatinib; or a mutation in the HER2 gene resulting in resistance to HER2 inhibitors lapatinib, BI1810631 (zongertinib), and / or trastuzumab-deruxtecan; or a mutation in the MET gene resulting in resistance to MET inhibitors tepotinib and capmatinib; or a mutation in the MEK gene resulting in resistance to MEK inhibitors binimetinib (MEK162), Cotellic, Koselugo, Mekinist, Mektovi, cobimetinib or XL518, selumetinib, trametinib (GSK1120212), PD- 325901, CI-1040, PD035901, and / or TAK-733; or a mutation in the AKT gene resulting in resistance to AKT inhibitor ipatasertib; or a mutation in the ERK gene resulting in resistance to ERK inhibitor ulixertinib; or a mutation in the CDK4 / 6 gene resulting in resistance to CDK4 / 6 inhibitor palbociclib; or a mutation in the BRAF gene resulting in resistance to BRAF inhibitors dabrafenib, vemurafenib, and / or encorafenib; or a mutation in the FGFR gene resulting in resistance to FGFR inhibitors Balversa, anlotinib, erdafitinib, infigratinib, pemazyre, and / or pemigatinib; wherein the CAR-T cell specifically binds to and eliminates the NSCLC cells having increased ALPG / ALPPL2; and wherein the CAR-T cell comprises a sequence set forth in SEQ ID NOs:1-18.

[0088] Also provided is a method for treating treatment-resistant non-small-cell lung cancer (NSCLC) in a patient in need thereof comprising: administering a bispecific T-cell engager (BiTE) targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from the group consisting of an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, and an FGFR mutation.

[0089] Also provided is a method for treating treatment-resistant non-small-cell lung cancer (NSCLC) in a patient in need thereof comprising: administering a chimeric antigen receptor (CAR) cell therapy or antibody drug conjugate (ADC) targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, aAttorney Docket No. MDA0083-401-PC MET mutation, a MEK mutation, an AKT mutation, an ERK mutation, a CDK4 / 6 mutation, a BRAF mutation, and an FGFR mutation.

[0090] Also provided is a method for treating treatment-resistant non-small-cell lung cancer (NSCLC) in a patient in need thereof comprising: identifying a genetic mutation in a NSCLC cell from a biological sample obtained from the patient; detecting and / or quantifying placental alkaline phosphatase (ALPP) and / or ALPPL2 cell surface expression in the NSCLC cell; administering a chimeric antigen receptor (CAR) cell therapy targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting the genetic mutation; wherein the genetic mutation is a mutation selected from an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, an AKT mutation, an ERK mutation, a CDK4 / 6 mutation, a BRAF mutation, and an FGFR mutation.

[0091] Also provided is a method for treating drug-tolerant persister non-small-cell lung cancer (NSCLC) cells or drug-resistant NSCLC cells in a patient in need thereof comprising: identifying a genetic mutation in a NSCLC cell from a biological sample obtained from the patient; detecting and / or quantifying ALPP and / or ALPPL2 cell surface expression in the NSCLC cell; administering a chimeric antigen receptor (CAR) cell therapy targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting the genetic mutation; wherein the genetic mutation is a mutation selected from an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, an AKT mutation, an ERK mutation, a CDK4 / 6 mutation, a BRAF mutation, and an FGFR mutation.

[0092] Also provided is a method of preventing emergence of resistance of non-small- cell lung cancer (NSCLC) to a drug therapy comprising administering a chimeric antigen receptor (CAR) cell therapy targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, an AKT mutation, an ERK mutation, a CDK4 / 6 mutation, a BRAF mutation, and an FGFR mutation.Attorney Docket No. MDA0083-401-PC

[0093] Also provided is a method for treating treatment-resistant non-small-cell lung cancer (NSCLC) in a patient in need thereof comprising administering a bispecific T-cell engager (BiTE) targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, an AKT mutation, an ERK mutation, a CDK4 / 6 mutation, a BRAF mutation, and an FGFR mutation.

[0094] In some embodiments, the non-small-cell lung cancer (NSCLC) cells comprise increased cell surface expression of ALPP and / or ALPPL2 relative to healthy lung cells.

[0095] In some embodiments, the non-small-cell lung cancer (NSCLC) cells comprise increased cell surface expression of ALPG / ALPPL2 relative to healthy lung cells.

[0096] In some embodiments, the EGFR mutation comprises an L858R point mutation, an exon 19 deletion, a T790M mutation, a C797S mutation, or an exon 20 insertion mutation.

[0097] In some embodiments, the KRAS mutation comprises a mutation in codon 12 or codon 13, such as a G12C mutation, a G12V mutation, or a G12D mutation.

[0098] In some embodiments, the KRAS mutation comprises a mutation in codon 12 or codon 13, such as a G12C mutation, a G12V mutation, a G12D mutation, or a G13D mutation.

[0099] In some embodiments, the RET mutation comprises a RET rearrangement or a RET fusion.

[0100] In some embodiments, the RET fusion is a CCDC6-RET fusion or a KIF5B-RET fusion.

[0101] In some embodiments, the ALK mutation comprises an ALK fusion.

[0102] In some embodiments, the ALK fusion comprises an EML4-ALK gene fusion.

[0103] In some embodiments, the HER2 mutation comprises an exon 20 YVMA insertion, an exon 20 GSP insertion, an exon 20 VC insertion, a tyrosine kinase domain mutation, or a mutation in the transmembrane domain, e.g., a V659 mutation.

[0104] In some embodiments, the MET mutation comprises a MET amplification or an exon 14 skipping mutation.

[0105] In some embodiments, wherein the MEK mutation comprises a mutation that involves the allosteric drug binding pocket or α-helix C, a mutation in a MEK1 codon located within or abutting the N-terminal negative regulatory helix (helix A), a MEK1(P124L) mutation, a MEK1(Q56P) mutation, or any mutation in the MEK gene that results in or confers resistance to a MEK inhibitorAttorney Docket No. MDA0083-401-PC

[0106] In some embodiments, the MEK mutation comprises a mutation that involves the allosteric drug binding pocket or α-helix C, a mutation in a MEK1 codon located within or abutting the N-terminal negative regulatory helix (helix A), a MEK1(P124L) mutation, a MEK1(Q56P) mutation, or any mutation in the MEK gene that results in or confers resistance to a MEK inhibitor.

[0107] In some embodiments, the BRAF mutation is BRAF V600E.

[0108] In some embodiments, the FGFR mutation comprises a gene fusion, an amplification, or a point mutation involving one or more of FGFR1, FGFR2, FGFR3, and FGFR4.

[0109] In some embodiments, the FGFR fusion comprises an FGFR2-ERC1 fusion, an FGFR3-TACC3 fusion, an FGFR2-INA fusion, a FGFR4-RAPGEFL1 fusion, and a fusion of the FGFR1 and SLC20A25’-untranslated regions.

[0110] In some embodiments, the FGFR amplification comprises an FGFR1 amplification, an FGFR2 amplification, an FGFR3 amplification, or an FGFR4 amplification.

[0111] In some embodiments, the FGFR point mutation comprises R248C, S249C, G370C, S371C, Y373C, G380R, W290C, S320C, or K660E / N.

[0112] In some embodiments, the non-small-cell lung cancer having an EGFR mutation is resistant to an inhibitor targeting EGFR; or the non-small-cell lung cancer having a KRAS mutation is resistant to an inhibitor targeting KRAS; or the non-small-cell lung cancer having a RET mutation is resistant to an inhibitor targeting RET; or the non-small-cell lung cancer having an ALK mutation is resistant to an inhibitor targeting ALK; or the non-small-cell lung cancer having a HER2 mutation is resistant to an inhibitor targeting HER2; or the non-small- cell lung cancer having a MET mutation is resistant to an inhibitor targeting MET; or the non-small-cell lung cancer having a MEK mutation is resistant to an inhibitor targeting MEK; or the non-small-cell lung cancer having an FGFR mutation is resistant to an inhibitor targeting FGFR.

[0113] In some embodiments, the non-small-cell lung cancer having an EGFR mutation is resistant to an inhibitor targeting EGFR; or the non-small-cell lung cancer having a KRAS mutation is resistant to an inhibitor targeting KRAS; or the non-small-cell lung cancer having a RET mutation is resistant to an inhibitor targeting RET; or the non-small-cell lung cancer having an ALK mutation is resistant to an inhibitor targeting ALK; or the non-small-cell lung cancer having a HER2 mutation is resistant to an inhibitor targeting HER2; or the non-small- cell lung cancer having a MET mutation is resistant to an inhibitor targeting MET; or the non-small-cell lung cancer having a MEK mutation is resistant to an inhibitor targeting MEK;Attorney Docket No. MDA0083-401-PC or the non-small-cell lung cancer having an AKT mutation is resistant to an inhibitor targeting AKT; or the non-small-cell lung cancer having an ERK mutation is resistant to an inhibitor targeting ERK; the non-small-cell lung cancer having an CDK4 / 6 mutation is resistant to an inhibitor targeting CDK4 / 6; the non-small-cell lung cancer having an BRAF mutation is resistant to an inhibitor targeting BRAF; or the non-small-cell lung cancer having an FGFR mutation is resistant to an inhibitor targeting FGFR.

[0114] In some embodiments, the non-small-cell lung cancer (NSCLC) cell has acquired resistance to a targeted inhibitor of an oncogene gene chosen from EGFR, KRAS, RET, ALK, HER2, MET, MEK, and FGFR, and wherein the NSCLC cell is a drug-tolerant persister cell (DTPC) or a drug-resistant cell (DRC).

[0115] In some embodiments, the treatment-resistant non-small-cell lung cancer (NSCLC) cell is a drug-tolerant persister cell (DTPC) or a drug-resistant cell (DRC).

[0116] In some embodiments, the administration of the chimeric antigen receptor (CAR) cell therapy targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or the therapy targeting the genetic mutation prevents the drug-tolerant persister cell (DTPC) from developing into a drug-resistant cell (DRC).

[0117] In some embodiments, the administration of the chimeric antigen receptor (CAR) cell therapy targeting ALPG / ALPPL2 in the NSCLC cell, and / or the therapy targeting the genetic mutation prevents the drug-tolerant persister cell (DTPC) from developing into a drug-resistant cell (DRC).

[0118] In some embodiments, antibody-drug conjugates are used to target ALPP and / or ALPPL2.

[0119] In some embodiments, the ALPP / ALPPL2 targeting molecule comprises a bi- specific T cell engager (BiTE) or a tri-specific natural killer cell engager therapy (TriNKET).

[0120] In some embodiments, the method treats drug-tolerant persister cells (DTPCs) and drug-resistant cells (DRCs) to prevent the emergence of resistance to EGFR tyrosine kinase inhibitors, KRAS inhibitors, RET inhibitors, ALK inhibitors, HER2 inhibitors, MET inhibitors, MEK inhibitors, or FGFR inhibitors.

[0121] In some embodiments, the method treats drug-tolerant persister cells (DTPCs) and drug-resistant cells (DRCs) to prevent the emergence of resistance to EGFR tyrosine kinase inhibitors, KRAS inhibitors, RET inhibitors, ALK inhibitors, HER2 inhibitors, MET inhibitors, MEK inhibitors, AKT inhibitors, ERK inhibitors, CDK4 / 6 inhibitors, BRAF inhibitors, or FGFR inhibitors.Attorney Docket No. MDA0083-401-PC

[0122] In some embodiments, detecting and / or quantifying placental alkaline phosphatase (ALPP) and / or ALPPL2 cell surface expression, or ALPG / ALPPL2 cell surface expression, in the NSCLC cell comprises histological analysis, immunohistochemical (IHC) staining for ALPP protein, a blood-based test, a tissue-based test, or imaging techniques.

[0123] In some embodiments, the tissue-based test comprises a tissue biopsy, flow cytometry, immunohistochemistry (IHC), western blot (WB), polymerase chain reaction (PCR), or immunofluorescence (IF).

[0124] In some embodiments, the tissue-based test comprises a Mammaprint + Blueprint® test or an Oncotype DX® test.

[0125] In some embodiments, the blood-based test comprises Galleri®, circulating tumor cell (CTC) test, a complete blood count (CBC), or a test or assay for measuring circulating proteins, autoantibodies, cell-free circulating DNA, or extracellular vesicle-derived proteins.

[0126] In some embodiments, the tissue biopsy is analyzed by hematoxylin and eosin (H&E) staining and / or microscopy.

[0127] In some embodiments, the chimeric antigen receptor (CAR) cell therapy targeting ALPP and / or ALPPL2, or targeting ALPG / ALPPL2, in the NSCLC cell comprises a CAR-T cell, a CAR-NK cell, or a combination thereof.

[0128] In some embodiments, the therapy targeting the genetic mutation comprises one or more targeted inhibitor and / or one or more immunotherapy agent.

[0129] In some embodiments, the one or more targeted inhibitor comprises one or more of a small molecule drug, a tyrosine kinase inhibitor (TKI), an angiogenesis inhibitor, a monoclonal antibody, a proteosome inhibitor, and / or a signal transduction inhibitor.

[0130] In some embodiments, the CAR-T cell or CAR-NK cell comprises a chimeric antigen receptor (CAR) that is specific for ALPP or ALPG / ALPPL2.

[0131] In some embodiments, the CAR specific for ALPP comprises a sequence chosen from SEQ ID NOs:1-18.

[0132] In some embodiments, the therapy chimeric antigen receptor (CAR) targeting ALPP and / or ALPPL2, or targeting ALPG / ALPPL2, is administered in combination with the one or more targeted inhibitors and / or the one or more immunotherapy agents.

[0133] In some embodiments, the one or more targeted inhibitors comprises an EGFR inhibitor, a KRAS inhibitor, a RET inhibitor, an ALK inhibitor, a MET inhibitor, a MEK inhibitor, a HER2 inhibitor, and / or an FGFR inhibitor.

[0134] In some embodiments, the one or more targeted inhibitors comprises an EGFR inhibitor, a KRAS inhibitor, a RET inhibitor, an ALK inhibitor, a MET inhibitor, a MEKAttorney Docket No. MDA0083-401-PC inhibitor, a HER2 inhibitor, an AKT inhibitor, an ERK inhibitor, a CDK4 / 6 inhibitor, a BRAF inhibitor, and / or an FGFR inhibitor.

[0135] In some embodiments, the EGFR inhibitor is selected from osimertinib, mobocertinib, amivantamab, CLN081, and / or DZD9008.

[0136] In some embodiments, the KRAS inhibitor is selected from adagrasib, sotorasib, ARS1323, and / or AZD4785.

[0137] In some embodiments, the KRAS inhibitor is selected from adagrasib, sotorasib, ARS1323, MRTX849, MRTX1257, MRTX1133, and / or AZD4785.

[0138] In some embodiments, the RET inhibitor is selected from selpercatinib and / or pralsetinib.

[0139] In some embodiments, the ALK inhibitor is selected from crizotinib, alectinib, brigatinib, and / or lorlatinib.

[0140] In some embodiments, the HER2 inhibitor is selected from lapatinib, BI1810631 and / or trastuzumab-deruxtecan.

[0141] In some embodiments, the MET inhibitor is selected from tepotinib and capmatinib.

[0142] In some embodiments, the MEK inhibitor is selected from binimetinib (MEK162), Cotellic, Koselugo, Mekinist, Mektovi, cobimetinib or XL518, selumetinib, trametinib (GSK1120212), PD-325901, CI-1040, PD035901, and / or TAK-733.

[0143] In some embodiments, the FGFR inhibitor is selected from Balversa, anlotinib, erdafitinib, infigratinib, pemazyre, and / or pemigatinib.

[0144] In some embodiments, the AKT inhibitor is ipatasertib.

[0145] The method of claim 34, wherein the ERK inhibitor is selected from ulixertinib and SCH772984.

[0146] The method of claim 34, wherein the CDK4 / 6 inhibitor is palbociclib.

[0147] The method of claim 34, wherein the BRAF inhibitor is selected from dabrafenib, vemurafenib, and encorafenib.

[0148] In some embodiments, the one or more immunotherapy agents comprises nivolumab, ipilumumab, pembrolizumab, cemiplimab, durvalumab, tremilumumab, and / or atezolizumab.

[0149] In some embodiments, the chimeric antigen receptor (CAR) cell therapy targeting ALPP and / or ALPPL2, or targeting ALPG / ALPPL2, in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from the group consisting of an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, aAttorney Docket No. MDA0083-401-PC MEK mutation, and an FGFR mutation are administered to the patient in combination with a radionuclide.

[0150] In some embodiments, the chimeric antigen receptor (CAR) cell therapy targeting ALPP and / or ALPPL2, or targeting ALPG / ALPPL2, in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, an AKT mutation, an ERK mutation, a CDK4 / 6 mutation, a BRAF mutation, and an FGFR mutation are administered to the patient in combination with a radionuclide.

[0151] In some embodiments, the radionuclide comprises an alpha particle selected from Polonium-210, Bismuth-213, and Uranium-238.

[0152] In some embodiments, the radionuclide comprises a beta particle selected from Strontium-90, Thallium-201, Carbon-14, and Tritium.

[0153] In some embodiments, the radionuclide comprises a gamma particle selected from Barium-133, Cadmium-109, Cobalt-57, Cobalt-60, Europium-152, Manganese-54, Sodium- 22, Zinc-65, and Technetium-99m.

[0154] In some embodiments, the radionuclide comprises a combination of alpha, beta, and / or gamma particles, selected from Cesium-137 and Americum-241.

[0155] In some embodiments, the chimeric antigen receptor (CAR) therapy targeting ALPP and / or ALPPL2, or targeting ALPG / ALPPL2, in the NSCLC cell, and / or the therapy targeting the genetic mutation are administered simultaneously.

[0156] In some embodiments, the therapy targeting ALPP and / or ALPPL2, or targeting ALPG / ALPPL2, in the NSCLC cell, and / or the therapy targeting the genetic mutation are administered sequentially.

[0157] In some embodiments, the CAR-T cell comprises a sequence chosen from SEQ ID NOs:1, 10, 13, 15, 16, and / or 18.

[0158] In some embodiments, the non-small-cell lung cancer (NSCLC) is mesenchymal NSCLC.

[0159] In some embodiments, the bispecific T-cell engager comprises a sequence set forth in SEQ ID NOs:23-27.

[0160] Alkaline phosphatase, placental type (ALPP) is a membrane-bound glycosylated dimeric enzyme that was first detected in the serum during pregnancy and shown to originate from the placenta. There are four different isotypes of alkaline phosphatase: placental-type (ALPP), placental-type 2 (ALPPL2), also referred to as alkaline phosphatase, germ cell (ALPG) or ALPPL2, intestinal ALPP (ALPI), and tissue-nonspecific ALPP (ALPL). Of theseAttorney Docket No. MDA0083-401-PC four isotypes, ALPP and ALPG / ALPPL2 are found to be associated with a large number of human cancers, such as including, but not limited to, lung cancer, testicular seminoma, ovarian cancer, and endometrial cancer, among others. Other than placental trophoblasts, ALPP and ALPG / ALPPL2 expression on normal tissues is virtually absent, providing an excellent opportunity for developing therapies that require a high degree of tumor specificity. Antibodies directed against ALPP and / or ALPPL2 can be conjugated with other classes of drugs such as DNA crosslinking agents or radionuclides (e.g., alpha particles). Antibody-drug conjugates described herein and / or known in the art can be used to target ALPP and / or ALPG / ALPPL2 as described herein.

[0161] A number of characteristics make ALPP and / or ALPG / ALPPL2 an attractive candidate for antigen-targeting immunotherapy: (1) ALPP is a membrane-bound protein and therefore is an accessible cell surface target for specific binding molecules, such as antibodies. (2) Limited expression of ALPP and / or ALPG / ALPPL2 in healthy tissues, but increased expression in malignant tumors suggests that it might serve as a tumor-specific antigen with low off-tumor expression. (3) Alkaline phosphatase activity is reported to induce tumor progression in different cancers, such as prostate cancer, head and neck squamous cell carcinoma, and ovarian cancer. Thus, targeting or co-targeting ALPP and / or ALPG / ALPPL2 as a cancer therapy target may also enhance tumor control by reducing tumor-derived alkaline phosphatase activity.

[0162] As described herein, increased or elevated cell surface expression of ALPP and / or ALPG / ALPPL2 in many cancer types provides a novel opportunity for treatment of cancers exhibiting elevated expression of one or both of these proteins. For those cancers in which the expression of ALPP and / or ALPG / ALPPL2 is elevated, not only can these proteins themselves serve as a cancer therapy target, but additionally, the expression of ALPP and / or ALPG / ALPPL2 can occur in combination with a mutation in a gene, such as EGFR, KRAS, RET, ALK, HER2, MET, MEK, or FGFR. Increased or enhanced expression of ALPP and / or ALPG / ALPPL2 may also occur in combination with a gene implicated in initiating or maintaining cancer, e.g., an oncogenic driver mutation. Expression of ALPP and / or ALPPL2 can be evaluated by detecting and / or quantifying the cell surface expression levels of ALPP and / or ALPG / ALPPL2 using any methods known or available in the art. Baseline expression of ALPP and / or ALPG / ALPPL2 may provide useful information relating to disease severity and prognosis for treatment of the cancer in the individual. Evaluating baseline cell surface expression levels of ALPP and / or ALPPL2 can determine whether ALPP and / or ALPPL2 is present in the cancer cell in an increased amount, which can serve as a useful first step inAttorney Docket No. MDA0083-401-PC determining treatment for the particular cancer, for treating a cancer that has acquired resistance to a targeted therapy, or for treating a treatment-resistant or drug-resistant cancer cell, such as a drug-tolerant persister cell (DTPC). In some embodiments, increased cell surface expression of ALPP and / or ALPG / ALPPL2 increases the susceptibility of cancer cells to therapies directed against ALPP and / or ALPG / ALPPL2. As described herein, the methods of the present disclosure enable prevention of the emergence of resistance of a cancer cell, such as a NSCLC cell, to a drug therapy. In addition, the methods described herein allow for the use of ALPG / ALPPL2 quantification and / or targeting as a way to select a patient for whom treatment of cancer with a targeted inhibitor may be beneficial.

[0163] Thus, in some embodiments, a method described herein for treating a cancer expressing elevated levels of ALPP and / or ALPG / ALPPL2 may initially utilize a step wherein the baseline levels of ALPP and / or ALPG / ALPPL2 are determined before initiating treatment of the cancer. For cancers having elevated levels of ALPP and / or ALPG / ALPPL2, a targeted drug treatment may be administered to target the cell surface ALPP and / or ALPG / ALPPL2 protein. In addition to a drug treatment targeting the ALPP and / or ALPG / ALPPL2 protein as described herein, a standard-of-care inhibitor treatment or an anti- proliferative agent targeting ALPP and / or ALPG / ALPPL2 may be administered for treatment of the cancer.

[0164] Thus, detecting and / or quantifying ALPP and / or ALPG / ALPPL2 cell surface expression in the cancer cell may help determine an appropriate treatment plan for the individual having cancer. Methods of detecting and / or quantifying ALPP and / or ALPG / ALPPL2 cell surface expression levels are well-known in the art, and can include, but are not limited to, histological analysis, immunohistochemical (IHC) staining for ALPP protein, electron microscopy, mass spectrometry analysis, immunofluorescence, a blood- based test, a tissue-based test, or imaging techniques. Any method capable of determining ALPP and / or ALPG / ALPPL2 levels in a biological sample from an individual can be employed and are intended to be encompassed within the scope of the present disclosure.

[0165] A blood-based test may 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 for measuring circulating proteins, autoantibodies, cell-free circulating DNA, or extracellular vesicle-derived proteins. In some embodiments, a blood-based assay or test to detect expression of or levels of ALPP and / or ALPG / ALPPL2 may include the use of a labeled ligand or antibody.Attorney Docket No. MDA0083-401-PC

[0166] A tissue-based test described herein may be any tissue-based test known or available in the art, such as a tissue 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., staining) and can detect and quantify the amount of ALPP and / or ALPG / ALPPL2 protein present in a biological sample using, e.g., antibodies or any other specific method for determining cell surface expression of ALPP and / or ALPG / ALPPL2. For example, specific tissue-based tests known in the art include, but are not limited to, Mammaprint + Blueprint® test, a Signatara™ test, an Altera Tumor Genomic Profile Test, or an Oncotype DX® test. In some embodiments, the tissue-based test comprises a Mammaprint + Blueprint® test or an Oncotype DX® test.

[0167] A biological sample appropriate for the methods described herein can be any biological sample, for example, a blood sample, or a tissue biopsy, or a cell culture sample. Depending on the cancer type, certain biological samples may be more advantageous, e.g., a tissue biopsy for a solid cancer, or a blood-based sample for a hematological cancer, however any biological sample may be useful with the methods described herein.

[0168] In some embodiments, the tissue biopsy is analyzed by hematoxylin and eosin (H&E) staining and / or microscopy.

[0169] Development of resistance to a certain drug treatment can result in the need for alternate therapies or drug treatments for cancer. For example, as described herein, genetic mutation can occur in a cancer cell that results in the cancer cell no longer responding to a specific drug treatment, e.g., a mutation in a gene including, but not limited to, EGFR, KRAS, RET, ALK, HER2, MET, MEK, and / or FGFR. Such mutation(s) may result in a lack of expression of the protein or gene product, an aberrant or misfolded protein, or may result in a fusion of two or more gene products together.

[0170] As described herein, a genetic mutation in EGFR may include, but is not limited to, L858R, exon 19 deletion, T790M, C797S, exon 20 insertion mutations. A genetic mutation in KRAS may include, but is not limited to, a G12C or G12D mutations. A genetic mutation in RET may include, but is not limited to, a fusion of the RET gene with another gene to create a RET rearrangement or RET fusion. Non-limiting examples of a RET fusion or rearrangement include, but are not limited to, a point mutation, such as a CCDC6-RET fusion or a KIF5b-RET fusion. A genetic mutation in ALK may include, but is not limited to, a fusion of ALK with another protein, such as an EML4-ALK gene fusion. A genetic mutation in HER2 may include, but is not limited to, exon 20 YVMA insertion, exon 20 VC insertion, exon 20 GSP insertion. A genetic mutation in MET may include, but is not limitedAttorney Docket No. MDA0083-401-PC to, a MET amplification, wherein extra copies of the MET gene are present in the body, and an exon 4 skipping mutation, which prevents degradation of the MET protein and promotes cancer. A genetic mutation in MEK may include, but is not limited to, a mutation that involves the allosteric drug binding pocket or α-helix C, a mutation in a MEK1 codon located within or abutting the N-terminal negative regulatory helix (helix A), a MEK1(P124L) mutation, a MEK1(Q56P) mutation, or any mutation in the MEK gene that results in or confers resistance to a MEK inhibitor. A genetic mutation in FGFR may include, but is not limited to, an FGFR fusion, such as an FGFR2-ERC1 fusion, an FGFR3-TACC3 fusion, an FGFR2-INA fusion, a FGFR4-RAPGEFL1 fusion, and a fusion of the FGFR1 and SLC20A2 5’-untranslated regions, an FGFR amplification, such as an FGFR1 amplification, an FGFR2 amplification, an FGFR3 amplification, or an FGFR4 amplification, or an FGFR point mutation, such as an R248C, S249C, G370C, S371C, Y373C, G380R, W290C, S320C, or K660E / N point mutation.

[0171] Targeting of a genetic mutation as a cancer target antigen may be performed in accordance with the present disclosure either alone, or in combination with targeting of ALPP and / or ALPG / ALPPL2. In some embodiments, when referring to treatment of lung cancer by targeting a genetic mutation described herein in combination with targeting of ALPP and / or ALPG / ALPPL2, ALPP and / or ALPG / ALPPL2 may be targeted for treatment sequentially with a genetic mutation described herein, or may be simultaneously targeted in a treatment of the present disclosure. In some embodiments, a genetic mutation described herein, e.g., an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, and / or an FGFR mutation is directly targeted by a chimeric antigen receptor (CAR) cell therapy targeting ALPP and / or ALPG / ALPPL2 in a cancer cell, such as a NSCLC cell. A genetic mutation described herein may also be directly targeted by using other therapies that allow targeting of a genetic mutation, e.g., an inhibitor molecule or a chemotherapeutic treatment described herein or known in the art.

[0172] The specific genetic mutation to be targeted in a method of treating non-small-cell lung cancer (NSCLC) can be selected by a practitioner or clinician for individualized treatment for NSCLC. The present disclosure is not intended to be limited to the list of genetic mutations described herein, but rather to exemplify the methods of treatment for cancers expressing ALPP and / or ALPG / ALPPL2 and / or having a genetic mutation described herein. In some embodiments, the cancer expresses ALPG / ALPPL2 at increased levels compared to a non-cancer cell.Attorney Docket No. MDA0083-401-PC

[0173] The most common way to classify cancer is to divide it into 4 categories based on whether it can be removed with surgery and where it has spread: resectable, borderline resectable, locally advanced, or metastatic. Resectable cancer can be surgically removed. The cancerous tumor may be located only in a specific area or organ, or extends beyond it, but it has not grown into important arteries or veins in the area. There is no evidence that the tumor has spread to areas outside of the area. Borderline resectable describes a tumor that may be difficult, or not possible, to remove surgically when it is first diagnosed, but if chemotherapy and / or radiation therapy is able to shrink the tumor first, it may be able to be removed later with negative margins. A negative margin means that no visible cancer cells are left behind. Locally advanced cancer is still located only in the immediate surrounding area around the tumor, but it cannot be surgically removed because it has grown into nearby arteries or veins or to nearby organs. However, there are no signs that it has spread to any distant parts of the body. Metastatic means the cancer has spread beyond the area of the tumor and to other organs, such as the liver or distant areas of the abdomen.

[0174] Options for treatment of NSCLC are established in the literature and can include surgery for partial or complete surgical removal of tumors or cancerous tissue, or can involve administering one or more cancer therapies described herein (e.g., CAR-T cell therapies, antibody-drug conjugates, chemotherapeutic drugs, immunotherapy drugs, therapeutic radiation, etc.).

[0175] Chemotherapeutic drugs approved for treatment of cancer are numerous and well known in the art. A number of these are described herein, however it is noted that the present disclosure is not limited to the cancer types or drugs described herein. One of skill in the art would understand that the present disclosure can be extended to any appropriate cancer type and any chemotherapeutic drugs or treatments. For example, any cancer type that expresses ALPP and / or ALPG / ALPPL2, or expresses increased cell surface levels of ALPP and / or ALPG / ALPPL2 is within the scope of the present methods. Likewise, any cancer type that has a genetic mutation in a specific gene capable of being targeted by a CAR-T cell therapy or an antibody-drug conjugate, e.g., an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, MET mutation, a MEK mutation, and / or an FGFR mutation is within the scope of the present disclosure. A cancer cell in accordance with the present disclosure may be a lung cancer cell, such as a non-small-cell lung cancer cell. A cancer cell may be a specific type of non-small-cell lung cancer, e.g., mesenchymal non- small-cell lung cancer.Attorney Docket No. MDA0083-401-PC

[0176] For example, techniques useful for determination of cell surface expression of ALPP and / or ALPG / ALPPL2, such as detecting and / or quantifying ALPP and / or ALPG / ALPPL2 cell surface expression, can include staining techniques, immunohistochemistry, cell viability assays, microscopy, phosphatase assays, flow cytometry, cell surface biotinylation, proton or chymotrypsin sensitivity assay, antibody labeling assays, ELISA, radioligand binding, cAMP enzyme immunoassays, bioluminescence resonance energy transfer (BRET), CREB phosphorylation assay, live cell staining, imaging, or any others known or available in the art.

[0177] Staining techniques are known in the art and can involve any stain appropriate for measuring protein, e.g., a fluorescein dye, 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 any other cell staining techniques or dyes known or available in the art.

[0178] In some embodiments, any NSCLC tumor or cancerous tissue that exhibits expression of ALPP and / or ALPG / ALPPL2, or exhibits increased expression of ALPP and / or ALPG / ALPPL2, may be treated according to the methods described herein. In some embodiments, ALPP and / or ALPG / ALPPL2 expression may be increased to a statistically significant degree or level, or may be increased to varying magnitudes, depending on the cancer type.

[0179] As would be understood by one of skill in the art, the present treatment methods may be appropriate for any cancer type, such as including, but not limited to, ovarian, breast, endometrial, pancreatic, gastric, colorectal, lung, urothelial, brain, testicular, seminoma, and mesothelioma. In some embodiments, the lung cancer is non-small cell lung cancer.

[0180] Provided herein are methods of treating a cancer that expresses ALPP and / or ALPG / ALPPL2, typically at elevated levels. Also provided herein is a method of treating a cancer that co-expresses one or more placental alkaline phosphatase (ALPP) proteins and possesses a genetic mutation selected from an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a MET mutation, a MEK mutation, a HER2 mutation, and an FGFR mutation. Such methods include administering to a patient a treatment regimen, wherein the treatment regimen comprises administration of a CAR-T cell therapy or CAR NK cell therapy or an antibody-drug conjugate to target ALPP and / or ALPG / ALPPL2 on the cell surface. In some embodiments, prior to administration of the treatment regimen, theAttorney Docket No. MDA0083-401-PC method further comprises assaying a biological sample obtained from the patient for cell surface expression levels of one or more ALPP proteins. It may also be useful to simultaneously target or sequentially target ALPP and / or ALPG / ALPPL2, along with a genetic mutation selected from an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, and an FGFR mutation. In some embodiments, the method comprises administration of a treatment regimen, e.g., a CAR-T cell therapy and / or an antibody-drug conjugate, or alternate therapies known in the art for targeting a genetic mutation.

[0181] As described herein, treatment methods for non-small-cell lung cancer may be selected from a CAR-T cell or CAR-NK therapy, or an antibody drug conjugate (ADC) useful for targeting ALPP and / or ALPG / ALPPL2, and a therapy targeting a genetic mutation, such as an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, and / or an FGFR mutation. Some embodiments of the present invention provide for use of a chemotherapeutic agent in addition to the CAR- T or CAR-NK cell therapy, or the antibody-drug conjugate, targeting ALPP and / or ALPG / ALPPL2, and the therapy targeting a genetic mutation, e.g., an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, and / or an FGFR mutation.

[0182] Chemotherapy is widely used as a standard-of-care treatment for cancers and acts to destroy cancer cells and keep them from growing, dividing, and producing more cancer cells. Some chemotherapeutic drugs act by damaging DNA and preventing cell replication, resulting in the death of the cancer cells. As cancer cells typically grow and divide faster than normal, non-cancerous cells, these chemotherapeutic drugs have more of an effect on those actively dividing cancer cells.

[0183] As described herein, a treatment regimen that may be used to increase cell-surface expression of ALPP and / or ALPG / ALPPL2 may be any standard-of-care inhibitor or anti- proliferative agent, and includes any chemotherapeutic drug known in the art for treatment of cancer, such as including, 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 Disperz (Everolimus), Alkeran (Melphan), Alpelisib, Altretamine (Hexalen®), Anastrozole, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane), Atezolizumab, Avastin (Bevacizumab), Bevacizumab (Avastin), Capecitabine (Xeloda®),Attorney Docket No. MDA0083-401-PC Carboplatin, Cisplatin, Cyclophosphamide (Cytoxan®), Docetaxel (Taxotere®), Doxorubicin Hydrochloride, Doxil (Doxorubicin Hydrochloride Liposome), Ellence (Epirubicin Hydrochloride), Enhertu® (Fam-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), Margenza (Margetuximab-cmkb), Margetuximab-cmkb, Megestrol Acetate, Melphalan, Methotrexate Sodium, mitomycin, Neratinib Maleate, Nerlynx (Neratinib Maleate), Niraparib Tosylate Monohydrate, Olaparib, oxorubicin (Adriamycin®), Oxaliplatin, Paclitaxel (Taxol®), Paclitaxel Albumin-stabilized Nanoparticle Formulation, Palbociclib, Pamidronate Disodium, Pembrolizumab, 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), Talazoparib Tosylate, Talzenna (Talazoparib 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), Venclexta (venetoclax), Verzenio (Abemaciclib), Vinblastine Sulfate, Xeloda (Capecitabine), Vinorelbine (Navelbine®), Zejula (Niraparib Tosylate Monohydrate), Zoladex (Goserelin Acetate). As would be understood by one of skill in the art, certain cancers benefit from certain chemotherapeutic drugs, or combinations thereof, and therefore the individual treatment plan for a specific cancer may be altered as deemed appropriate by a clinician or practitioner.

[0184] In some embodiments, a CAR-T cell therapy or CAR NK cell therapy targeting ALPP and / or ALPG / ALPPL2, as well as a therapy targeting a genetic mutation in EGFR, KRAS, RET, ALK, HER2, MET, MEK, or FGFR may also be combined with one or moreAttorney Docket No. MDA0083-401-PC standard-of-care inhibitors or anti-proliferative agents, or in combination with one or more targeted inhibitors and / or the one or more immunotherapy agents. A “targeted inhibitor” as described herein may be an EGFR inhibitor, a KRAS inhibitor, a RET Inhibitor, an ALK inhibitor, a HER2 inhibitor, a MET inhibitor, a MEK inhibitor, and / or an FGFR inhibitor. EGFR inhibitors are known in the art, and include, but are not limited to, osimertinib, mobocertinib, amivantamab, CLN081, and / or DZD9008. KRAS inhibitors are known in the art and can include, but are not limited to, adagrasib, sotorasib, ARS1323, and / or AZD4785. RET inhibitors are known in the art and include, but are not limited to, selpercatinib and / or pralsetinib. ALK inhibitors are known in the art and can include, but are not limited to, crizotinib, alectinib, brigatinib, and / or lorlatinib. HER2 inhibitors are known in the art and include, but are not limited to, lapatinib, BI1810631, and / or trastuzumab-deruxtecan. MET inhibitors are known in the art and include, but are not limited to, tepotinib and capmatinib. MEK inhibitors are known in the art and include, but are not limited to, binimetinib (MEK162), Cotellic, Koselugo, Mekinist, Mektovi, cobimetinib or XL518, selumetinib, trametinib (GSK1120212), PD-325901, CI-1040, PD035901, and / or TAK- 733.FGFR inhibitors are known in the art and include, but are not limited to, Balversa, anlotinib, erdafitinib, infigratinib, pemazyre, and / or pemigatinib.

[0185] In some embodiments, an immunotherapy agent as described herein may include, but is not limited to, nivolumab, ipilumumab, pembrolizumab, cemiplimab, durvalumab, tremilumumab, and / or atezolizumab.

[0186] In some embodiments, one or more standard-of-care inhibitors or anti- proliferative agents that increase cell surface expression of ALPP and / or ALPG / ALPPL2 are combined together in a combination treatment. Combination treatments may include any combination of treatments for cancer as deemed appropriate by a clinician or physician. For example, cancer treatment options useful for the present methods include one or more of chemotherapeutic drugs, radiation therapies, immunotherapy drugs, DNA cross-linking agents, antibody-drug conjugates, hormone therapies, targeted drug therapies, radionuclides, cryoablation therapies, surgical procedures, or the like.

[0187] In some embodiments, the one or more standard-of-care inhibitor or anti- proliferative agents are chosen from Trastuzumab (Herceptin®), Cisplatin, Etoposide (VP- 16), Bleomycin, Ifosfamide (Ifex®), Paclitaxel (Taxol®), Carboplatin, Vinblastine, oxorubicin (Adriamycin®), liposomal Doxorubicin (Doxil®), Docetaxel (Taxotere®), Albumin bound Paclitaxel (nab-Paclitaxel, Abraxane®), Altretamine (Hexalen®), Capecitabine (Xeloda®), Cyclophosphamide (Cytoxan®), Gemcitabine (Gemzar®),Attorney Docket No. MDA0083-401-PC 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.

[0188] Combination treatments known in the art include, but are not limited to, one or more standard-of-care inhibitor or anti-proliferative agents comprises 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 and Paclitaxel and Doxorubicin; a combination of Ifosfamide (Ifex®) and Cisplatin; a combination of Ifosfamide (Ifex®) and Paclitaxel; a combination of Trifluridine and Tipiracil (Lonsurf); a combination of Oxaliplatin and 5-FU / leucovorin (FOLFOX); a combination of Oxaliplatin and capecitabine (CAPOX); a combination of 5-FU / leucovorin, Oxaliplatin, and Docetaxel; a combination of Docetaxel or Paclitaxel and either 5-FU or capecitabine; a combination of Cisplatin and either 5-FU or capecitabine; or a combination of Paclitaxel and Carboplatin.

[0189] In some embodiments, an antibody-drug conjugate may be used to treat a cancer that overexpresses ALPG / ALPPL2 as described herein. In some embodiments, the antibody- drug conjugate specifically targets ALPG / ALPPL2. Antibody-drug conjugates consist of three main components: an antibody drug specific for a particular cancer protein, a cytotoxic chemotherapy drug, and a linker protein connecting the two. In some embodiments, an antibody-drug conjugate useful in accordance with the present disclosure comprises an antibody targeting ALPG / ALPPL2 conjugated to a chemotherapeutic drug. Any chemotherapeutic drug disclosed herein may be useful for use in an antibody-drug conjugate. Typically administered intravenously, the antibody portion (e.g., an M25 antibody or humanized H17E2 antibody described herein) targets the specific cancer protein and is taken up by the cancer cell, where the cytotoxic cancer drug is released and kills the cancer cell. A number of antibody-drug conjugates are known in the art. One example is SGN-ALPV, a novel investigational vedotin antibody–drug conjugate composed of a humanized anti- ALPP / ALPPL2 monoclonal antibody, a protease-cleavable linker, and the microtubule disrupting agent monomethylauristatin E (MMAE). In some embodiments, an ADC useful as described herein may be an M25 antibody, e.g., provided in Tables 7 (M25 heavy chain) and 8 (M25 light chain), conjugated to MMAE, MMAF, DM1, Duocarmycin DM (DMDM), Deruxtecan (DXd), PNU159682, Amanitin, and PBD. SGN-ALPV is undergoing phase I clinical trials in patients with solid tumors, such as ovarian neoplasms, endometrialAttorney Docket No. MDA0083-401-PC neoplasms, non-small cell lung carcinoma, uterine cervical neoplasms, and testicular neoplasms. Other useful antibody-drug conjugates include Tivdak®, which is used to treat cervical cancer, Brentuximab vedotin (Adcetris®), used for treatment of relapsed Hodgkin and systemic anaplastic large cell lymphomas, 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, Inotuzumab ozogamicin (Besponsa®), which targets the CD22 receptor and is approved for relapsed B-cell precursor acute lymphoblastic leukemia, Polatuzumab vedotin-piiq (Polivy®), which targets the CD79b receptor and is approved in combination with certain chemotherapy regimens 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 cancer that expresses 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®.

[0190] In some embodiments, the antibody-drug conjugate comprises SGN-ALPV, Adcetris®, Kadcyla®, Besponsa®, Mylotarg®, Polivy®, Padcev®, Enhertu®, Trodelvy®, Blenrep®, Zynlonta™, Akalux®, Aidixi®, and Tivdak®. One of skill in the art would understand that any appropriate antibody-drug conjugate would be 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.

[0191] In some embodiments, certain drugs described herein and known in the art, or combinations of drugs or compounds, may be used to treat specific types of cancers that express or overexpress ALPP and / or ALPG / ALPPL2, either alone, or in combination with a genetic mutation in one or more of EGFR, KRAS, RET, ALK, HER2, MET, MEK, and / or FGFR.

[0192] Radionuclide therapy uses radioactive substances called radiopharmaceuticals to treat cancer. Radionuclides are introduced into the body by various means and localize to specific locations, organs, or tissues depending on their properties and administration routes. Radionuclides can be provided in a variety of particle types, including alpha, beta, gamma, or combinations of these.

[0193] In some embodiments, a 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.Attorney Docket No. MDA0083-401-PC

[0194] In some embodiments, a radionuclide is a beta particle, such as Strontium-90, Thallium-201, Carbon-14, and Tritium. In some embodiments, a 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, a radionuclide may be a combination of these particles, such as Cesium-137 and Americum- 241. Any radionuclides known or available in the art may be used in accordance with the methods described herein.

[0195] In some embodiments, combinations of radionuclides can be used, wherein each radionuclide in a combination radionuclide emits radiation at a different wavelength such that each individual radionuclide is separately distinguishable.

[0196] In some embodiments, a drug treatment described herein may be administered in a clinical setting or may be administered in an alternate setting as deemed appropriate by a clinician or practitioner. In some embodiments, the CAR-T cell therapy or CAR-NK cell therapy or antibody-drug conjugate targeting ALPP and / or ALPPL2, along with the therapy targeting a genetic mutation, e.g., an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, and / or an FGFR mutation are administered simultaneously or sequentially.

[0197] Any of the treatments described herein for treatment of a cancer that expresses ALPP and / or ALPG / ALPPL2, either alone or in combination with a genetic mutation, e.g., an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, and an FGFR mutation can be administered to a patient in need thereof (e.g., a patient that has cancer), or may be combined with (i.e., by co- administration or sequential administration) a further therapeutic treatments or drugs. In one embodiment, the further therapeutic treatments or drugs are included in a pharmaceutical composition as described herein. In other embodiments, the additional therapeutic treatments or drugs are co-administered, administered concurrently, or administered sequentially in separate or distinct compositions.

[0198] As would be understood by one of skill in the art, a treatment described herein is administered in any form necessary or useful to the subject for treatment of cancer, for example, a liquid (e.g., injectable and infusible solutions), a semi-solid, a solid, an aqueous solution, a suspension, an emulsion, a gel, a magma, a mixture, a tincture, a powder, a capsule, a dispersion, a tablet, a pellet, a pill, a powder, a liposome, a lozenge, a troche, a liniment, an ointment, a lotion, a paste, a suppository, a spray, an inhalant, or the like. In some embodiments, a drug as described herein for treatment of cancer may be administeredAttorney Docket No. MDA0083-401-PC in a liquid or aqueous form for injection into a patient, or in a pill or tablet form for oral administration. The dosage form of a drug described herein can depend on the intended mode of administration and therapeutic application. Typically, dosage forms for the drug treatments described herein are in the form of injectable or infusible solutions, or in the form of a pill for oral administration.

[0199] In some embodiments, a drug as described herein for treatment of cancer in a patient may be administered by any route or mode of administration, such as intraperitoneal, intravenous, oral, sublingual, rectal, vaginal, ocular, otic, nasal, cutaneous, enteral, epidural, intra-arterial, intravascular, nasal, respiratory, subcutaneous, topical, transdermal, intramuscular, or the like. In other embodiments, a second drug regimen, such as a chemotherapeutic drug regimen, may be in the form of an aqueous solution for intravenous administration.

[0200] Unless otherwise specified herein, the methods described herein can be performed in accordance with the procedures exemplified herein or routinely practiced methods well known in the art. The following sections provide additional guidance for practicing the methods of the present disclosure.

[0201] In some embodiments, a cancer therapy to be administered to an individual for treatment of lung cancer may be provided as a composition. In some embodiments, the methods described herein may involve compositions to be administered as a single composition. In some embodiments, each drug may be administered separately (while still being administered concurrently), i.e., in separate solutions or drug forms as described herein. Pharmaceutical formulation is well established and known in the art.

[0202] In some embodiments, a drug or composition for use in the methods described herein may be formulated with excipient materials, such as sodium citrate, sodium dibasic phosphate heptahydrate, sodium monobasic phosphate, Tween-80, and / or a stabilizer. The drug or composition for use in the methods described herein can be provided, for example, in a buffered solution at a suitable concentration and can be stored at an appropriate temperature to maintain the efficacy of the drug(s), for example a temperature of 2-8°C. In some other embodiments, the pH of the composition is between about 5.5 and 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).

[0203] A pharmaceutical composition for the described methods can also include agents that reduce aggregation of the drug when formulated. Examples of aggregation reducing agents include one or more amino acids selected from methionine, arginine, lysine, aspartic acid, glycine, and glutamic acid. The pharmaceutical compositions can also include a sugarAttorney Docket No. MDA0083-401-PC (e.g., sucrose, trehalose, mannitol, sorbitol, or xylitol) and / or a tonicity modifier (e.g., sodium chloride, mannitol, or sorbitol) and / or a surfactant (e.g., polysorbate-20 or polysorbate-80).

[0204] As described above for a cancer treatment of the present disclosure, compositions for use with the described methods can be administered by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). The phrases “parenteral administration” and “administered parenterally” as used herein mean modes of administration, usually by injection, and include, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection, and infusion.

[0205] A composition for use with the described methods can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration. Sterile injectable solutions can be prepared by incorporating an agent described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating an agent described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze drying that yield a powder of an agent described herein plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0206] In certain embodiments, compositions may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York (1978).

[0207] In some embodiments, a composition is formulated in sterile distilled water or phosphate buffered saline. The pH of the pharmaceutical formulation may be between aboutAttorney Docket No. MDA0083-401-PC 5.5 and 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).

[0208] A cancer treatment for treating a cancer that expresses ALPP and / or ALPG / ALPPL2 or overexpresses ALPP and / or ALPG / ALPPL2, either alone or in combination with a genetic mutation, e.g., an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, and an FGFR mutation, can be provided in a kit. In one embodiment, the kit includes (a) a container that contains the individual cancer therapies as described herein, and optionally (b) informational material. The informational material can be descriptive, instructional, marketing, or other material that relates to the methods described herein and / or the use of the agents for therapeutic benefit.

[0209] In some embodiments, the kit also includes a further agent (e.g., a chemotherapeutic or immunotherapy drug described herein) for treating cancer described herein. For example, the kit includes a first container that contains a CAR-T cell therapy, or an antibody-drug conjugate therapy, and a second container that contains a therapy targeting a genetic mutation, e.g., an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, and an FGFR mutation. In some embodiments, a further container may be included to contain a standard-of-care inhibitor or anti-proliferative agent, and a chemotherapeutic or immunotherapy drug or combination of drugs.

[0210] The informational material of the kits is not limited in its form. In one embodiment, the informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to methods of administering the cancer treatment, as well as the chemotherapeutic or immunotherapy drug, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein), to treat a subject who has cancer. The information can be provided in a variety of formats, including printed text, computer readable material, video recording, or audio recording, or information that provides a link or address to substantive material, e.g., on the internet.

[0211] In addition to the cancer treatments or agents, the kit can include materials or reagents necessary for determining the cell surface expression of ALPP and / or ALPG / ALPPL2, along with other ingredients, such as a solvent or buffer, a stabilizer, or a preservative. In some embodiments, the kit can further include materials or reagentsAttorney Docket No. MDA0083-401-PC necessary for identifying a genetic mutation in the patient, e.g., The cancer treatments can be provided in any form described herein, e.g., liquid, dried or lyophilized form, substantially pure and / or sterile. In some embodiments, when the agents are provided in a liquid solution, the liquid solution is an aqueous solution. When the agents are provided as a lyophilized product, the lyophilized powder is generally reconstituted by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer (e.g., PBS), can optionally be provided in the kit.

[0212] The kit can 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 informational material. For example, the cancer treatments can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the cancer treatments or agents may be contained in a bottle, vial, or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of the agents. The containers can include a combination unit dosage, e.g., a unit that includes both the cancer treatment(s) in a desired ratio. For example, the kit may include a plurality of syringes, ampules, foil packets, blister packs, or medical devices, e.g., each containing a single combination unit dose. The containers of the kits can be air-tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or light-tight.

[0213] The kit optionally includes a device suitable for administration of the cancer treatments, e.g., a syringe or other suitable delivery device. The device can be provided pre-loaded with one or both of the agents or can be empty, but suitable for loading.

[0214] Chimeric Antigen Receptor (CAR) Cell Therapy

[0215] Also provided herein are chimeric antigen receptors (CARs; and transgenic T-cell receptors, TCRs) comprising polypeptides as disclosed herein, e.g. as disclosed in Tables 1-6 and SEQ ID NOs:1-18, and immune effector cells expressing them. A CAR-T cell useful in accordance with the present disclosure may comprise a sequence chosen from SEQ ID NOs:1, 10, 13, 15, 16, and / or 18. An immune effector cell useful in accordance with the present disclosure may be a T cell or a natural killer (NK) cell. that is specific for ALPP and / or ALPG / ALPPL2. A CAR is a recombinant fusion protein comprising: 1) an extracellular ligand-binding domain, i.e., an antigen-recognition domain, 2) a hinge domain,Attorney Docket No. MDA0083-401-PC 3) a transmembrane domain, and 4) a cytoplasmic signaling domain, 5) and optionally, a co- stimulatory domain.

[0216] Methods for CAR design, delivery and expression, and the manufacturing of clinical-grade CAR-T cell populations are known in the art. CAR designs are generally tailored to each cell type.

[0217] The extracellular ligand-binding domain of a chimeric antigen receptor recognizes and specifically binds an antigen, typically a surface-expressed antigen of a malignant cell, such as ALPP and / or ALPG / ALPPL2. The extracellular ligand-binding domain specifically binds an antigen when, for example, it binds the antigen with an affinity constant or affinity of interaction (KD) between about 0.1 pM to about 10 μM, or about 0.1 pM to about 1 μM, or about 0.1 pM to about 100 nM. Methods for determining the affinity of interaction are known in the art. An extracellular ligand-binding domain can also be said to specifically bind a first polymorphic variant of an antigen when it binds it selectively over a second polymorphic variant of the same antigen.

[0218] An extracellular ligand-binding domain suitable for use in a CAR may be any antigen-binding polypeptide, a wide variety of which are known in the art. In some instances, the extracellular ligand-binding domain is a single chain Fv (scFv). Other antibody-based recognition domains (cAb VHH (camelid antibody variable domains) and humanized versions thereof, lgNAR VH (shark antibody variable domains) and humanized versions thereof, sdAb VH (single domain antibody variable domains) and “camelized” antibody variable domains are suitable for use. In some instances, T-cell receptor (TCR) based recognition domains such as single chain TCR (scTv, single-chain two-domain TCR containing VαVβ) are also suitable for use. In some embodiments, the extracellular ligand- binding domain is constructed from a natural binding partner, or a functional fragment thereof, to a target antigen.

[0219] The targeted antigen to which the CAR binds via its extracellular ligand-binding domain may be an antigen that is expressed on a lung cancer cell as described herein, T cell or, other cell. As used herein, “antigen” refers to the target protein or fragment thereof that binds to a T cell receptor. An antigen expressed on a malignant lung cancer cell can include ALPP and / or ALPG / ALPPL2.

[0220] Typically, the extracellular ligand-binding domain is linked to the intracellular domain of the chimeric antigen receptor by a transmembrane (TM) domain. A peptide hinge connects the extracellular ligand-binding domain to the transmembrane domain. A transmembrane domain traverses the cell membrane, anchors the CAR to the T cell surface,Attorney Docket No. MDA0083-401-PC and connects the extracellular ligand binding to the cytoplasmic signaling domain, thus impacting expression of the CAR on the T cell surface.

[0221] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. For example, the transmembrane region may be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, and PAG / Cbp. Alternatively, the transmembrane domain can be synthetic and comprise predominantly hydrophobic amino acid residues (e.g., leucine and valine). In some cases, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the transmembrane domain is derived from the T-cell surface glycoprotein CD8 alpha chain isoform 1 precursor (NP_001139345.1) or CD28. A short oligo- or polypeptide linker, such as between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the endoplasmic domain of the CAR. In some embodiments, the CAR has more than one transmembrane domain, which can be a repeat of the same transmembrane domain, or can be different transmembrane domains.

[0222] NK cells express a number of transmembrane (TM) adapters that signal activation, that are triggered via association with activating receptors. This provides an NK cell specific signal enhancement via engineering the TM domains from activating receptors, and thereby harness endogenous adapters. The TM adapter can be any endogenous TM adapter capable of signaling activation. In some embodiments, the TM adapter may be chosen from FceR1γ (ITAMx1), CD3ζ (ITAMx3), DAP12 (ITAMx1), or DAP10 (YxxM / YINM), NKG2D, FcγRIIIa, NKp44, NKp30, NKp46, actKIR, NKG2C, CD8α, and IL15Rb.

[0223] The CAR can further comprise a hinge region between extracellular ligand- binding domain and said transmembrane domain. The term “hinge region” (equivalently,Attorney Docket No. MDA0083-401-PC “hinge” or “spacer”) generally means any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. In particular, the hinge region is used to provide more flexibility and accessibility for the extracellular ligand-binding domain, and can confer stability for efficient CAR expression and activity. A hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. Hinge region may be derived from all or parts of naturally-occurring molecules such as CD28, 4-1BB (CD137), OX-40 (CD134), CD3ζ, the T cell receptor α or β chain, CD45, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, ICOS, CD154 or from all or parts of an antibody constant region. In some embodiments, for example, the hinge sequence is derived from a CD8a molecule or a CD28 molecule. Alternatively, the hinge region may be a synthetic sequence that corresponds to a naturally-occurring hinge sequence or the hinge region may be an entirely synthetic hinge sequence.

[0224] After antigen recognition, the cytoplasmic signaling domain transmits a signal to the immune effector cell, activating at least one of the normal effector functions of the immune effector cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. While usually the entire cytoplasmic signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the cytoplasmic signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function.

[0225] Cytoplasmic signaling sequences that regulate primary activation of the TCR complex that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of ITAM containing cytoplasmic signaling sequences include those derived from CD8, CD3ζ, CD3δ, CD3γ, CD3ε, CD32 (Fc gamma RIIa), DAP10, DAP12, CD79a, CD79b, FcγRIγ, FcγRIIIγ, FcεRIβ (FCERIB), and FcεRIγ (FCERIG).

[0226] First-generation CARs typically have the cytoplasmic signaling domain from the CD3 chain, which is the primary transmitter of signals from endogenous TCRs. Second- generation CARs add cytoplasmic signaling domains from various co-stimulatory protein receptors (e.g., CD28, 4-1BB, ICOS) to the cytoplasmic signaling domain of the CAR to provide additional signals to the T cell.Attorney Docket No. MDA0083-401-PC

[0227] A “costimulatory domain” is derived from the intracellular signaling domains of costimulatory proteins that enhance cytokine production, proliferation, cytotoxicity, and / or persistence in vivo. Preclinical studies have indicated that the second generation of CAR designs improves the antitumor activity of T cells. More recent, third-generation, and later generation, CARs combine multiple costimulatory domains to further augment potency. T cells grafted with these CARs have demonstrated improved expansion, activation, persistence, and tumor-eradicating efficiency independent of costimulatory receptor / ligand interaction.

[0228] For example, the cytoplasmic signaling domain of the CAR can be designed to comprise the signaling domain (e.g., CD3ζ) by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR. For example, the cytoplasmic domain of the CAR can comprise a signaling domain (e.g., CD3ζ) chain portion and a costimulatory signaling region. The co-stimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a co-stimulatory molecule. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D.

[0229] In some embodiments, the cytoplasmic signaling domain is a CD3 zeta (CD3ζ) signaling domain. In some embodiments, the co-stimulatory domain comprises the cytoplasmic domain of CD28, 4-1BB, or a combination thereof. In some cases, the co- stimulatory signaling region contains 1, 2, 3, or 4 cytoplasmic domains of one or more intracellular signaling and / or co-stimulatory molecules.

[0230] The co-stimulatory signaling domain(s) may contain one or more mutations in the cytoplasmic domains of CD28 and / or 4-1BB that enhance signaling.

[0231] In some embodiments, the disclosed CARs comprise one or more 4-1BB domains with mutations that enhance binding to specific TRAF proteins, such as TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, or any combination thereof. In some cases, the 41BB mutation enhances TRAF1- and / or TRAF2-dependent proliferation and survival of the T-cell, e.g. through NF-kB. In some cases, the 4-1BB mutation enhances TRAF3-dependent antitumor efficacy, e.g. through IRF7 / INFβ. Therefore, the disclosed CARs can comprise cytoplasmic domain(s) of 4-1BB having at least one mutation in these underligned sequences that enhance TRAF-binding and / or enhance NFκB signaling.-

[0232] Also as disclosed herein, TRAF proteins can in some cases enhance CAR T cell function independent of NFκB and 4-1BB. For example, TRAF proteins can in some casesAttorney Docket No. MDA0083-401-PC enhance CD28 co-stimuation in T cells. Therefore, also disclosed herein are immune effector cells co-expressing CARs with one or more TRAF proteins, such as TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, or any combination thereof. In some cases, the CAR is any CAR that targets a tumor antigen. For example, first-generation CARs typically had the intracellular domain from the CD3 chain, while second-generation CARs added intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 4-1BB, ICOS) to the cytoplasmic signaling domain of the CAR to provide additional signals to the T cell, while third-generation CARs contain two costimulatory domains. In some cases, the CAR is the disclosed CAR with enhanced 4-1BB activation.

[0233] Variations on CAR components may be advantageous, depending upon the type of cell in which the CAR is expressed.

[0234] For example, in NK cells, in some embodiments, the transmembrane domain can be a sequence associated with NKG2D, FcγRIIIa, NKp44, NKp30, NKp46, actKIR, NKG2C, or CD8α. In certain embodiments, the NK cell is a ML-NK or CIML-NK cell and the TM domain is CD8α (also referred to as CD8a). Certain TM domains that do not work well in NK cells generally may work in a subset; CD8α, for example, works in ML-NKs but not NK cells generally.

[0235] Similarly, in NK cells, in some embodiments, the intracellular signaling domain(s) can be any co-activating receptor(s) capable of functioning in an NK cell, such as, for example, CD28, CD137 / 41BB (TRAF, NFkB), CD134 / OX40, CD278 / ICOS, DNAM-1 (Y- motif), NKp80 (Y-motif), 2B4 (SLAMF) :: ITSM, CRACC (CS1 / SLAMF7) :: ITSM, CD2 (Y-motifs, MAPK / Erk), CD27 (TRAF, NFkB), or integrins (e.g., multiple integrins).

[0236] Similarly, in NK cells, in some embodiments, an intracellular signaling domain can be a cytokine receptor capable of functioning in an NK cell. For example, a cytokine receptor can be a cytokine receptor associated with persistence, survival, or metabolism, such as IL-2 / 15Rbyc :: Jak1 / 3, STAT3 / 5, PI3K / mTOR, MAPK / ERK. As another example, a cytokine receptor can be a cytokine receptor associated with activation, such as IL-18R :: NFkB. As another example, a cytokine receptor can be a cytokine receptor associated with IFN-γ production, such as IL-12R :: STAT4. As another example, a cytokine receptor can be a cytokine receptor associated with cytotoxicity or persistence, such as IL-21R :: Jak3 / Tyk2, or STAT3. As another example, an intracellular signaling domain can be a TM adapter, such as FceR1γ (ITAMx1), CD3ζ (ITAMx3), DAP12 (ITAMx1), or DAP10 (YxxM / YINM). As another example, CAR intracellular signaling domains (also known as endodomains) can beAttorney Docket No. MDA0083-401-PC derived from costimulatory molecules from the CD28 family (such as CD28 and ICOS) or the tumor necrosis factor receptor (TNFR) family of genes (such as 4-1BB, OX40, or CD27). The TNFR family members signal through recruitment of TRAF proteins and are associated with cellular activation, differentiation and survival. Certain signaling domains that may not work well in all NK cells generally may work in a subset; CD28 or 4-1BB, for example, work in ML-NKs.

[0237] Methods of Making CARs and CAR-Bearing Cells

[0238] The chimeric antigen receptor (CAR) construct, which encodes the chimeric receptor can be prepared in conventional ways. Since, for the most part, natural sequences are employed, the natural genes are isolated and manipulated, as appropriate (e.g., when employing a Type II receptor, the immune signaling receptor component may have to be inverted), so as to allow for the proper joining of the various components. Thus, the nucleic acid sequences encoding for the N-terminal and C-terminal proteins of the chimeric receptor can be isolated by employing the polymerase chain reaction (PCR), using appropriate primers which result in deletion of the undesired portions of the gene. Alternatively, restriction digests of cloned genes can be used to generate the chimeric construct. In either case, the sequences can be selected to provide for restriction sites that are blunt-ended, or those having complementary overlaps.

[0239] The various manipulations for preparing the chimeric construct can be carried out in vitro and in particular embodiments the chimeric construct is introduced into vectors for cloning and expression in an appropriate host using standard transformation or transfection methods. Thus, after each manipulation, the resulting construct from joining of the DNA sequences is cloned, the vector isolated, and the sequence screened to ensure that the sequence encodes the desired chimeric receptor. The sequence can be screened by restriction analysis, sequencing, or the like.

[0240] A chimeric construct can be introduced into immune effector cells as naked DNA or in a suitable vector. Methods of stably transfecting immune effector cells by electroporation using naked DNA are known in the art. Naked DNA generally refers to the DNA encoding a chimeric receptor contained in a plasmid expression vector in proper orientation for expression.

[0241] Alternatively, a viral vector (e.g., a retroviral vector, adenoviral vector, adeno- associated viral vector, or lentiviral vector) can be used to introduce the chimeric construct into immune cell, e.g., T cells. Suitable vectors are non-replicating in the immune effector cells of the subject. A large number of vectors are known which are based on viruses, whereAttorney Docket No. MDA0083-401-PC the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell. Illustrative vectors include the pFB-neo vectors (STRATAGENE™) as well as vectors based on HIV, SV40, EBV, HSV or BPV. Once it is established that the transfected or transduced immune effector cell is capable of expressing the chimeric receptor as a surface membrane protein with the desired regulation and at a desired level, it can be determined whether the chimeric receptor is functional in the host cell to provide for the desired signal induction (e.g., production of Rantes, Mip1-alpha, GM-CSF upon stimulation with the appropriate ligand).

[0242] Engineered CARs may be introduced into CAR-bearing immune effector cells using retroviruses, which efficiently and stably integrate a nucleic acid sequence encoding the chimeric antigen receptor into the target cell genome. Other methods known in the art include, but are not limited to, lentiviral transduction, transposon-based systems, direct RNA transfection, and CRISPR / Cas systems (e.g., type I, type II, or type Ill systems using a suitable Cas protein such Cas3, Cas4, Cas5, Cas5e (or CasD), Cash, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas1 Od, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3,Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, etc.). Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) may also be used. See, e.g., Shearer RF and Saunders DN, “Experimental design for stable genetic manipulation in mammalian cell lines: lentivirus and alternatives,” Genes Cells 2015 January; 20(1):1-10.

[0243] In accordance with the present disclosure, a CAR construct useful for targeting ALPP and / or ALPG / ALPPL2 may have a CD8a signal peptide, an M25 scFv (VH-linker-VL), a spacer, IgG2 CH2 and CH3 domain with an N297Q mutation, a CD28 transmembrane and intracellular domain, a 4-1BB, and CD3z. In some embodiments, a CAR construct useful for targeting ALPP and / or ALPG / ALPPL2 may have a CD8a signal peptide, an H2 scFv (VH- linker-VL), a spacer, IgG2 CH2 and CH3 domain with an N297Q mutation, a CD28 transmembrane and intracellular domain, a 4-1BB, and CD3z. In some embodiments, a CAR construct useful for targeting ALPP and / or ALPG / ALPPL2 may have a CD8a signal peptide, an M25 scFv (VH-linker-VL), a CD8 spacer and transmembrane domain, a 4-1BB, and CD3z. In some embodiments, a CAR construct useful for targeting ALPP and / or ALPG / ALPPL2 may have a CD8a signal peptide, an H2 scFv (VH-linker-VL), a CD8 spacer and transmembrane domain, a 4-1BB, and CD3z. These constructs are provided in Tables 1-6.Attorney Docket No. MDA0083-401-PC

[0244] Definitions

[0245] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range.

[0246] Unless defined otherwise, 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 herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Any discrepancy between the disclosure of a reference discussed herein and the present disclosure shall be resolved in favor of the present disclosure. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0247] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the disclosure pertains. Some specific terminology relevant to the description of the present disclosure is defined below.

[0248] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the,” along with similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims), can be construed to cover both the singular and the plural, unless specifically noted otherwise. Thus, for example, “an active agent” refers not only to a single active agent, but also to a combination of two or more different active agents, “a dosage form” refers to a combination of dosage forms, as well as to a single dosage form, and the like. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.Attorney Docket No. MDA0083-401-PC

[0249] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being 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 mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon 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 presented 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 herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. In some embodiments, “about” refers to a specified value + / - 10%.

[0250] The terms “comprise,” “have,” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes,” and “including,” are also open-ended. For example, any method that “comprises,” “has,” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has,” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0251] As used herein, “co-administration” refers to the combined administration of one or more drugs with another. In some embodiments, both drugs are administered at the same time. Co-administration may also refer to any particular time period of administration of either drug, or both drugs. For example, as described herein, a drug may be administered hours or days before administration of another drug and still be considered to have been co- administered. In some embodiments, co-administration may refer to any time of administration of either drug such that both drugs are present in the body of a patient at the same. In some embodiments, either drug may be administered before or after the other, soAttorney Docket No. MDA0083-401-PC long as they are both present within the patient for a sufficient amount of time that the patient received the intended clinical or pharmacological benefits.

[0252] As used herein, “anti-cancer therapy” refers to any therapy for treatment or prevention of cancer, e.g., NSCLC. For example, an anti-cancer therapy as described herein may refer to a chemical such as a chemotherapeutic drug or compound, or combination of drugs or compounds for cancer treatment. In some embodiments, an anti-cancer therapy may refer to a drug or compound, or combination of drugs or compounds, that leads to induced immunogenicity of the cell (e.g., a cancer cell, or a drug-resistant cancer cell, or a drug- tolerant persister cell). In some embodiments, inducing immunogenicity of a cancer cell can include, for example, any means for inducing the cancer cell(s) to progress to a more differentiated state associated with a more limited capacity to proliferate and a finite lifespan. This can be achieved by, e.g., upregulation or enhancement of expression of self and tumor antigens that make such cells more susceptible to immune-based therapies., or a drug- resistant cancer cell, or a drug-tolerant persister cell).

[0253] As used herein, a “DNA-damaging agent” refers to an agent or compound that introduces damage to the DNA of a cancer cell, through a number of mechanisms. For example, DNA damage can result from errors in DNA replication or prevention of DNA repair machinery, among others, which can include double-strand break (DSB) repair through the homologous recombination (HR) and non-homologous end joining (NHEJ) pathways. DNA-damaging agents are widely used in oncology to treat both hematological and solid cancers. Some commonly used modalities include, without limitation, ionizing radiation, platinum drugs (e.g., Cisplatin, Oxaliplatin, and Carboplatin), cyclophosphamide, chlorambucil, Doxorubicin, and temozolomide. Other commonly used drugs for treatment of cancers are described in detail herein. A “DNA-damaging agent” as used herein may refer to a chemotherapy agent or regimen, or may refer to any agent known or available in the art capable of introducing DNA damage to a cancer cell.

[0254] As used herein, a “targeted inhibitor” refers to a small molecule drug, a tyrosine kinase inhibitor (TKI), an angiogenesis inhibitor, a monoclonal antibody, a proteosome inhibitor, and / or a signal transduction inhibitor that can be used to specifically target a tumor. For example, an EGFR inhibitor is used to treat tumors expressing EGFR and / or having acquired resistance to an EGFR inhibitor, and an FGFR inhibitor is used to treat tumors expressing FGFR and / or having acquired resistance to an FGFR inhibitor. An inhibitor of a gene described herein may refer to any type of inhibitor known in the art that affects, i.e., inhibits, the pathway in which the specific gene is involved. For example, in someAttorney Docket No. MDA0083-401-PC embodiments, a targeted inhibitor may be a tyrosine kinase inhibitor or a JAK inhibitor. In some embodiments, an inhibitor may refer to an antibody that specifically inhibits some step in the pathway of a gene described herein, such as EGFR, KRAS, RET, ALK, HER2, MET, MEK, and / or FGFR. Such an antibody may be a monoclonal antibody.

[0255] In some embodiments, any of the above targeted inhibitors may be combined with a means for targeting ALPP and / or ALPG / ALPPL2. In some embodiments, a means for targeting ALPP and / or ALPG / ALPPL2 can be any useful method or technology known in the art. For example, ALPP and / or ALPG / ALPPL2 could be targeted by a small molecule, a blocking antibody, an antibody-drug conjugate (ADC), a bispecific antibody, a chimeric antigen receptor (CAR)-T cell, a CAR-natural killer (NK) cell, a bi-specific T-cell engager (BiTE), a or tri-specific NK cell engager therapy (TriNKET). In some embodiments, a CAR capable of targeting ALPP / ALPPL may comprise a sequence set forth in SEQ ID NOs: 1-18. In some embodiments, an antibody-drug conjugate (ADC) capable of targeting ALPP / ALPPL comprises a sequence set forth in SEQ ID NOs: 19-22. In some embodiments, a bispecific T- cell engager (BiTE) comprises a sequence set forth in SEQ ID NOs:23-27. As would be understood by one of skill in the art, specific elements in each of the constructs (e.g., linkers, signal peptides, spacers, transmembrane regions, co-stimulations domains, or the like) described herein may be used in more than one construct without deviating from the scope of the present disclosure.

[0256] As used herein, “treatment-resistant non-small-cell lung cancer” refers to a cell that is resistant or tolerant to a particular drug treatment. A treatment-resistant NSCLC cell can be a drug-tolerant persister cell (DTPC) or a drug-resistant cell (DRC).

[0257] As used herein, a cancer “having acquired resistance to a targeted inhibitor” refers to a cell that is resistant or tolerant to a particular drug treatment. A treatment-resistant NSCLC cell, or a NSCLC cell having acquired resistance to a targeted inhibitor, can be a drug-tolerant persister cell (DTPC) or a drug-resistant cell (DRC).

[0258] As used herein, a “drug-resistant cell” or “DRC” refers to a cell that has developed resistance to a particular drug, e.g., a targeted inhibitor as described herein.

[0259] As used herein, a “drug-tolerant persister cell” or “DTPC” refers to a cell that exhibits a reversible phenotype. DTPCs can resume proliferation and drug sensitivity after discontinuation of a drug treatment. DTPCs can eventually acquire several types of drug- resistant mechanisms under continuous treatment. The methods described herein prevent a drug-tolerant persister cell (DTPC) from developing into a drug-resistant cell (DRC). The methods described herein also treat drug-tolerant persister cells (DTPCs) and drug-resistantAttorney Docket No. MDA0083-401-PC cells (DRCs) by preventing the emergence of resistance to EGFR tyrosine kinase inhibitors, KRAS inhibitors, RET inhibitors, ALK inhibitors, HER2 inhibitors, MET inhibitors, MEK inhibitors, or FGFR inhibitors. In some embodiments, the methods described herein treat cells with EGFR TKI resistance that is mediated by MET upregulation.

[0260] As used herein, a “mutation” refers to a change in the DNA of a particular gene or amino acid sequence of an encoded protein. A mutation in a gene described herein may be an insertion, where additional nucleotides are inserted into a DNA sequence, a deletion, where one or more nucleotides are missing or removed, and a point mutation, where a single nucleotide is altered. Mutations in accordance with the present disclosure may be any mutation in a gene described herein, e.g., EGFR, KRAS, RET, ALK, HER2, MET, MEK, and / or FGFR. Common mutations known to be involved in the development or maintenance of cancer, e.g., lung cancer, in these genes include point mutations, frameshift mutations, activating mutations, gene amplifications, and / or gene fusions, which may occur at the nucleic acid or protein level.

[0261] As used herein, “EGFR” refers to the EGFR gene, also known as the “Epidermal Growth Factor” gene and “HER1.” A number of mutations in EGFR are known to be associated with cancer, including NSCLC. For example, EGFR mutations found in lung cancer include, but are not limited to, a T790M mutation, a C797S mutation, exon 19 deletions, an L858R point mutation, and exon 20 insertions. EGFR tyrosine kinase inhibitor (TKI) resistance in some NSCLC cells can also be mediated by MET gene upregulation.

[0262] As used herein, “KRAS” refers to the KRAS gene, also known as “Kirsten Rat Sarcoma Virus” gene. A number of mutations in KRAS are known to be associated with cancer, including NSCLC. For example, KRAS mutations found in lung cancer include, but are not limited to, mutations in codons 12 and 13, with the most common subtypes including G12C, G12V, and G12D.

[0263] As used herein, RET refers to the RET gene, also known as “Rearranged During Transfection” gene. Numerous types of RET mutations are known in the art to be associated with different types of cancer, including NSCLC. For example, RET mutations found in lung cancer include, but are not limited to, a fusion of the RET gene with another gene to create a RET rearrangement or RET fusion. Non-limiting examples of a RET fusion or rearrangement include, but are not limited to, a point mutation, such as a CCDC6-RET fusion or a KIF5b- RET fusion

[0264] As used herein, “ALK” refers to the ALK gene, also known as “Anaplastic Lymphoma Kinase” gene. A number of ALK mutations are known to be associated withAttorney Docket No. MDA0083-401-PC cancer, including NSCLC. For example, ALK mutations found in lung cancer include, but are not limited to, a fusion of ALK with another protein, such as an EML4-ALK gene fusion.

[0265] As used herein, “HER2” refers to the HER2 gene, also known as “human epidermal growth factor receptor 2” or “ERBB2.” A number of mutations in HER2 are known to be associated with cancer, including NSCLC. For example, HER mutations found in lung cancer include, but are not limited to, an exon 20 YVMA insertion, an exon 20 GSP insertion, an exon 20 VC insertion, a tyrosine kinase domain mutation, or a V659 mutation.

[0266] As used herein, “MET” refers to the MET gene, also known as “mesenchymal epithelial transition” gene. A number of MET mutations are known to be associated with cancer, including NSCLC. For example, a MET mutation described herein includes, but is not limited to, a MET amplification, wherein extra copies of the MET gene are present in the body, and an exon 4 skipping mutation, which prevents degradation of the MET protein and promotes cancer.

[0267] As used herein, “MEK” refers to the MEK gene, also known as “mitogen- activated protein kinase kinase” gene, MAP2K, MEK, or MAPKK. A number of MEK mutations are known to be associated with cancer, including NSCLC. For example, a MEK mutation described herein includes, but is not limited to, a mutation that involves the allosteric drug binding pocket or α-helix C, a mutation in a MEK1 codon located within or abutting the N-terminal negative regulatory helix (helix A), a MEK1(P124L) mutation, a MEK1(Q56P) mutation, or any mutation in the MEK gene that results in or confers resistance to a MEK inhibitor.

[0268] As used herein, “FGFR” refers to the FGFR gene, also known as “fibroblast growth factor receptor” gene. A number of FGFR mutations are known to be associated with cancer, including NSCLC. For example, an FGFR mutation described herein includes, but is not limited to, an FGFR fusion, an FGFR amplification, and / or an FGFR point mutation. In some embodiments, an FGFR fusion includes, but is not limited to, an FGFR2-ERC1 fusion, an FGFR3-TACC3 fusion, an FGFR2-INA fusion, a FGFR4-RAPGEFL1 fusion, and a fusion of the FGFR1 and SLC20A25’-untranslated regions. In some embodiments, An FGFR amplification comprises an FGFR1 amplification, an FGFR2 amplification, an FGFR3 amplification, or an FGFR4 amplification. In some embodiments, an FGFR point mutation comprises R248C, S249C, G370C, S371C, Y373C, G380R, W290C, S320C, or K660E / N point mutations.

[0269] For any of the genetic mutations described herein, e.g., a mutation in EGFR, KRAS, RET, ALK, HER2, MET, MEK, and / or FGFR, the genetic mutation in questionAttorney Docket No. MDA0083-401-PC renders the cancer cell resistant to an inhibitor. For example, a non-small-cell lung cancer having an EGFR mutation is resistant to an inhibitor targeting EGFR. In other embodiments, the non-small-cell lung cancer having a KRAS mutation is resistant to an inhibitor targeting KRAS. In other embodiments, the non-small-cell lung cancer having a RET mutation is resistant to an inhibitor targeting RET. In other embodiments, the non-small-cell lung cancer having an ALK mutation is resistant to an inhibitor targeting ALK. In other embodiments, the non-small-cell lung cancer having a HER2 mutation is resistant to an inhibitor targeting HER2. In other embodiments, the non-small-cell lung cancer having a MET mutation is resistant to an inhibitor targeting MET. In other embodiments, the non-small-cell lung cancer having a MEK mutation is resistant to an inhibitor targeting MEK. In other embodiments, the non-small-cell lung cancer having an FGFR mutation is resistant to an inhibitor targeting FGFR.

[0270] A pharmaceutical composition(s) comprising one or more cancer treatments described herein may include a “therapeutically effective amount” of the cancer treatments as described herein. The term “therapeutically effective amount,” “pharmacologically effective dose,” “pharmacologically effective amount,” or simply “effective amount” may be used interchangeably and refers to that amount of an agent effective to produce the intended pharmacological, therapeutic or preventive result, e.g., a reduction of cancerous cells or lessened cancer cell burden (i.e., reduction in number of cancer cells), tumor size, tumor density, lymph node involvement, metastases, cancer recurrence or relapse, or associated symptoms in the patient. The pharmacologically effective amount results in the amelioration of one or more symptoms of a disorder (e.g., a hematological cancer), or prevents the advancement of a disorder, or causes the regression of the disorder, or prevents the disorder. Such effective amounts can be determined based on the effect of the administered agent, e.g., cancer treatment described herein, or the combinatorial effect of agents if more than one agent is used, e.g., a cancer treatment described followed by a second cancer treatment or agent described herein. A therapeutically effective amount of an agent may also vary according to factors such as the disease stage, state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual, e.g., amelioration of at least one disorder parameter or amelioration of at least one symptom of the disorder. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. In some examples, an “effective amount” is one that treats (including prophylaxis) one or more symptoms and / or underlying causes of cancer. In some examples, an effective amount is aAttorney Docket No. MDA0083-401-PC therapeutically effective amount. In some examples, an effective amount is an amount that prevents one or more signs or symptoms of a particular disease or condition from developing.

[0271] As used herein, “gene expression” or “expression” refers to the process of gene transcription, translation, and post-translational modification. In some embodiments, gene expression includes epigenetic modifications, which may refer to mechanisms or processes in a cell that affect the expression of a gene without altering the primary DNA sequence. Such epigenetic modifications may include, but are not limited to, DNA methylation, histone modifications, and small non-coding microRNAs (miRNAs). Epigenetic modification may lead to altered gene function and malignant cellular transformation.

[0272] As used herein, an “immunotherapy drug” refers to a drug or compound that stimulates or suppresses the immune system to help the body fight cancer, infection, and / or other diseases. Immunotherapy drugs can be antibodies, e.g., a monoclonal antibody. In some embodiments, an immunotherapy drug may be an immune checkpoint inhibitor drug, e.g., a drug that targets the programmed cell death protein (PD-1) or its ligand, PDL-1, and / or cytotoxic T lymphocyte antigen 4 (CTLA-4) receptors. For example, useful immunotherapy drugs 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®). Immunotherapy drugs targeting PD-1 include, but are not limited to, Pembrolizumab (Keytruda®), Nivolumab (Opdivo®), and Cemiplimab (Libtayo®). Immunotherapy drugs targeting PD-L1 include, but are not limited to, Atezolizumab (Tecentriq®), Avelumab (Bavencio®), and Durvalumab (Imfinzi®). Immunotherapy drugs targeting CTLA-4 include, but are not limited to, Ipilimumab (Yervoy®). Additional immunotherapy drugs targeting these checkpoint inhibitors are known and available in the art, and are encompassed within the scope of the present disclosure.

[0273] By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effects or interacting in a deleterious manner 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 is implied that the carrier or excipient has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration (FDA) or comparable foreign regulatory agencies. “Pharmacologically active” (or simply “active”) asAttorney Docket No. MDA0083-401-PC in a “pharmacologically active” (or “active”) derivative or analog, refers to a derivative or analog having the same type of pharmacological activity as the parent compound and approximately equivalent in degree. Some pharmacologically active derivatives may have improved pharmacological activity. The term “pharmaceutically acceptable salts” includes acid addition salts which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, 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.

[0274] As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The composition can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt.

[0275] As used herein, “reducing” refers to a lowering or lessening, such as reducing cancer cell burden. In some embodiments, administration of a cancer treatment as described herein may result in “reduced” or lessened cancer cell burden (i.e., reduction in number of cancer cells), tumor size, tumor density, blast cell involvement, proliferation, proportion of quiescent (G0) cells, lymph node involvement, metastases, or associated symptoms in the patient compared to a patient not having been administered such drugs. “Reducing” may also refer to a reduction in disease symptoms as a result of a treatment as described herein, either alone, or co-administered with another drug.

[0276] As used herein, the term “cancer” refers to a malignant neoplasm characterized by the abnormal proliferation of cells, the growth of which cells exceeds and is uncoordinated with that of the normal tissues around it. A cancer useful with the present methods may be any type of cancer described herein, e.g., lung cancer, or more specifically, non-small-cell lung cancer or mesenchymal non-small-cell lung cancer. A cancer as described herein may refer to a primary cancer or a secondary cancer, e.g., a metastasis or a recurrence of a cancer in the same or a new location.

[0277] As used herein, “healthy” refers to an individual who does not have cancer. A patient of the present disclosure having cancer may be compared to a healthy individual, at least in terms of symptom severity, disease course, expression of a protein disclosed herein, e.g., ALPP and / or ALPG / ALPPL2, or the like. Comparison may also be made to a “healthy” lung cell, e.g., a lung cell that does not have cancer.Attorney Docket No. MDA0083-401-PC

[0278] As used herein, the term “ELISA” refers to enzyme-linked immunosorbent assay. This assay generally involves contacting a fluorescently tagged sample of proteins with antibodies having specific affinity for those proteins. Detection of these proteins can be accomplished with a variety of means, including, but not limited to, laser fluorimetry.

[0279] As used herein, “subject” or “individual” or “patient” refers to any patient for whom or which therapy is desired, and generally refers to the recipient of the therapy. A “subject” or “patient” refers to any animal classified as a mammal, e.g., human and non- human mammals. Examples of non-human animals include dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Unless otherwise noted, the terms “patient” or “subject” are used herein interchangeably. In some embodiments, a subject amenable for therapeutic applications may be a primate, e.g., human and non-human primates. As used herein, a “subject” or “individual” or “patient” may be one with cancer, such as lung cancer and for whom further treatment can be provided. Lung cancer may refer to non-small-cell lung cancer, or mesenchymal non-small-cell lung cancer.

[0280] The terms “treating” and “treatment” or “alleviating” as used herein refer to reduction or lessening in severity and / or frequency of symptoms, elimination of symptoms and / or underlying cause, and improvement or remediation of damage. In certain aspects, the term “treating” and “treatment” as used herein refer to the prevention of the recurrence of symptoms. In other aspects, the term “treating” and “treatment” as used herein refer to the prevention of the underlying cause of symptoms associated with a disease or condition, such as lung cancer. The phrase “administering to a patient” refers to the process of introducing a composition or drug into the patient via an art-recognized means of introduction. “Treating” or “alleviating” also includes the administration of compounds or agents to a subject to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease (e.g., cancer), alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. Subjects in need of treatment include those already suffering from the disease or condition, those previously suffering from the disease or condition and at risk of recurrence, as well as those being at risk of developing the disease or condition.

[0281] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language inAttorney Docket No. MDA0083-401-PC the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0282] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability.

[0283] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples. EXAMPLES

[0284] The following examples are included to demonstrate embodiments of the disclosure. The following examples are presented only by way of illustration and to assist one of ordinary skill in using the disclosure. The examples are not intended in any way to otherwise limit the scope of the disclosure. Those of ordinary skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. Example 1

[0285] While cancer patients with activating mutations in driver oncogenes such as epidermal growth factor receptor (EGFR), Kirsten rat sarcoma virus (KRAS), rearranged during transfection (RET), anaplastic lymphoma kinase (ALK), and human epidermal growth factor 2 (HER2) are initially responsive to agents targeting these specific molecules, resistant disease inevitably emerges. Novel targeting approaches are sorely needed to combat resistant disease and to prevent the emergence of resistance.

[0286] To this end, an unbiased bioinformatic strategy was performed to identify surface targets in EGFR tyrosine kinase inhibitor (TKI) drug-resistant cells (DRCs) and drug-tolerant persister cells (DTPCs) using public datasets from the GEO database (FIG.1A). ALPP was identified as one of the top candidate genes commonly upregulated in EGFR TKI DRCs and DTPCs (FIG.1B). Moreover, ALPP has minimal expression in normal tissues (FIG.1C). Moreover, ALPP mRNA was significantly upregulated in a panel of H1975 cells with acquired resistance to the EGFR TKI osimertinib (FIG.2A). ALPP cell surface expression was then evaluated by flow cytometry, which demonstrated that ALPP was highly expressedAttorney Docket No. MDA0083-401-PC on osimertinib resistant (OR) cells (FIG.2B-2F). It was then tested whether ALPP upregulation could be exploited in the cells with acquired EGFR-TKI resistance.

[0287] ALPP and ALPG RNA expression was then evaluated in EGFR mutant NSCLC clinical specimens before and after progression on osimertinib. ALPP (FIG.3A) and ALPG mRNA (FIG.3B) levels were increased in tumors at the time of progression as compared to pretreatment levels. By immunohistochemistry, ALPP was found to be upregulated in osimertinib refractory EGFR mutant tumors (FIG.3D) as compared to TKI naïve tumors (FIG.3C).

[0288] An ALPP-CAR construct was generated by composing the sequence of a single- chain variable fragment (scFv) derived from an ALPP-specific antibody into a third- generation CAR backbone (FIG.4A). ALPP CAR-T cells were then generated by retrovirally transducing T cells with this construct. The sequences for the CAR constructs are provided in Tables 1- 6 below. Table 1 – Sequence Information for ALPP / ALPPL2-CAR #1: M25-M228BB3z AA Sequence Note SEQ OAttorney Docket No. MDA0083-401-PC GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGAttorney Docket No. MDA0083-401-PC KMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPRAA Sequence Note SEQ ID NOAttorney Docket No. MDA0083-401-PC VTMSVDTSKNQFSLKLSSVTAADTAVYYCARPH H2 is YGSSYVGAMEYWGAGTTVTVSS humanizedTable 3 – Sequence Information for ALPP-CAR #1: M25-8BB3z AA Sequence Note SEQ OAttorney Docket No. MDA0083-401-PC GLEWVAVISYDGSNKYYADSVKGRFTISRDNSK NTLYLQMDSLRAEDTAVYFCAKEGDSSRWSYDLAttorney Docket No. MDA0083-401-PC 355- KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE 4-1BB 8 396 EEEGGCELq AA Sequence Note SEQ ID NOAttorney Docket No. MDA0083-401-PC H17E2 (US Pat. GGGGSGGGGSGGGGS Publ. No. 4

[0289] Using a firefly luciferase-based cytotoxicity system, it was demonstrated that ALPP CAR-T cells could specifically eliminate ALPP-expressing cells (FIG.4B). ALPP CAR-T cells killed ALPP overexpressing (H1975-ALPP) and ALPG overexpressing (H1975- ALPG) cells but not parental (ALPP negative) cells (FIG.4C). Moreover, it was observed that, while ALPP CAR-T cells did not kill untreated HCC4006 and H1975 parental cells (ALPP low), they effectively killed HCC4006 and H1975 OR cells (ALPP high) (FIG.4D and FIG.4E). It was determined that ALPP CAR-T cells (H2-M228BB3Z) killed HCC4006 OR cells but not HCC4006 parental cells (FIG.4F). ALPP CAR-T cells (H2-M228BB3Z) killed H1975 OR cells but not H1975 parental cells (FIG.4G). These data indicate that ALPP targeting approaches can eliminate tumors with acquired resistance to EGFR TKIs.

[0290] It was observed that ALPP expression could also be targeted by CAR-NK cells in EGFR TKI-resistant NSCLC cells. ALPP CAR-NK cells killed HCC4006 OR cells more effectively as compared to HCC4006 parental cells (FIG.5A). ALPP CAR-NK cells specifically killed HCC4006 OR cells by blocking natural cytotoxicity pathways (FIG.5B).

[0291] Next, it was evaluated whether upregulation of ALPP was an early event in the evolution of resistance to EGFR TKIs. EGFR mutant NSCLC cells (H1975, HCC827, andAttorney Docket No. MDA0083-401-PC HCC4006) were treated with osimertinib for 14 days, at which point the majority of cells were killed and only DTPCs remained. It was observed that ALPP is upregulated on EGFR TKI (osimertinib) drug tolerant persister cells (DTPCs). ALPP mRNA levels were increased in EGFR mutant NSCLC cell lines (H1975, HCC827, and HCC4006) after treatment with the EGFR inhibitor osimertinib for 14 days (FIG.6A). Flow cytometry data indicated upregulated ALPP expression on the cell surface of H1975 DTPCs, HCC827 DTPCs, and HCC4006 DTPCs as compared to untreated cells (FIG.6B&C). It was also observed that ALPP mRNA was upregulated in HCC827 cells treated with osimertinib for 2, 4, 8, 16, and 24 hours (FIG.6D). By immunohistochemistry it was determined that protein levels of ALPP are upregulated in H1975 xenograft tumors after treatment with osimertinib for two weeks in vivo (FIG.6E).

[0292] EGFR mutant NSCLC cells and osimertinib DTPCs were then co-cultured with ALPP CAR-T cells. It was then observed that ALPP CAR-T cells effectively killed osimertinib-tolerant persister cells (ALPP high) but did not kill parental cells (ALPP low) (FIG.7). These data indicate that ALPP targeting approaches can eliminate residual cells after initial treatment with EGFR TKIs.

[0293] Next, the in vivo activity of CAR NK cells targeting ALPP was tested in vivo. EGFR TKI (osimertinib) DTPCs were generated in vivo by treating tumor bearing mice with osimertinib to allow tumor shrinkage. ALPP CAR-NK cells inhibited H1975 xenograft growth when administrated with osimertinib at the same period (FIG.8A). Moreover, ALPP CAR-NK cells inhibited H1975 xenograft growth when administrated after osimertinib treatment for 2 weeks (FIG.8B).

[0294] It was next investigated whether tumor cells with driver oncogenes other than EGFR also upregulate ALPP during the evolution of resistance to targeted agents. KRAS mutant NSCLC cells (H358, H1373, and Calu1) were treated with adagrasib for 14 days, at which point ALPP mRNA was upregulated (FIG.9A). ALPP was also increased on the cell surface of KRAS inhibitor (adagrasib or sotorasib) DTPCs as determined by flow cytometry (FIG.9B&C). KRAS mutant cells were then cocultured with ALPP CAR-T cells. It was observed that ALPP CAR-T cells effectively killed adagrasib- and sotorasib-treated cells (ALPP high) but not untreated parental cells (ALPP low) (FIG.10). FIG.11A shows that mRNA levels of ALPP are upregulated in H23 and H358 cells treated with KRAS G12C inhibitor sotorasib and H23 and H358 cells with acquired resistance to KRAS G12C inhibitor sotorasib. The figure was generated from GEO dataset GSE229070. A panel of H358 cells with acquired resistance to the KRAS inhibitor adagrasib or sotorasib was then developed byAttorney Docket No. MDA0083-401-PC continuously culturing cells in adagrasib or sotorasib until resistant clones emerged. ALPP expression was evaluated by qPCR and flow cytometry, and it was determined that ALPP was highly expressed in the adagrasib and sotorasib resistant cells (FIG.11 B-F). These data indicate that upregulation of ALPP is an early event in the evolution of resistance to KRAS inhibitors and ALPP is a target on cells with acquired resistance to KRAS inhibitors. ALPP expression could be effectively targeted in NSCLC cells with acquired resistance to KRAS G12C inhibitors as demonstrated in FIG.12. ALPP-targeting CAR-T cells killed H358 sotorasib-resistant clones but not H358 parental cells (FIG.12).

[0295] It was next assessed whether ALPP was similarly upregulated on tumor cells bearing other activating mutations in driver oncogenes after treatment with targeted agents. TH1101 (KIF5b-RET fusion) or LC2 / AD cells (CCDC6-RET fusion) were treated with the RET inhibitor selpercatinib or pralsetinib for 14 days and evaluated mRNA levels by real time PCR. Consistent with previous findings, ALPP was significantly upregulated in selpercatinib-treated cells (FIG.13A-D). Moreover, LC2 / AD cells were developed with acquired resistance to selpercatinib or pralsetinib, and it was found that ALPP was highly expressed in the selpercatinib and pralsetinib resistant cells (FIG.13C – FIG.13F). It was next tested whether ALPP could be targeted on tumor cells bearing RET fusions. ALPP CAR T cells effectively killed LC2ad-selpercatinib resistant cells (ALPP high) but not LC2ad parental cells (ALPP low) (FIG.14A). Similarly ALPP CAR NK cells effectively killed LC2ad-selpercatinib resistant cells (ALPP high) but not LC2ad parental cells (ALPP low) (FIG.14B). ALPP CAR-NK cells specifically killed LC2 / AD selpercatinib-resistant cells by blocking natural cytotoxicity pathways (FIG.14C).

[0296] In addition, an ALPP-targeting Antibody Drug Conjugate (ADC) was prepared. Briefly, M25 antibody (Tables 7 and 8) was labeled with oYo-Link®VcMMAE (alphathera.com / product / antibody-drug-conjugation / oyo-link-vcmmae / ) by photo- crosslinking under 365 nm LED light for 2 hours, which was following the manual. It was found that the ADC killed more LC2 / AD selpercatinib-resistant cells compared to control ADC (FIG.14D).

[0297] Next, ALPP expression was assessed in ALK fusion-positive H3122 NSCLC cells treated with the ALK inhibitor crizotinib for 14 days. ALPP was significantly upregulated in ALK inhibitor treated cells (FIG.15A). Next transcriptomic data was evaluated from H2228 cells that harbor an EML4-ALK gene fusion and H2228 cells treated with the ALK inhibitors alectinib and lorlatinib for 9 days. Analysis revealed the upregulation of ALPP in both ALK inhibitors treated H2228 cells (FIG.15B). Consistently, ALPP was also significantlyAttorney Docket No. MDA0083-401-PC upregulated in alectinib-treated A925L cells that harbor an EML4-ALK gene fusion (FIG. 15C). Moreover, transcriptomic data from H2228 cells with acquired resistance to the ALK inhibitor alectinib was evaluated, and it was found that ALPP was upregulated in alectinib- resistant cells (FIG.15D). In addition, transcriptomic data from breast cancer cells (BT474, EFM192, HCC1419, and SKBR3) treated with the HER2 inhibitor lapatinib was evaluated, and the upregulation of ALPP in lapatinib-treated cells (FIG.16). Also, ALPP was observed to be upregulated in bladder cancer cells treated with the FGFR inhibitor infigratinib (FIG. 17). In KRAS mutant H358 cells, treatment with other KRAS inhibitors ARS1323 or AZD4785 or a MEK inhibitor, selumetinib, also increased ALPP expression (FIG.18). In HCC827 and PC9 EGFR mutant NSCLC cells, treatment with osimertinib in combination with trametinib also increased expression of ALPP (FIG.19). Next using HCC827 cells with acquired osimertinib resistance (OR7, OR6, OR4, OR3, OR2) that occurred though MET amplification (FIG.20A), treatment with the MET inhibitors tepotinib or capmatinib resulted in upregulation of ALPP at the RNA (FIG.20B) and protein levels (FIG.20C). Collectively, these data demonstrate ALPP to be a common target on tumor cells after treatment with targeted agents and tumor cells with acquired resistance to targeted agents. Example 2

[0298] While cancer patients with activating mutations in driver oncogenes such as epidermal growth factor receptor (EGFR), Kirsten rat sarcoma virus (KRAS), rearranged during transfection (RET), anaplastic lymphoma kinase (ALK), human epidermal growth factor 2 (HER2), and / or fibroblast growth factor receptor (FGFR), are initially responsive to agents targeting these specific molecules, resistant disease inevitably emerges. Novel targeting approaches are sorely needed to combat resistant disease and to prevent the emergence of resistance.

[0299] To this end, an unbiased bioinformatic strategy was performed to identify surface targets in EGFR tyrosine kinase inhibitor (TKI) drug-resistant cells (DRCs) and drug-tolerant persister cells (DTPCs) using public datasets from the GEO database (FIG.21A). ALPG / ALPPL2 was identified as one of the top candidate genes commonly upregulated in EGFR TKI DRCs and DTPCs (FIG.21B). Moreover, ALPG has minimal expression in normal tissues (FIG.21C). Moreover, ALPG mRNA was significantly upregulated in a panel of H1975 cells with acquired resistance to the EGFR TKI osimertinib (FIG.22A). ALPG cell surface expression was then evaluated by flow cytometry. Using ALPP-overexpression and ALPG-overexpression cells, it was found that the two flow cytometry antibodies, 8B6 andAttorney Docket No. MDA0083-401-PC H17E2, recognize both ALPP and ALPG (ALPPL2) (FIG.22B). Furthermore, ALPP / ALPG (ALPPL2) was highly expressed on osimertinib-resistant (OR) cells (FIG.22D-FIG.22G).

[0300] Using a patient cohort of matched EGFR mutant NSCLC tumors collected prior to osimertinib treatment and after progression, a significant increase in ALPP RNA was observed in the osimertinib refractory samples as compared to the pre-treated tumors, particularly in patients exhibiting long progression-free survival (PFS) (FIG.23A-FIG.23B). Protein levels of ALPG (ALPPL2) were next assessed by IHC in EGFR TKI naïve NSCLC clinical specimens and unmatched tumor specimens from EGFR mutant patients who progressed on osimertinib. ALPP / ALPPL2 expression was significantly elevated in osimertinib refractory tumors as compared to treatment naïve tumors (FIG.23C).

[0301] Next, it was tested whether ALPP / ALPG (ALPPL2) upregulation could be exploited in the cells with acquired EGFR-TKI resistance. An ALPP / ALPPL2-CAR construct was generated by composing the sequence of a single-chain variable fragment (scFv) derived from two ALPP / ALPPL2-specific antibodies, M25 and humanized H17E2, into a second- generation and a third-generation CAR backbone (FIG.24A). ALPP / ALPPL2 CAR-T cells were then generated by retrovirally transducing T cells with this construct. Using a firefly luciferase-based cytotoxicity system, it was demonstrated that ALPP / ALPPL2 CAR-T cells could specifically eliminate ALPP-expressing cells and ALPG (ALPPL2)-expressing cells (FIG.24B-FIG.24C). Moreover, it was observed that, while ALPP / ALPPL2 CAR-T cells did not kill untreated HCC4006 and H1975 parental cells (ALPP / ALPPL2 low), they effectively killed HCC4006 and H1975 osimertinib-resistant (OR) cells (ALPP / ALPPL2 high) (FIG. 24D-FIG.24G). The sequences for the CAR constructs used in the examples herein are provided in Tables 1-6.

[0001] Furthermore, ALPP / ALPPL2 CAR-NK cells effectively killed HCC4006 OR cells as compared to HCC4006 parental cells (FIG.25A). Additionally, blockade of both NKp30 and NKG2D assays showed that the ALPP-CAR NK cell approach is cytotoxic and specific, targeting drug-resistant cells while maintaining its effectiveness even when inhibiting the NK cell activating receptors (FIG.25B). These data indicate that ALPPL2 targeting approaches can eliminate tumors with acquired resistance to EGFR TKIs.

[0002] Next, it was assessed whether upregulation of ALPG (ALPPL2) was an early event in the evolution of resistance to EGFR TKIs. EGFR mutant NSCLC cells (H1975, HCC827, and HCC4006) were treated with osimertinib for 14 days, at which point the majority of cells have been killed, and only drug-tolerant persister cells (DTPCs) remain. ItAttorney Docket No. MDA0083-401-PC was found that osimertinib-derived DTPCs upregulated ALPG (ALPPL2) at both the mRNA level (FIG.26A) and on the cell surface (FIG.26B and FIG.26C).

[0003] It was then evaluated whether ALPP was upregulated on EGFR TKI DTPCs in vivo. H1975 cells were injected subcutaneously into immunodeficient mice. Once tumors reached 600 mm3, animals were treated with either vehicle or osimertinib for 14 days. Immunohistochemistry (IHC) analysis revealed that, while control-treated tumors were negative for ALPP, residual tumors after osimertinib treatment were positive for ALPP (FIG. 26D). Cells were then co-cultured with ALPP / ALPPL2 CAR-T cells. It was observed that ALPP / ALPPL2 CAR-T cells effectively killed osimertinib-tolerant persister cells (ALPP / ALPPL2 high), but did not kill parental cells (ALPP / ALPPL2 low) (FIG.27A-27C). These data indicate that ALPG (ALPPL2) targeting approaches can eliminate residual cells after initial treatment with EGFR TKIs.

[0004] To evaluate the antitumor activity of ALPP / ALPPL2-CAR T cells against DRCs in vivo, an osimertinib-resistant xenograft model H1975 OR17 cells was utilized. Animals were randomized to receive vehicle, ALPP / ALPPL2-CAR T cells, or ALPP / ALPPL2-CAR NK cells. Treatment with third-generation ALPP-CAR T cells H2-M228BB3z (Table 2), M25-H428BB3z (Table 5), and H2-H428BB3z (Table 6) resulted in significant inhibition of tumor growth (FIG.28A). Likewise, second-generation ALPP-CAR NK cells, H2-28BB3z (Table 4), significantly inhibited the OR17 tumor growth (FIG.28B). H1975 xenograft mice treated with osimertinib plus ALPP CAR-NK cells at the same period (osimertinib + ALPP- CAR NK cells) exhibited prolonged tumor regression compared to the osimertinib-treated group (FIG.28C). In addition, H1975 xenograft mice initially treated with osimertinib (DTPCs), and then ALPP-CAR-NK cells (osimertinib, then ALPP-CAR NK cells) also exhibited further reductions in tumor volumes (FIG.28D). Table 5 – Sequence Information for ALPP / ALPPL2-CAR #5: M25-H428BB3z AA Sequence Note SEQ OAttorney Docket No. MDA0083-401-PC PGKAPKVMIYDVTNRPSGVSNRFSGSKSGNTASL TISGLQAEDEADYYCSSYTSTSTLVVFGGGTKLTAttorney Docket No. MDA0083-401-PC KMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPRq AA Sequence Note SEQ ID NOAttorney Docket No. MDA0083-401-PC WYQQKPGKAPKLLIYNAKSLASGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQHHYVSPWTFGGGTTable 7 – Sequence Information for ALPP / ALPPL2-antibody: M25 heavy chain AA Sequence Note SEQ OAttorney Docket No. MDA0083-401-PC ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKTable 8 – Sequence Information for ALPP / ALPPL2-antibody: M25 light chain AA Sequence Note SEQ ID NO

[0005] Next, it was investigated whether tumor cells with driver oncogenes other than EGFR also upregulate ALPP / ALPPL2 during the evolution of resistance to targeted agents. KRAS mutant NSCLC cells (H358, H1373, and Calu1) were treated with adagrasib for 14 days, at which point ALPP / ALPPL2 was upregulated (FIG.29A-FIG.29B). Significant upregulation of ALPG (ALPPL2) was observed at the mRNA level in H358 cells treated with sotorasib for 1 or 3 days (FIG.29C). H358 DTPCs, H1373 DTPCs, and Calu1 DTPCs were then cocultured with ALPP / ALPPL2 CAR-T cells. It was observed that ALPP / ALPPL2 CAR-T cells effectively killed adagrasib- and sotorasib-treated cells (ALPP / ALPPL2 high) but not untreated parental cells (ALPP / ALPPL2 low) (FIG.30A-FIG.30F). In H23 and H358Attorney Docket No. MDA0083-401-PC with acquired resistance to sotorasib, significant upregulation of ALPG was observed at the mRNA level compared to their parental cells (FIG.31A). A panel of H358 cells with acquired resistance to the KRAS inhibitor adagrasib or sotorasib was then developed by continuously culturing cells in adagrasib or sotorasib until resistant clones emerged. ALPG (ALPPL2) expression was evaluated by western blot (WB) and flow cytometry, revealing that ALPG (ALPPL2) was highly expressed in the adagrasib- and sotorasib-resistant cells (FIG.31B-FIG.31D). Sotorasib-resistant cells were then co-cultured with ALPP / ALPPL2 CAR-T cells. It was observed that ALPP / ALPPL2 CAR-T cells effectively killed sotorasib- resistant cells but did not kill parental cells (FIG.32A). These data indicate that (1) upregulation of ALPG (ALPPL2) is an early event in the evolution of resistance to KRAS inhibitors, (2) ALPG (ALPPL2) is a target on cells with acquired resistance to KRAS inhibitors, and (3) ALPG (ALPPL2) targeting approaches can eliminate KRAS G12C DRCs and DTPCs.

[0006] It was next assessed whether ALPG (ALPPL2) was similarly upregulated on tumor cells bearing other activating mutations (e.g., a mutation in RET) in driver oncogenes after treatment with targeted agents. LC2 / ad cells with acquired resistance to selpercatinib were developed, and it was determined that ALPP / ALPPL2 was highly expressed in the selpercatinib-resistant cells (FIG.33A-FIG.33C). Cells were then co-cultured with ALPP / ALPPL2 CAR-T cells. It was observed that ALPP / ALPPL2 CAR-T cells effectively killed selpercatinib-resistant cells and selpercatinib DTPCs but did not kill parental LC2 / ad cells (FIG.34A). In addition, it was observed that ALPP / ALPPL2 CAR-NK cells effectively killed LC2 / ad selpercatinib-resistant cells as compared to LC2 / ad parental cells (FIG.34B- FIG.34C). Furthermore, an ALPP / ALPPL2-targeting antibody-drug conjugate (ADC), M25- MMAE (see Tables 7 and 8), also effectively killed LC2 / ad selpercatinib-resistant cells as compared to LC2 / ad parental cells (FIG.34D).

[0007] Next, transcriptomic data was evaluated from H2228 cells that harbor an EML4- ALK gene fusion and H2228 cells treated with the ALK inhibitors alectinib and lorlatinib for 9 days. The analysis revealed upregulation of ALPG (ALPPL2) in H2228 cells treated with both ALK inhibitors (FIG.35A). Consistently, ALPG (ALPPL2) was also significantly upregulated in alectinib-treated A925L cells that harbor an EML4-ALK gene fusion (FIG. 35B). Furthermore, it was observed that ALPG (ALPPL2) was significantly upregulated in crizotinib-treated H3122 cells that harbor an EML4-ALK gene fusion (FIG.35C). Moreover, transcriptomic data from H2228 cells with acquired resistance to the ALK inhibitor alectinibAttorney Docket No. MDA0083-401-PC was evaluated, revealing that ALPG (ALPPL2) was upregulated in alectinib-resistant cells (FIG.35D).

[0008] Next, transcriptomic data from breast cancer cells (BT474, EFM192, and SKBR3) treated with the HER2 inhibitor lapatinib was evaluated, revealing upregulation of ALPG (ALPPL2) in lapatinib-treated cells (FIG.36A). Transcriptomic data showed a significant increase in ALPG (ALPPL2) in stomach cancer N87 cells treated with the HER2 inhibitor lapatinib for 7 days (FIG.36B). A significant increase in ALPG (ALPPL2) in bladder cancer RT112 cells treated with the FGFR inhibitor infigratinib (BGJ398) for 7 days (FIG.37A). Moreover, ALPG (ALPPL2) was upregulated in H358 treated with KRAS inhibitors ARS1323 and AZD4785 and the MEK inhibitor selumetinib (FIG.38A), as well as in HCC827 and PC9 cells treated with osimertinib and the MEK inhibitor trametinib (FIG. 39A). In HCC827 cells with acquired resistance to osimertinib (HCC827 OR2) through MET amplification (FIG.40A), upregulation of ALPP / ALPPL2 after treatment with the MET inhibitors tepotinib and capmatinib was observed (FIG.40B).

[0009] It was next assessed whether ALPP / ALPG expression can be targeted in NSCLC cells with acquired resistance to EGFR TKIs by bispecific T cell engager (BiTE). An exemplary structure of ALPP / ALPG-targeting bispecific T cell engager (BiTE) and a proposed mechanism of action is shown in FIG.41A. BiTE revealed that ALPP / ALPG- targeting M25-BiTE was able to specifically kill H1650 cells when co-cultured with human peripheral blood mononuclear cells (PBMCs) at an effector:target (E:T) ratio of 10:1 for 72 hours (FIG.41B). ALPP / ALPG-targeting M25-BiTE also specifically killed H1975 OR5 and OR16 cells, and HCC4006 OR2 and OR7 cells, when co-cultured with human PBMCs at an E:T ratio of 10:1 for 72 hours (FIG.41C and FIG.41D, respectively). It was also shown that ALPP / ALPG-targeting M25-BiTE specifically killed LC2 / AD selpercatinib-resistant cells when co-cultured with human PBMCs at an E:T ratio of 10:1 for 72 hours (FIG.41E). Sequence information for the ALPP / ALPPL2-BiTE construct used herein is provided in Table 9 below. Table 9 – Sequence Information for ALPP / ALPPL2-BiTe: M25-BiTE AA Sequence Note SEQ OAttorney Docket No. MDA0083-401-PC KEGDSSRWSYDLWGRGTLVTVSSGGGGSGGGG SGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVAttorney Docket No. MDA0083-401-PC QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNY 27 PNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLvia a number of targeting molecules or methods described herein, on tumor cells acquiring resistance to targeted agents and tumor cells with acquired resistance to targeted agents.

[0011] The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to in this specification and / or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

[0012] These and other changes can be made to the embodiments in light of the above-detailed description. In general, 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 should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

Attorney Docket No. MDA0083-401-PC CLAIMS What is claimed is:

1. A method for treating treatment-resistant non-small-cell lung cancer (NSCLC) in a patient in need thereof comprising: administering a chimeric antigen receptor (CAR) cell therapy targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, an AKT mutation, an ERK mutation, a CDK4 / 6 mutation, a BRAF mutation, and an FGFR mutation.

2. A method for treating treatment-resistant non-small-cell lung cancer (NSCLC) in a patient in need thereof comprising: identifying a genetic mutation in a NSCLC cell from a biological sample obtained from the patient; detecting and / or quantifying placental alkaline phosphatase (ALPP) and / or ALPPL2 cell surface expression in the NSCLC cell; administering a chimeric antigen receptor (CAR) cell therapy targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting the genetic mutation; wherein the genetic mutation is a mutation selected from an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, an AKT mutation, an ERK mutation, a CDK4 / 6 mutation, a BRAF mutation, and an FGFR mutation.

3. A method for treating drug-tolerant persister non-small-cell lung cancer (NSCLC) cells or drug-resistant NSCLC cells in a patient in need thereof comprising: identifying a genetic mutation in a NSCLC cell from a biological sample obtained from the patient; detecting and / or quantifying ALPP and / or ALPPL2 cell surface expression in the NSCLC cell; administering a chimeric antigen receptor (CAR) cell therapy targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting the genetic mutation; wherein the genetic mutation is a mutation selected from an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEKAttorney Docket No. MDA0083-401-PC mutation, an AKT mutation, an ERK mutation, a CDK4 / 6 mutation, a BRAF mutation, and an FGFR mutation.

4. A method of preventing emergence of resistance of non-small-cell lung cancer (NSCLC) to a drug therapy comprising administering a chimeric antigen receptor (CAR) cell therapy targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, an AKT mutation, an ERK mutation, a CDK4 / 6 mutation, a BRAF mutation, and an FGFR mutation.

5. A method for treating treatment-resistant non-small-cell lung cancer (NSCLC) in a patient in need thereof comprising: administering a bispecific T-cell engager (BiTE) targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, an AKT mutation, an ERK mutation, a CDK4 / 6 mutation, a BRAF mutation, and an FGFR mutation.

6. The method of any one of claims 1 to 5, wherein the non-small-cell lung cancer (NSCLC) cells comprise increased cell surface expression of ALPP and / or ALPPL2 relative to healthy lung cells.

7. The method of any one of claims 1 to 6, wherein the EGFR mutation comprises an L858R point mutation, an exon 19 deletion, a T790M mutation, a C797S mutation, or an exon 20 insertion mutation.

8. The method of any one of claims 1 to 6, wherein the KRAS mutation comprises a mutation in codon 12 or codon 13, a G12C mutation, a G12V mutation, a G12D mutation, or a G13D mutation.

9. The method of any one of claims 1 to 6, wherein the RET mutation comprises a RET fusion or a RET rearrangement.Attorney Docket No. MDA0083-401-PC 10. The method of claim 9, wherein the RET fusion is a CCDC6-RET fusion or a KIF5B- RET fusion.

11. The method of any one of claims 1 to 6, wherein the ALK mutation comprises an ALK fusion.

12. The method of claim 11, wherein the ALK fusion comprises an EML4-ALK gene fusion.

13. The method of any one of claims 1 to 6, wherein the HER2 mutation comprises an exon 20 YVMA insertion, an exon 20 GSP insertion, an exon 20 VC insertion, a tyrosine kinase domain mutation, or a V659E mutation.

14. The method of any one of claims 1 to 6, wherein the MET mutation comprises a MET amplification or an exon 14 skipping mutation.

15. The method of any one of claims 1 to 6, wherein the MEK mutation comprises a mutation that involves the allosteric drug binding pocket or α-helix C, a mutation in a MEK1 codon located within or abutting the N-terminal negative regulatory helix (helix A), a MEK1(P124L) mutation, a MEK1(Q56P) mutation, or any mutation in the MEK gene that results in or confers resistance to a MEK inhibitor.

16. The method of any one of claims 1 to 6, wherein the BRAF mutation is BRAF V600E.

17. The method of any one of claims 1 to 6, wherein the FGFR mutation comprises a gene fusion, an amplification, or a point mutation involving one or more of FGFR1, FGFR2, FGFR3, and FGFR4.

18. The method of claim 17, wherein the FGFR fusion comprises an FGFR2-ERC1 fusion, an FGFR3-TACC3 fusion, an FGFR2-INA fusion, a FGFR4-RAPGEFL1 fusion, and a fusion of the FGFR1 and SLC20A25’-untranslated regions.Attorney Docket No. MDA0083-401-PC 19. The method of any one of claims 1 to 18, wherein: the non-small-cell lung cancer having an EGFR mutation is resistant to an inhibitor targeting EGFR; or the non-small-cell lung cancer having a KRAS mutation is resistant to an inhibitor targeting KRAS; or the non-small-cell lung cancer having a RET mutation is resistant to an inhibitor targeting RET; or the non-small-cell lung cancer having an ALK mutation is resistant to an inhibitor targeting ALK; or the non-small-cell lung cancer having a HER2 mutation is resistant to an inhibitor targeting HER2; or the non-small-cell lung cancer having a MET mutation is resistant to an inhibitor targeting MET; or the non-small-cell lung cancer having a MEK mutation is resistant to an inhibitor targeting MEK; the non-small-cell lung cancer having an AKT mutation is resistant to an inhibitor targeting AKT; the non-small-cell lung cancer having an ERK mutation is resistant to an inhibitor targeting ERK; the non-small-cell lung cancer having a CDK4 / 6 mutation is resistant to an inhibitor targeting CDK4 / 6; the non-small-cell lung cancer having a BRAF mutation is resistant to an inhibitor targeting BRAF; or the non-small-cell lung cancer having a FGFR mutation is resistant to an inhibitor targeting FGFR.

20. The method of any one of claims 1 to 19, wherein the treatment-resistant non-small- cell lung cancer (NSCLC) cell is a drug-tolerant persister cell (DTPC) or a drug-resistant cell (DRC).

21. The method of any one of claims 1 to 20, wherein the administration of the chimeric antigen receptor (CAR) cell therapy targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or the therapy targeting the genetic mutation prevents the drug-tolerant persister cell (DTPC) from developing into a drug-resistant cell (DRC).Attorney Docket No. MDA0083-401-PC 22. The method of any one of claims 1 to 21, wherein the method treats drug-tolerant persister cells (DTPCs) and drug-resistant cells (DRCs) to prevent the emergence of resistance to EGFR tyrosine kinase inhibitors, KRAS inhibitors, RET inhibitors, ALK inhibitors, HER2 inhibitors, MET inhibitors, MEK inhibitors, AKT inhibitors, ERK inhibitors, CDK4 / 6 inhibitors, BRAF inhibitors, or FGFR inhibitors.

23. The method of any one of claims 1 to 22, wherein detecting and / or quantifying placental alkaline phosphatase (ALPP) and / or ALPPL2 cell surface expression in the NSCLC cell comprises histological analysis, immunohistochemical (IHC) staining for ALPP protein, a blood-based test, a tissue-based test, or imaging techniques.

24. The method of claim 23, wherein the tissue-based test comprises a tissue biopsy, flow cytometry, immunohistochemistry (IHC), western blot (WB), polymerase chain reaction (PCR), or immunofluorescence (IF).

25. The method of claim 23, wherein the tissue-based test comprises a Mammaprint + Blueprint® test or an Oncotype DX® test.

26. The method of claim 23, wherein the blood-based test comprises Galleri®, circulating tumor cell (CTC) test, a complete blood count (CBC), or a test or assay for measuring circulating proteins, autoantibodies, cell-free circulating DNA, or extracellular vesicle- derived proteins.

27. The method of claim 21, wherein the tissue biopsy is analyzed by hematoxylin and eosin (H&E) staining and / or microscopy.

28. The method of any one of claims 1 to 27, wherein the chimeric antigen receptor (CAR) cell therapy targeting ALPP and / or ALPPL2 in the NSCLC cell comprises a CAR-T cell, a CAR-NK cell, or a combination thereof.

29. The method of any one of claims 1 to 28, wherein the therapy targeting the genetic mutation comprises one or more targeted inhibitor and / or one or more immunotherapy agent.Attorney Docket No. MDA0083-401-PC 30. The method of claim 29, wherein the one or more targeted inhibitor comprises one or more of a small molecule drug, a tyrosine kinase inhibitor (TKI), an angiogenesis inhibitor, a monoclonal antibody, a proteosome inhibitor, and / or a signal transduction inhibitor.

31. The method of claim 28, wherein the CAR-T cell or CAR-NK cell comprises a chimeric antigen receptor (CAR) that is specific for ALPP.

32. The method of claim 31, wherein the CAR specific for ALPP comprises a sequence chosen from SEQ ID NOs:1-18.

33. The method of any one of claims 1 to 32, wherein the therapy chimeric antigen receptor (CAR) targeting ALPP and / or ALPPL2 is administered in combination with the one or more targeted inhibitors and / or the one or more immunotherapy agents.

34. The method of claim 33, wherein the one or more targeted inhibitors are chosen from an EGFR inhibitor, a KRAS inhibitor, a RET inhibitor, an ALK inhibitor, a HER2 inhibitor, a MET inhibitor, a MEK inhibitor, an AKT inhibitor, an ERK inhibitor, a CDK4 / 6 inhibitor, a BRAF inhibitor, and an FGFR inhibitor.

35. The method of claim 34, wherein the EGFR inhibitor is selected from cetuximab, osimertinib, mobocertinib, amivantamab, CLN081, and DZD9008.

36. The method of claim 34, wherein the KRAS inhibitor is selected from adagrasib, sotorasib, ARS1323, MRTX849, MRTX1257, MRTX1133, and AZD4785.

37. The method of claim 34, wherein the RET inhibitor is selected from selpercatinib and pralsetinib.

38. The method of claim 34, wherein the ALK inhibitor is selected from crizotinib, alectinib, brigatinib, and lorlatinib.

39. The method of claim 34, wherein the HER2 inhibitor is selected from lapatinib, BI1810631, and trastuzumab-deruxtecan.Attorney Docket No. MDA0083-401-PC 40. The method of claim 34, wherein the MET inhibitor is selected from tepotinib and capmatinib.

41. The method of claim 34, wherein the MEK inhibitor is selected from binimetinib (MEK162), Cotellic, Koselugo, Mekinist, Mektovi, cobimetinib or XL518, selumetinib, trametinib (GSK1120212), PD-325901, CI-1040, PD035901, and TAK-733.

42. The method of claim 34, wherein the FGFR inhibitor is selected from Balversa, anlotinib, erdafitinib, infigratinib, pemazyre, and / or pemigatinib.

43. The method of claim 34, wherein the AKT inhibitor is ipatasertib.

44. The method of claim 34, wherein the ERK inhibitor is selected from ulixertinib and SCH772984.

45. The method of claim 34, wherein the CDK4 / 6 inhibitor is palbociclib.

46. The method of claim 34, wherein the BRAF inhibitor is selected from dabrafenib, vemurafenib, and encorafenib.

47. The method of any one of claim 1 to 46, wherein the one or more immunotherapy agents comprises nivolumab, ipilumumab, pembrolizumab, cemiplimab, durvalumab, tremilumumab, and / or atezolizumab.

48. The method of any one of claims 1 to 47, wherein the chimeric antigen receptor (CAR) cell therapy targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or a therapy targeting a genetic mutation selected from an EGFR mutation, a KRAS mutation, a RET mutation, an ALK mutation, a HER2 mutation, a MET mutation, a MEK mutation, an AKT mutation, an ERK mutation, a CDK4 / 6 mutation, a BRAF mutation, and an FGFR inhibitor are administered to the patient in combination with a radionuclide.Attorney Docket No. MDA0083-401-PC 49. The method of claim 48, wherein the radionuclide comprises an alpha particle selected from Polonium-210, Bismuth-213, and Uranium-238.

50. The method of claim 48, wherein the radionuclide comprises a beta particle selected from Strontium-90, Thallium-201, Carbon-14, and Tritium.

51. The method of claim 48, wherein the radionuclide comprises a gamma particle selected from Barium-133, Cadmium-109, Cobalt-57, Cobalt-60, Europium-152, Manganese- 54, Sodium-22, Zinc-65, and Technetium-99m.

52. The method of claim 48, wherein the radionuclide comprises a combination of alpha, beta, and / or gamma particles, selected from Cesium-137 and Americum-241.

53. The method of any of claims 1 to 52, wherein the chimeric antigen receptor (CAR) therapy targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or the therapy targeting the genetic mutation are administered simultaneously.

54. The method of any of claims 1 to 53, wherein the therapy targeting ALPP and / or ALPPL2 in the NSCLC cell, and / or the therapy targeting the genetic mutation are administered sequentially.

55. The method of claim 5, wherein the bispecific T-cell engager comprises a sequence set forth in SEQ ID NOs:23-27.

56. A method for treating non-small-cell lung cancer (NSCLC) that is resistant to a targeted inhibitor in a patient in need thereof comprising: administering a CAR-T cell targeting ALPP and / or ALPPL2 on the surface of the NSCLC cells; wherein the NSCLC cells exhibit increased expression of ALPP and / or ALPPL2 relative to a healthy lung cell; wherein the CAR-T cell targeting ALPP and / or ALPPL2 comprises a single-chain variable-fragment (scFv) derived from an ALPP-specific antibody; wherein the NSCLC cells comprise:Attorney Docket No. MDA0083-401-PC • a mutation in the EGFR gene resulting in resistance to EGFR tyrosine kinase inhibitor osimertinib; or • a mutation in the KRAS gene resulting in resistance to KRAS inhibitors adagrasib and / or sotorasib; or • a mutation in the RET gene resulting in resistance to RET inhibitor selpercatinib; or • a mutation in the ALK gene resulting in resistance to ALK inhibitors alectinib and / or lorlatinib; or • a mutation in the HER2 gene resulting in resistance to HER2 inhibitor lapatinib; or • a mutation in the MET gene resulting in resistance to MET inhibitors tepotinib and / or capmatinib; • a mutation in the MEK gene resulting in resistance to MEK inhibitors binimetinib (MEK162), Cotellic, Koselugo, Mekinist, Mektovi, cobimetinib or XL518, selumetinib, trametinib (GSK1120212), PD- 325901, CI-1040, PD035901, and / or TAK-733; • a mutation in the AKT gene resulting in resistance to AKT inhibitor ipatasertib; • a mutation in the ERK gene resulting in resistance to ERK inhibitor ulixertinib; • a mutation in the CDK4 / 6 gene resulting in resistance to CDK4 / 6 inhibitor palbociclib; • a mutation in the BRAF gene resulting in resistance to BRAF inhibitors dabrafenib, vemurafenib, and / or encorafenib; • a mutation in the FGFR gene resulting in resistance to FGFR inhibitors Balversa, anlotinib, erdafitinib, infigratinib, pemazyre, and / or pemigatinib; wherein the CAR-T cell specifically binds to and eliminates the NSCLC cells having increased ALPP and / or ALPPL2; and wherein the CAR-T cell comprises a sequence set forth in SEQ ID NOs:1-18.

57. The method of claim 56, wherein the CAR-T cell comprises a sequence chosen from SEQ ID NOs:1, 10, 13, 15, 16, and / or 18.Attorney Docket No. MDA0083-401-PC 58. The method of any of claims 1 to 57, wherein the non-small-cell lung cancer (NSCLC) is mesenchymal NSCLC.

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