Combination therapy comprising bispecific antibodies comprising nrp1 binding domains

Abstract

A method of enhancing the therapeutic effect of a therapeutic agent in a subject suffering from cancer resistant to the therapeutic agent, the method comprising administering to the subject (i) a therapeutic agent and (ii) a target protein degrading agent that synergistically increases the therapeutic activity of the therapeutic agent by reversing or reducing the resistance of cancer to the therapeutic agent; and thereby enhancing the therapeutic effect of the therapeutic agent in the subject.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 423,456, filed on November 7, 2022, the entire contents of which are incorporated herein by reference. Background Art

[0003] Targeted therapies have advanced rapidly and initially demonstrated benefit in patients with certain cancer-causing mutations. However, despite initial clinical responses, long-term efficacy is currently limited due to acquired resistance to targeted therapies, which hinders their effectiveness. Effective treatments are needed. Summary of the Invention

[0004] The present disclosure relates to cancer therapy and preventing or reducing drug resistance in cancer. Specifically, the present disclosure relates to combination therapies using targeted protein degraders and one or more therapeutic agents. The present disclosure relates to a method for enhancing the therapeutic effect of a therapeutic agent in a subject suffering from a cancer resistant to the therapeutic agent, wherein the therapeutic agent is administered in combination with a targeted protein degrader.

[0005] Thus, in one aspect, the present disclosure relates to a method of enhancing the effect of a therapeutic agent in a subject having a cancer that is resistant or refractory to the therapeutic agent, the method comprising:

[0006] administering to a subject (i) a therapeutic agent; and (ii) a target protein degrader such that the effect of the therapeutic agent is enhanced compared to administration of the therapeutic agent alone;

[0007] The target protein degrader includes a bispecific binding molecule, which comprises:

[0008] (a) a target protein binding domain that specifically binds to a target protein on a cancer cell in a subject; and

[0009] (b) An NRP1 binding domain that binds to neuropilin-1 (NRP1), comprising an antibody or an NRP-1 binding fragment thereof.

[0010] In one embodiment, the target protein is a receptor tyrosine kinase (RTK). In other embodiments, the receptor tyrosine kinase is selected from epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), Met receptor tyrosine kinase (MET) and vascular endothelial growth factor (VEGFR). In one embodiment, the receptor tyrosine kinase is EGFR. In one embodiment, the receptor tyrosine kinase is cMET. In one embodiment, the receptor tyrosine kinase is HER2. In one embodiment, the receptor tyrosine kinase is IGF1R.

[0011] In one embodiment, the target cell is a cancer cell, such as a cancer cell selected from the group consisting of lung cancer, breast cancer, colon and rectal cancer, head and neck cancer, esophageal and gastric cancer, liver cancer, glioblastoma, prostate cancer, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, and pancreatic cancer. In one embodiment, the cancer cell is a non-small cell lung cancer (NSCLC) cell.

[0012] In one embodiment, the target protein binding domain and the NRP1 binding domain are each independently selected from IgG, half antibody, single domain antibody, nanobody, Fab, monospecific Fab2, Fc, scFv, minibody, IgNAR, V-NAR, hcIgG, VHH domain, camel antibody and antibody-polypeptide conjugate (peptibody).

[0013] In one embodiment, the NRPl binding domain comprises:

[0014] (i) an antibody heavy chain variable (VH) domain comprising CDR1, CDR2, and CDR3 regions (HCDR1, HCDR2, and HCDR3, respectively), wherein HCDR1 consists of the sequence set forth in SEQ ID NO: 79, HCDR2 consists of the sequence set forth in SEQ ID NO: 80, and HCDR3 consists of the sequence set forth in any one of SEQ ID NOs: 81-84; and

[0015] (ii) an antibody light chain variable (VL) domain comprising CDR1, CDR2, and CDR3 regions (LCDR1, LCDR2, and LCDR3, respectively), wherein LCDR1 consists of the sequence set forth in any one of SEQ ID NOs: 85-87, LCDR2 consists of the sequence set forth in SEQ ID NO: 88, and LCDR3 consists of the sequence set forth in SEQ ID NO: 89.

[0016] In one embodiment,

[0017] (i) HCDR1 consists of the sequence shown in SEQ ID NO: 79, HCDR2 consists of the sequence shown in SEQ ID NO: 80, and HCDR3 consists of the sequence shown in any one of SEQ ID NO: 84; and

[0018] (ii) LCDR1 consists of the sequence shown in SEQ ID NO: 85, LCDR2 consists of the sequence shown in SEQ ID NO: 88, and LCDR3 consists of the sequence shown in SEQ ID NO: 89.

[0019] In one embodiment, the enhanced effect comprises increased tumor growth inhibition. In one embodiment, the enhanced effect comprises increased median survival time.

[0020] In one embodiment, the therapeutic agent targets the EGFR pathway, non-limiting examples of which are disclosed herein. In one embodiment, the therapeutic agent targeting the EGFR pathway is osimertinib.

[0021] In one embodiment, the therapeutic agent targets the cMET pathway, non-limiting examples of which are disclosed herein. In one embodiment, the therapeutic agent targeting the cMET pathway is crizotinib.

[0022] In one embodiment, the therapeutic agent targets a KRAS protein (e.g., a mutant KRAS protein), non-limiting examples of which are disclosed herein. In one embodiment, the therapeutic agent targeting a KRAS protein is sotolacib.

[0023] In other embodiments, the therapeutic agent targets a protein selected from, for example, HER2, IGF1R, ALK, Braf, VEGF, and PDGF.

[0024] In one embodiment, the method further comprises administering to the subject a second therapeutic agent, wherein the protein target degrader enhances the effect of the second therapeutic agent compared to administration of the second therapeutic agent alone. In one embodiment, the therapeutic agent targets EGFR and the second therapeutic agent targets cMET, for example, in one embodiment, the therapeutic agent is osimertinib and the second therapeutic agent is sotolacib.

[0025] In another aspect, the present disclosure relates to a method of enhancing the effect of a receptor tyrosine kinase (RTK) inhibitor in a subject having a cancer that is resistant or refractory to the RTK inhibitor, the method comprising:

[0026] administering to a subject (i) an RTK inhibitor; and (ii) a target protein degrader such that the effect of the RTK inhibitor is enhanced compared to administration of the RTK inhibitor alone;

[0027] The target protein degrader includes a bispecific binding molecule, which comprises:

[0028] (a) a target protein binding domain that specifically binds to an RTK on a cancer cell in a subject; and

[0029] (b) An NRP1 binding domain that binds to neuropilin-1 (NRP1), comprising an antibody or an NRP-1 binding fragment thereof.

[0030] In another aspect, the present disclosure relates to a method of enhancing the effect of an epidermal growth factor receptor (EGFR) inhibitor in a subject having a cancer that is resistant or refractory to the EGFR inhibitor, the method comprising:

[0031] administering to a subject (i) an EGFR inhibitor; and (ii) a target protein degrader, such that the effect of the EGFR inhibitor is enhanced compared to administration of the EGFR inhibitor alone;

[0032] The target protein degrader includes a bispecific binding molecule, which comprises:

[0033] (a) a target protein binding domain that specifically binds to EGFR on a cancer cell in a subject; and

[0034] (b) An NRP1 binding domain that binds to neuropilin-1 (NRP1), comprising an antibody or an NRP-1 binding fragment thereof.

[0035] The present disclosure provides methods for enhancing the therapeutic effect of a therapeutic agent in a subject having a cancer that is resistant to the therapeutic agent: (i) administering a target protein degrader and a therapeutic agent to the subject, which synergistically increases the therapeutic activity of the therapeutic agent by reversing or reducing the resistance of the cancer to the cancer therapeutic agent in the subject.

[0036] In some embodiments, the cancer is resistant to chemotherapeutic agents.

[0037] The present disclosure provides a method for sensitizing a subject's cancer that is resistant to a therapeutic agent to a therapeutic agent. The method comprises administering to the subject a target protein degrading agent that degrades a target protein, thereby inducing tumor regression in the subject.

[0038] In some embodiments, the method includes a pharmaceutical composition comprising: a bispecific binding molecule that degrades a target protein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the bispecific binding molecule and a pharmaceutically acceptable carrier or diluent.

[0039] In some embodiments, the pharmaceutical is provided as a kit, and the kit further comprises a package insert containing instructions for use of the pharmaceutical composition comprising a therapeutically effective amount of the bispecific binding molecule and a pharmaceutically acceptable carrier or diluent.

[0040] In some embodiments, the bispecific binding molecule comprises an EGFR degrader.In some embodiments, the bispecific binding molecule comprises a first polypeptide having the sequence of SEQ ID NO: 11 and a second polypeptide having the sequence of SEQ ID NO: 12.

[0041] In some embodiments, cancer includes lung cancer, breast cancer, colon and rectal cancer, head and neck cancer, esophageal and gastric cancer, liver cancer, glioblastoma, prostate cancer, cervical cancer, ovarian cancer, bladder cancer, kidney cancer and pancreatic cancer. In some embodiments, the cancer is non-small cell lung cancer (NSCLC).

[0042] In some embodiments, therapeutic agents include receptor tyrosine kinases, including epidermal growth factor receptor (EGFR) inhibitors, ERBB inhibitors, c-MET inhibitors, fibroblast growth factor receptor (FGFR) and platelet-derived growth factor receptor (PDGFR) inhibitors, CSF1R inhibitors, cKIT inhibitors, FTL3 inhibitors, and receptor vascular endothelial growth factor (VEGFR) inhibitors, TGFβ1R and TGFβ2R inhibitors, integrin inhibitors, and IGF1R and IR inhibitors.

[0043] In some embodiments, the EGFR inhibitor is selected from lazertinib, osimertinib (AZD9291), WZ4002, cyasterone, erlotinib (OSI-774) hydrochloride, gefitinib (ZD1839), lapatinib (GW-572016) ditosylate, afatinib (BIBW2992), saracatinib (AZD0530), vandetanib (ZD6474), neratinib (HKI-272), canertinib (CI-1033), lapatinib (GW-572016), AG-490 (tyrosine phosphorylation inhibitor B42), CP-724714, Dacomitinib (PF-00299804), sabutinib (AZD8931), CUDC-101, AG-1478 (tyrosine phosphorylation inhibitor AG-1478), PD153035 hydrochloride, pelitinib (EKB-569), AC480 (BMS-599626), AEE788 (NVP-AEE788), AP26113-analog (ALK-IN-1), OSI-420, WZ3146, HER2-inhibitor-1, WZ8040, alitinib tosylate, rociletinib (CO-1686), genistein (NPI 031L), valitinib, TQB33804 (EGFR-IN-7), icotinib (BPI-2009H), TAK-285, daphnetin, tyrosine phosphorylation inhibitor 9, AG-18, AG 555, AZ5104, CL-387785 (EKI-785), tyrosine phosphorylation inhibitor AG-258, AG-556, tucatinib, erlotinib (OSI-774), gefitinib-based PROTAC 3, zolitinib (AZD 3759), ErbB2, AV-412 free base, AST-1306, JND3229, BI-4020, cilitinib (HMPL-309), BDTX-189, rifirafenib (BGB-283), pyrotinib (SHR-1258), O-desmethyl gefitinib, epatinib hydrochloride, SU5214, avitinib (AC0010), AG 494 and poziotinib (HM781-36B). In certain embodiments, the EGFR inhibitor is N-(2-{2-dimethylaminoethyl-methylamino}-4-methoxy-5-{[4-(1-methylindol-3-yl)pyridin-2-yl]amino}phenyl)prop-2-enamide methanesulfonate (osimertinib).

[0044] In some embodiments, the therapeutic agent comprises an ERBB inhibitor. In some embodiments, the ERBB inhibitor is selected from tucatinib, HER2 inhibitor-1, afatinib (BIBW2992), neratinib (HKI-272), CP-724714, muritinib (TAK 165), AC480 (BMS-599626), AEE778, TAK-285, tyrosine phosphorylation inhibitor AG 879, tyrosine phosphorylation inhibitor AG-528, SU5204, poziotinib (HM781-36B), TAS0728, BDTX-189, pyrotinib and epatitinib hydrochloride.

[0045] In some embodiments, the therapeutic agent comprises a cMET inhibitor. In some embodiments, the cMET inhibitor is selected from crizotinib, cabozantinib, foretinib, PHA-665752, SU11274, SGX-523, BMS-777607, tivatinib, JNJ-38877605, PF-04217903, amvatinib (MP-470), MGCD-265 analogs, capmatinib, BMS-754807, BMS-794833, AMG-208, MK-2461, govatinib, AMG-458, NVP-BVU972, AMG 337, meritinib, JNJ-38877618, crizotinib hydrochloride, ningetinib, AMG-1, UNC2025, Pamufetinib, atelatinib, NPS-1304, and savotinib.

[0046] In some embodiments, the therapeutic agent includes a PDGFR inhibitor and an FGFR inhibitor. In some embodiments, the PDGFR inhibitor and the FGFR inhibitor are selected from ponatinib (AP24534), infigratinib (BGJ398), nintedanib (BIBF 1120), pazopanib hydrochloride (GW786034HC1), pazopanib, AZD4547, tyrosine phosphorylation inhibitor AG1296, SSR128129E, LY2874455, derazentinib (ARQ-087), SU5402, ODM-203, pemigatinib (INCB054828), derazentinib (E3810) hydrochloride, ferulic acid, masitinib mesylate, fisorinib (BLU-554), PRN1371, ON123300, FIIN-3, robitinib (FGF401), fobatinib (TAS-120), FIIN-2, zoligratinib (Debio-1347), nintedanib essylate, BLU9931, surufatinib, and combinations thereof.

[0047] In some embodiments, the therapeutic agent comprises a CSF1R inhibitor. In some embodiments, the CSF1R inhibitor is selected from CSF1R-IN-1, Ki20227, pazopanib, elzovantinib (TPX-0022), ARRY-382, PRN1371, ENMD-2076, PF-477736, surufatinib, and combinations thereof.

[0048] In some embodiments, the therapeutic agent comprises a cKIT inhibitor. In some embodiments, the cKIT inhibitor is selected from dasatinib (BMS-354825), sorafenib (BAY 43-9006) mesylate, imatinib (STI571) mesylate, sunitinib (SU11248) malate, ponatinib (AP24534), axitinib (AG 013736), imatinib (STI571), nintedanib (BIBF 1120), regorafenib (BAY 73-4506), pazopanib hydrochloride (GW786034HC1), linifanib (ABT-869), clelanib (CP-868596), masitinib (AB1010), amovatinib (MP-470), olectinib (SU6668), CP-673451, telatinib, PP121, pazopanib, tyrosine phosphorylation inhibitor AG 1296, tyrosine phosphorylation inhibitor 9, SU14813, regorafenib hydrochloride, tyrosine phosphorylation inhibitor AG1433, sunitinib (SU11248), ripretinib (DCC-2618), masitinib mesylate, AZD3229, ON123300, afatinib (BLU-285), serlatinib (GB002), AZD2932, JNJ-10198409, nintedanib essylate, flumatinib (HH-GV-678), regorafenib (BAY-734506) monohydrate, and combinations thereof.

[0049] In some embodiments, the therapeutic agent comprises a FLT3 inhibitor. In some embodiments, the FLT3 inhibitor is selected from pacutinib (SB1518), TCS 359, linifanib (ABT-869), zotirapecxib, cediranib (AZD2171), dovitinib (TKI258) lactate, UNC2025 HCl, SU5614, FLT3-IN-2, FLT3-IN-4, FF-10101, meritinib (LY2801653), emavusertib (CA-4948), tandutinib (MLN518), R406 (free base), 5'-fluorodiisobutylketone oxime, tauzetib, quizartinib (AC220), R406, AST-487 (NVP-AST487), sorafenib (BAY 43-9006) mesylate, BMS-794833, sorafenib (BAY 43-9006), 4SC-203, dovitinib (TKI-258), isoguanosine, TAK-659, ATH686, SGI-1776 free base, MK-2461, fostamatinib (R788) disodium, MRX-2843, brigatinib (AP26113), GW2580, rebastinib (DCC-2036), BMS-754807, UNC2025, fidartinib (TG101348), FLT3-IN-3, G-749, cleralinib (CP-868596), BPR1K871, PHA-680632, entuximab (GS-9973), HPK1-IN-2, SP600125, SKLB477 1(FLT3-IN-1), KW-2449, gilteritinib (ASP2215), CCT241736, PRT062607(P505-15)HC1, ENMD-2076, FN-1501, ceritinib (LDK378), cimisatinib (CX-4945), AZD2932, fostamatinib (R788), tivozanib (AV-951), PF-477736, BPR1J-097, pegidatinib (PLX3397), Go6976, HM43239, OSI-930, TG101209, PLX5622, amvatinib (MP-470), GNF-2, midostaurin (PKC412), AMG 925 and ENMD-2076 L-(+)-tartaric acid.

[0050] In some embodiments, the therapeutic agent comprises a VEGFR1, VEGFR2, VEGFR3, and VEGFR4 inhibitor. In some embodiments, the VEGFR1, VEGFR2, VEGFR3, or VEGFR4 inhibitor is selected from sorafenib (BAY 43-9006) mesylate, sunitinib (SU11248) malate, lenalidomide (CC-5013), cabozantinib (BMS-907351), ponatinib (AP24534), axitinib (AG 013736), forlatinib (GSK1363089), vandetanib (ZD6474), nintedanib (BIBF 1120), regora ... 73-4506), pazopanib hydrochloride (GW786034HC1), cediranib (AZD2171), PD173074, dovitinib (TKI-258), linifanib (ABT-869), vatalanib (PTK787) 2HC1, RAF265 (CHIR-265), tivozanib (AV-951), motesanib diphosphate (AMG-706), lenvatinib (E7080), brivanib (BMS-540215), MGCD-265 analog, AEE788 (NVP-AEE788), ENMD-2076, OSI-930, CYC116, Ki8751, telatinib, PP121, pazopanib, KRN 633, SAR131675, BMS-794833, apatinib (YN968D1) mesylate, sorafenib (BAY 43-9006), cabozantinib malate, brivanib alanine salt (BMS-582664), govatinib (E7050), semaxanib (SU5416), ZM 306416, ZM 323881HC1, ENMD-2076L-(+)-tartrate, LY2874455, BAW2881 (NVP-BAW2881), WHI-P180, SU14813, ZD-4190, SU1498, SU5402, PDGFR inhibitor 1, Ki20227, dovitinib (TKI258) lactate, tocinib phosphate, cedirimib Nibuvir maleate, apatinib, BFH772, lenvatinib (E7080) mesylate, SU5614, regorafenib hydrochloride, SU5204, SU5208, fruquintinib (HMPL-013), hVEGF-IN-1, ODM-203, erdafitinib (JNJ-42756493), tyrosine phosphorylation inhibitor AG1433, MAZ51, SKLB610, sunitinib (SU11248), 4SC-203, sitritinib (MGCD516), R1530, donafenib (sorafenib D3), envodostat (PTC299), AG-13958, SKLB1002, motesanib (AMG-706), 4,4'-bis(4-aminophenoxy)biphenyl, deritinib (E3810) hydrochloride, ogifanil, sioroni, ningetinib, X-82 (voronib), pamufetinib (TAS-1 15), Cassia seed extract, CS-2660 (JNJ-38158471), WAY-340935, (20R)-protopanaxadiol, litchi seed extract, atelatinib, vitamin E, SU5408, AZD2932, anlotinib (AL3818) dihydrochloride, nintedanib ethanesulfonate, chebulic acid, SU5205, SU5214, regorafenib (BAY-734506) monohydrate, taxifolin (dihydroquercetin), surufatinib, XL092, and combinations thereof.

[0051] In some embodiments, the therapeutic agent includes a TGFβ1R and TGFβ2R inhibitor. In some embodiments, the TGFβ1R or TGFβ2R inhibitor is selected from SD-208, GW788388, A-83-01, Desistat (P144), SRI-011381, TP0427736 HCl, LY2109761, Ophiopogonin D, SB505124, SIS3 HCl, BIBF-0775, LY 3200882, LSKL, thrombospondin (TSP-1) inhibitor, Galunisertib (LY2157299), ginsenoside Rh4, LDN-193189, A77-01, LDN-193189 2HCl, Vactosertib (TEW-7197), halofuginone hydrobromide, halofuginone, sulfasalazine (NSC 667219), BMS-986260, XAV-939, LY364947, oxymatrine, pirfenidone (S-7701), hypoaconitine, SB525334, ITD-1, and bufastatin, TA-02, PD 169316, and combinations thereof.

[0052] In some embodiments, the therapeutic agent comprises an integrin inhibitor. In some embodiments, the integrin inhibitor is selected from cilengitide trifluoroacetate, RGD (Arg-Gly-Asp) peptide, A-205804, SB273005, cilengitide, RGD peptide (GRGDNP), OSU-T315, ILK-IN-3, Cyclo (-RGDfK), A286982, Cyclo (RGDyK), A286982 and combinations thereof.

[0053] In some embodiments, the therapeutic agent includes an IGF1R inhibitor and an IR inhibitor. In some embodiments, the IGF1R inhibitor or the IR inhibitor is selected from lumispivir (NVP-AUY922), linsitinib (OSI-906), NVP-AEW541, GSK1904529A, NVP-ADW742, BMS-536924, ceritinib (LDK378), AG-1024, GSK1838705A, BMS-754807, PQ 401, ZD3463, nordihydroguaiaretic acid (NDGA), NT157, insulin (human), ceritinib dihydrochloride, Rhizoma Dioscoreae extract, MID-1, brigatinib (AP26113), picropodophyllin (PPP), MSDC-0160, degludec, chromium picolinate, SBI-477, XL228 and a combination thereof.

[0054] In some embodiments, the therapeutic agent comprises a neuropilin-associated receptor inhibitor. In some embodiments, neuropilin-associated receptor inhibitors include receptor tyrosine kinases, receptor serine / threonine kinases, G protein-coupled receptors, ion channel receptors, CXCR, and immune checkpoint regulators.

[0055] In some embodiments, the therapeutic agent comprises a PARP inhibitor. In some embodiments, the PARP inhibitor is selected from PJ34 HCl, AZD2461, Olaparib (AZD2281), Veliparib (ABT-888), XAV-939, Rucaparib (AG-014699) phosphate, Iniparib (BSI-201), Talazoparib (BMN 673), AG-14361, 3-aminobenzamide, A-966492, Niraparib (MK-4827), UPF 1069, ME0328, Licorice Chalcone D, DR2313, MN 64, 4',5,7-trimethoxyflavone, Rucaparib, M2912, GeA-69, BYK204165, BGP-15 2HC1, Atamparib (RBN-2397), Vinadaparib (IDX-1197), Niraparib (MK-4827) mesylate, NU1025, Rucaparib camphorsulfonate, Berberine hydrochloride (NSC 646666), Pamiparib (BGB-290), Fluzopanib (SHR-3162), G007-LK, NVP-TNKS656, Berberine hydrochloride hydrate, HI-TOPK-032, Stenoparib (E7449), 4-Hydroxyquinazoline, NMS-P118, WIKI4, RBN012759, AZD5305, AZD-9574, RK-287107, Benzamide, JW55, Picolinamide, and combinations thereof.

[0056] In some embodiments, the therapeutic agent comprises a Raf inhibitor. In some embodiments, the Raf inhibitor is selected from vemurafenib (PLX4032), B-Raf inhibitor 1 (Compound 13) dihydrochloride, Raf inhibitor 1, Raf inhibitor 2, sorafenib (BAY 43-9006) mesylate, PLX-4720, dabrafenib (GSK2118436), regorafenib (BAY 73-4506), damanimide (BIRB 796), GDC-0879, RAF265 (CHIR-265), AZ 628, NVP-BHG712, SB590885, ZM336372, sorafenib (BAY 43-9006), GW5074, TAK-632, agrafenib (RXDX-105), conagrafenib (LGX818), BAW2881 (NVP-BAW2881), PLX8394, TBAP-001, regorafenib hydrochloride, MCP110, naborafenib (LXH254), B-Raf IN 1. Donafenib (sorafenib D3), CCT196969, RAF709, rifolafenib (BGB-283), L-779450, PLX7904, LY3009120, dabrafenib mesylate, RO5126766 (CH5126766), AZ304, Belvarafenib (HM95573), regorafenib (BAY-734506) monohydrate, tovorafenib (MLN2480), and their combinations.

[0057] In some embodiments, the therapeutic agent comprises an autophagy activator. In some embodiments, the autophagy activator is selected from the group consisting of enzalutamide (MDV3100), obaclava mesylate (GX15-070), SRT1720 HCl, fulvestrant (ICI-182780), bicalutamide (ICI-176334), resveratrol (SRT501), forskolin, rosiglitazone (BRL-49653) maleate, rosiglitazone (BRL 49653), mifepristone (RU486), purmorphamine, clemastine (HS-592) fumarate, GW4064, chloroquine diphosphate, ivermectin (MK-933), loperamide hydrochloride, melatonin (NSC 113928), methylprednisolone (NSC-19987), clonidine hydrochloride, flubendazole, fenofibrate (NSC-281319), montelukast sodium, LYN-1604, EN6, Eprenetapopt (APR-246), 3BDO, MHY1485, methylprednisolone acetate, QX77, anisomycin, β-elemene, spermidine trihydrochloride, troglitazone (CS-045), conosine, obeticholic acid, SMER28, BC1618, monomethyl fumarate, PCNA-I1, CA77.1, spermidine, xylitol, isorhynchophylline, MPP+iodide, and combinations thereof.

[0058] In some embodiments, the therapeutic agent comprises an autophagy inhibitor. In some embodiments, the autophagy inhibitor is selected from MK-2206 2HC1, bortezomib (PS-341), olaparib (AZD2281), vemurafenib (PLX4032), vorinostat (SAHA), ABT-737, Y 276322HC1, daclis (BEZ235), sorafenib (BAY 43-9006) mesylate, dasatinib (BMS-354825), rapamycin (AY-22989), crizotinib (PF-02341066), erlotinib (OSI-774) hydrochloride, everolimus (RAD001), gefitinib (ZD1839), veliparib (ABT-888), entinostat (MS-275), BI 2536, Pitilipsin (GDC-0941), Laduviglusib (CHIR-99021) HC1, LY294002, Trametinib (GSK1120212), Ruxolitinib (INCB018424), Panobinostat (LBH589), Imatinib (STI571) mesylate, KU-55933 (ATM kinase inhibitor), Alecetin (MLN8237), Afatinib (BIBW2992), U0126-EtOH, Idelalisib, Tauzacertib, AZD8055, Sacatinib (AZD0530), Paclitaxel (NSC 125973), SP600125, ponatinib (AP24534), tanespiramycin (17-AAG), YM155 (sapentronium bromide), DAPT (GSI-IX), cisplatin (NSC119875), imatinib (STI571), nilotinib (AMN-107), temsirolimus (CCI-779), PI-103, lumispivir (NVP-AUY922), vandetanib (ZD6474), gemcitabine (LY-188011) hydrochloride, moxistat (MGCD0103), regorafenib (BAY 73-4506), SRT1720 HC1, pazopanib hydrochloride (GW786034HC1), bosutinib (SKI-606), cediranib (AZD2171), belinostat (PXD101), GSK690693, SB202190 (FHPI), carfilzomib (PR-171), fulvestrant (ICI-182780), SB216763, SU11274, linifanib (ABT-869), pemetrexed (LY-231514) disodium, tocotinib (PP242), etoposide (VP-16), wortmannin (KY12420), LY2109761, daruselutib (PHA-739358), cimisatinib (CX-4945), venetoclax (ABT-199), flavopiridol (L86-8275), SGI-1776 free base, lapatinib (GW-572016), vincristine (NSC-67574) sulfate, temozolomide (CCRG81045), tacrolimus (FK506), metformin hydrochloride, fasudil (HA-1077) hydrochloride, oxaliplatin (NSC266046), rabusertib (LY2603618), 3-methyladenine (3-MA), flavopiridol (L86-8275) hydrochloride, (+)-JQ1, zoledronic acid (ZOL 446), momelotinib (CYT387), ixazomib citrate (MLN9708) analog, tamoxifen (ICI 46474) citrate, letrozole (CGS20267), topotecan (NSC609699) hydrochloride, Torin1, omelisabet (GSK2126458), Degrasyn (WP1130), 2-methoxyestradiol (2-MeOE2), azacitidine (5-azacytidine), BX-795, OSI-027, ixazomib (MLN2238), TWS119, apellisib (GDC-0980), BI-D1870, resveratrol (SRT501), dexamethasone (MK-125), cytarabine (U-19920A), simvastatin (MK 733), Vistusertib (AZD2014), CCT128930, Idarubicin hydrochloride, Gemcitabine (LY-188011), Verteporfin (CL318952), PF-4708671, Torin 2, Streptozotocin (STZ), H 89 2HC1, Brefeldin A, Geldanamycin (NSC 122750), MK-5108 (VX-689), Forskolin, Valproic acid (NSC 93819) sodium salt, pitavastatin (NK-104) calcium, doxormorphine (Compound C) dihydrochloride, lovastatin (MK-803), rosiglitazone (BRL-49653) maleate, necrostatin-1, pazopanib, nocodazole (R17934), dapoline, clofarabine, cabazitaxel (XRP6258), atorvastatin calcium, (R)-(-)-gossypol acetic acid, pifitarin-α (PFTα) hydrobromide, SN-38, GSK343, BIX 01294, nutrin-3a, tigecycline (GAR-936), STF-62247, binitinib (MEK162), itraconazole (R 51211), mircini (PHA-848125), sorafenib (BAY43-9006), 10058-F4, YM201636, C646, hydroxyurea (NSC-32065), bardoxolone methyl, sodium butyrate, PR-619, linagliptin (BI-1356), niclosamide (BAY2353), nitazoxanide (NSC697855), UNC1999, heparin sodium, Dynasore, curcumin, celastrol (NSC 70931), GSK2606414, honokiol (NSC 293100), laduviglusib (CHIR-99021), GANT61, omeprazole, amiodarone (NSC 85442) hydrochloride, dexamethasone sodium phosphate, aspirin (NSC 27223), AZD3463, GSK2656157, carbamazepine, bafilomycin Al (Baf-A1), azithromycin (CP-62993), nimodipine, IU1, sulfasalazine (NSC 667219), pifetaline-μ, PF-543 hydrochloride, tubastatin A, nitrendipine, DC661, KB-R7943 mesylate, tubastatin A TFA, PFK15, nordihydroguaiaretic acid (NDGA), nilotinib hydrochloride monohydrate, thalidomide, neferine, emetine hydrochloride, SBI-0206965, apatinib, EAD1, lanatoside C, ruxolitinib phosphate, lopimod (GW856553X), STO-609, IITZ-01, Vacuolin-1, entrectinib (RXDX-101), sunitinib (SU11248), valproic acid (VPA), URMC-099, spautin-1, erlotinib (OSI-774), pemetrexed disodium hydrate, crizotinib hydrochloride, atorvastatin, berberine hydrochloride (NSC 646666), quercetin (NSC 9221), VLX600, afatinib (BIBW2992) dimaleate, FL-411, pemetrexed, Autophinib, cryptotanshinone, nortriptyline hydrochloride, dihydroartemisinin (DHA), KN-93 phosphate, tiraginostat (CB-839), purvalanol A, brevicornulin A, chloroquine (NSC-187208), PFK158, sulfacetamide sodium hydrate, OTS964, sinomenine hydrochloride, MRT67307 HC1, Lys05, sulfacetamide sodium, resatovir (TAK-242), DMH1, hydroxychloroquine sulfate (NSC 4375), MRT68921HC1, PHY34, vinorelbine ditartrate (KW-2307), CA-5f, VPS34-IN1, DMOG, ICCB-19 hydrochloride, AS1842856, SAR405, PIK-III, ULK-101, vinblastine (NSC-49842) sulfate, SP2509, ROC-325, Codonopsis pilosula extract, ABTL-0812, ML-9HC1, AZD1208, lucanthone, leonurine, VER155008, RA-190, LY3009120, 4-phenylbutyric acid (4-PBA), NSC 185058, E260, daurisoline, doxormorphine (Compound C), tamoxifen (ICI 46474), deferoxamine mesylate (Ba 33112), pepstatin A, trametinib DMSO solvate, and concanavalin A.

[0059] In some embodiments, the therapeutic agent comprises an mTOR inhibitor. In some embodiments, the mTOR inhibitor is selected from mTOR inhibitor-1, everolimus (RAD001), KU-0063794, daclis (BEZ235), rapamycin (AY-22989), AZD8055, temsirolimus (CCI-779), PI-103, NU7441 (KU-57788), tocotinib (PP242), lidaformolimus (rapamycin, MK-86 69), sabasetib (MLN0128), voxtalisib (XL765) analogs, Torin1, omelisib (GSK2126458), OSI-027, PF-04691502, apellisib (GDC-0980), GSK1059615, gidalisib (PKI-587), WYE-354, vistusertib (AZD2014), Torin 2, WYE-125132 (WYE-132), BGT226 (NVP-BGT226) maleate, Palomid 529 (P529), PP121, WYE-687, Nitazoxanide (NSC 697855), WAY-600, ETP-46464, GDC-0349, XL388, 4EGI-1, JR-AB2-011, ferric acid, lanatoside C, compound 401, astragaloside IV, ginkgolide K, CC-115, zotarolimus (ABT-578), Paxalisib (GDC-0084), CZ415, SF2523, bimilisib (PQR309), voxtalisib (XL765), rhubarb acid, onatasertib (CC 223), 3-hydroxyanthranilic acid, Samotolisib (LY3023414), MTI-31, ABTL-0812, PQR620, MHY-1685, GNE-477, GNE-493, and combinations thereof.

[0060] In some embodiments, the therapeutic agent comprises a PI3K activator and a PI3K inhibitor. In some embodiments, the PI3K activator is selected from demethylcoclaurine, cinobufagin, resifugine, 740YP (PDGFR 740Y-P), erucic acid, picrogentosinate, YS-49 and a combination thereof. In some embodiments, the PI3K inhibitor is selected from PI-103, XL147 analogs, 3-methyladenine (3-MA), apellis (GDC-0980), TG100713, taselixis (GDC 0032), daclisi (BEZ235), pitilixis (GDC-0941), LY294002, idelalisib, buparisib (BKM120), NU7441 (KU-57788), TGX-221, IC-87114, wortmannin (KY 12420), ZSTK474, Apellisib (BYL719), AS-605240, PIK-75HC1, Regseti (ON-01910), A66, Voxtalisib (XL765) analogs, Omelisse (GSK2126458), PIK-90, AZD6482, PF-04691502, GSK1059615, Duvilisse (IPI-145), Gidalisib (PKI-587), TG100-115, AS-252424, NU7026, BGT226 (NVP-BGT226) maleate, Feminostat (CUDC-907), PIK-294, AS-604850, GSK2636771, Kupanisib (BAY 80-6946), YM201636, CH5132799, CAY10505, PIK-293, PKI-402, VS-5584 (SB2343), KU-0060648, CZC24832, demethylcoclaurine, oroxylin B, homosalate, AMG319, cinobufagin, lipobufogenin, homoplanin, GSK2292767, lanatoside C, zeaxanthin, desitatin (P144), cafestol, Paxalisib (GDC-0084), SKI-V, MTX-211, selelisib (UCB-5857), Trichosanthes peel extract, Rhizoma Dioscoreae extract, GDC-0326, 740YP (PDGFR 740Y-P), PIK-108, Pasalisib (INCB050465) hydrochloride, HS-173, SF2523, Lenilixib (CDZ 173), Lupetin, Cerelisib (TAK-117), Eganelisib (IPI-549), Quercetin (NSC9221), Bimilisib (PQR309), VPS34 inhibitor 1 (Compound 19), IHMT-PI3K6-372, Voxtalisib (XL765), Autophinib, GNE-317, (E)-Akt inhibitor-IV, Panax notoginseng saponin R1, Tenalisib (RP6530), Austrosolamine, Alizarin Violet, Cynarin, Inalisib (GDC-0077), SRX3207, α-linolenic acid, Erbulisib (TGR-1202), acalisib (GS-9820), ME-401, 3-hydroxyanthranilic acid, nano Lisebate, Samotolisib (LY3023414), VPS34-IN1, Ailanthone, Tripterygium wilfordii extract, IPI-3063, SAR405, PIK-III, IPI-3063, Pasalisib (INCB050465), Quercetin dihydrate, Pilaralisib (XL147), SPP-86, AZD8835, Trigonelline, Deguelin, Selective PI3Kδ inhibitor 1 (Compound 7n), Loureirin A, PF-4989216, AZD8186, GNE-477, GNE-493, and combinations thereof.

[0061] In some embodiments, the therapeutic agent comprises a proteasome inhibitor. In some embodiments, the proteasome inhibitor is selected from the group consisting of: salinosporamide A (NPI-0052), bortezomib (PS-341), MG132, carfilzomib (PR-171), ixazomib citrate (MLN9708), ixazomib (MLN2238), ONX-0914 (PR-957), oprozomib (ONX 0912), delanzomib (CEP-18770), tripterine (NSC 70931), epoxomicin (BU-4061T), shikonin (CI 75535), VR23, isoginkgetin, RA-190, and PI-1840.

[0062] In some embodiments, the therapeutic agent comprises a JNK inhibitor. In some embodiments, the JNK inhibitor is selected from the group consisting of JNK inhibitor VIII, JNK inhibitor IX, JNK-IN-8, BI-78D3, SP600125, damarimod (BIRB 796), metformin hydrochloride, DB07268, 3'-hydroxypterostilbene, KB-R7943 mesylate, JNK-IN-7, luurein B, astragaloside IV, cucurbitacin IIb, trans-zeatin, etamostat, IQ 3, berberine hydrochloride (NSC 646666), SU3327, bemamod (AS602801), indirubin-3'-oxime, falcarindiol, tanzisetti (CC-930), NDMC101, morulin A, C-401 hydrochloride, RPI-1, ginsenoside Re, IQ-1S, urolithin B, and combinations thereof.

[0063] In some embodiments, the therapeutic agent comprises an NF-κB inhibitor. In some embodiments, the NF-κB inhibitor is selected from the group consisting of phosphorylated IKBα (S32) rabbit recombinant monoclonal antibody, ophiopogonin D, cornus officinalis glycosides, rubiacein 1-methyl ether, SM-7368, NF-κB-IN-1, chitosan oligosaccharide, Cynanchi Atrat extract, IAXO-102, Adjudin, CBL0137, IQ 3. Erythrina pine, apigenin isoliquiritigenin, cyanidin, bortezomib (PS-341), urolithin B, sulfasalazine (NSC667219), acetylcysteine (N-acetylcysteine), erdosteine, curcumin, andrographolide, dihydroartemisinin (DHA), indole-3-methanol, magnolol, (-)-parthenolide, evodiamine, chondroitin sulfate, TPCA-1, sodium salicylate, methylthiouracil, articaine salt Salt, L-quercetin, UCB-9260, zeaxanthin, bud yellow yellow, mace lignan, gardenia glycoside, N-methyl cytisine (Caulophylline) (N-methyl cytisine), demethyl berberine, 3-hydroxyanthranilic acid, (E / Z)-IT-603, triptolide (PG490), smilax glabra sapogenin, berbamine dihydrochloride, ammonium pyrrolidine dithiocarbamate, stachydrine hydrochloride, sodium aescin, Stachydrine, tyrosol, mangiferin, baicalin, ginsenoside Re, dehydroevodiamine, (E)-cardamomine, musk ketone, guaiacol, curcumenol, schisandrin A, hederagenin, astragaloside IV, ginsenoside Rgl, ginsenoside Rbl, ginsenoside Rd, 4'-methoxyresveratrol, (+)-α-lipoic acid, sodium 4-aminosalicylate, IMM-H007, diethyl maleate, 4-hydroxychalcone, benfotiamine, Q NZ (EVP4593), 4'-hydroxychalcone, neferine, vanillic acid, hyperoside, chelidonic acid, caffeic acid ethyl ester, sulforaphane, (R)-(-)-ibuprofen, SN50, C25140, INH14, licorice chalcone D, SC75741, JSH-23, caffeic acid phenethyl ester, melinamide, APX-3330, DTP3, omasolone (RTA-408), bardoxolone methyl, shikonin (CI 75535), TAK-243 (MLN7243), CBL0137HC1, withaferin A, NIK SMI1, arobuse (GS-5829), anthatin, maslinic acid, morulin A, eleutheroside E, berbamine, chrysanthemoside, aristolochic acid A, ginsenoside Rb3, 8-O-acetyl shanzhioside methyl ester, dauricine, 2',5'-dihydroxyacetophenone, (+)-peucedanin A, homoplanin, madecassic acid, BTYNB, and combinations thereof.

[0064] In some embodiments, the therapeutic agent comprises an HSP90 inhibitor. In some embodiments, the HSP90 inhibitor is selected from Lumispir (NVP-AUY922), Tanspiramycin (17-AAG), Aspiramycin (17-DMAG) hydrochloride, Ganetespib (STA-9090), Elisemol (STA-4783), BIIB021, Tamoxifen (ICI 46474) citrate, Onaspi (AT13387), NVP-BEP800, Geldanamycin (NSC 122750), SNX-2112 (PF-04928473), PF-04929113 (SNX-5422), KW-2478, XL888, Pifitrine-μ, NMS-E973, Zelavespib (PU-H71), Teprenone, Dimethylenastron, Apolazole, KRIBB11, Tebuconazole, TRC051384, DTHIB, Methamide, KNK437, VER-49009, HA15, Pimetinib (TAS-116), CH5138303, VER-50589, YUM70, Triptolide (PG490), VER155008, JG98, Tamoxifen (ICI 46474), NPX800, HSP990 (NVP-HSP990), and combinations thereof.

[0065] In some embodiments, the therapeutic agent comprises an E3 ligase inhibitor. In some embodiments, the E3 ligase inhibitor is selected from lenalidomide (CC-5013), pomalidomide (CC-4047), thalidomide (K17), NSC 207895, TAME, PRT4165, CC-885, dCBP-1, iberdomide (CC220), BC-1215, CC-90009, avadomide (CC-122), VL285, (S,R,S)-AHPC (MDK7526), Smurfl-IN-A01, (S,R,S)-AHPC-PEG4-NH2 hydrochloride, SZL P1-41, VH298, thalidomide-OH, MuRF1-IN-1, Skp2 inhibitor Cl (SKPin C1), GMB-475, mezigmid (CC-92480), isotype PROTAC Cereblon degrader 1, THAL-SNS-032, NSC232003, Apcin, thalidomide-O-COOH (Cereblon ligand 3), and combinations thereof.

[0066] In some embodiments, the therapeutic agent comprises a KRAS inhibitor. In some embodiments, the KRAS inhibitor is selected from MRTX1133, Sotolacib (AMG510), Adalasib (MRTX849), LC-2, Deltarasin, BAY-293, ARS-1620, BI-2852, ARS-853 (ARS853), BI-3406, ASP2453, Sotolacib (AMG510) racemate, Pan-RAS-IN-1, MRTX-1257, Zoledronic acid (ZOL 446), lonafarnib (SCH66336), K-Ras (G12C) inhibitor 9, salirexetine, Alamandine, (Rac)-antineoplaston A10, K-Ras-IN-1, MCP110, 6H05, K-Ras (G12C) inhibitor 12, Kobe0065, K-Ras (G12C) inhibitor 6, BQU57, Kobe2602, NAV-2729, antineoplaston A10, fendiline hydrochloride, KRpep-2d, perillyl alcohol, RBC8, KY1220, CID-1067700, zoledronic acid monohydrate, and combinations thereof.

[0067] In some embodiments, the protein targeted and inhibited by the therapeutic agent is the same protein bound and degraded by the target protein degrader.

[0068] In certain embodiments, use of a target protein degrader and an EGFR inhibitor in the manufacture of a medicament for treating cancer in a subject when administered in combination with an EGFR inhibitor.

[0069] In some embodiments, the therapeutic agent is an EGFR inhibitor and the target protein is an EGFR degrader. In some embodiments, the EGFR inhibitor is osimertinib. In some embodiments, the cancer is NSCLC that is resistant to osimertinib.

[0070] In some embodiments, the target protein degrader is administered intraperitoneally.

[0071] In some embodiments, the method comprises administering to a patient an amount of a target protein degrader that increases the therapeutic activity of the therapeutic agent by at least 2-fold. The method of claim 1 comprises administering to a patient an amount of a target protein degrader that increases the therapeutic activity of the therapeutic agent by at least 5-fold. The method of claim 1 comprises administering to a patient an amount of a target protein degrader that increases the therapeutic activity of the therapeutic agent by at least 10-fold. The method of claim 1 comprises administering to a patient an amount of a target protein degrader that increases the therapeutic activity of the therapeutic agent by at least 20-fold.

[0072] In some embodiments, the cancer is resistant to immunotherapeutic antibodies. In certain embodiments, the immunotherapeutic antibodies include anti-EGFR monoclonal antibodies. In certain embodiments, the anti-EGFR monoclonal antibodies include ervantumab, cetuximab, dextrolimus, martin-dextrolimus, dugolubuvir, vortuximab, GC1118, imatinizumab, matuzumab, necituzumab, nimotuzumab, panitumumab, zalutumumab, and HumMR1.

[0073] In some embodiments, the subject is a human.

[0074] The present disclosure further provides various methods of administering a pharmaceutical composition comprising a bispecific antibody to a subject. In some embodiments, administration is intravenous. In some embodiments, administration is intraperitoneal, intrathecal, intraventricular, or intraparenchymal.

[0075] The following drawings are provided by way of example only and are not intended to limit the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 is a schematic diagram illustrating the acquisition of resistance following targeted therapy.

[0077] Figure 2 It was shown that existing EGFR blockers cannot achieve sustained responses due to the lack of EGFR degradation.

[0078] Figure 3 The combination of osimertinib and an EGFR degrader demonstrated enhanced anti-tumor activity in osimertinib-resistant mouse xenografts (H1975-OR). The H1975-OR mouse xenograft model was treated with either the EGFR signaling-blocking antibody panitumumab (10 mg / kg) or an EGFR degrader (13.74 mg / kg) in combination with a high dose of osimertinib (10 mg / kg). Tumor volume was quantified to determine the therapeutic effect of the EGFR degrader.

[0079] Figure 4 Co-treatment with a target protein and a target protein degrader is shown to prevent or minimize acquired resistance to target protein inhibitors in cancers that are resistant to the target protein.

[0080] Figures 5A-5B Graphs showing results from H1975-NRP1 OE xenograft studies with the indicated treatments. Figure 5A Shown are the mean tumor volumes over time. Figure 5B Displays the probability of survival over time.

[0081] Figures 6A-6BGraphs showing results from H1975-HGF xenograft studies with the indicated treatments. Figure 6A Results are shown for combination with osimertinib. Figure 6B Results are shown for combinations with osimertinib and / or crizotinib.

[0082] Figure 7 Figure 2 is a graph showing results from a mutant KRAS xenograft study using the indicated treatments. Results show mean tumor volume over time. DETAILED DESCRIPTION

[0083] Provided herein are methods and compositions for treating cancer and preventing resistance to antiproliferative drugs or enhancing their therapeutic effects in subjects. Using the H1975 xenograft mouse model resistant to osimertinib, we found that treatment with the EGFP degrader EGFP×NRP1 antibody can sensitize cancers resistant to osimertinib to continued treatment with osimertinib. EGFP degraders (EGFP×NRP1 antibodies) can be used alone or in combination with any EGFR inhibitor to prevent or minimize acquired resistance to cancer drugs. We demonstrate that combined treatment with a target protein degrader and a target protein inhibitor is an effective therapeutic approach for treating cancers that have developed or are about to develop resistance to target protein inhibitors.

[0084] 1. Definition

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Accordingly, the following terms are intended to have the following meanings:

[0086] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0087] As used herein, the terms "includes," "comprising," "having," "having," "may," "containing," and variations thereof as used herein are intended as open transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures.

[0088] As used herein, "administration" of a disclosed polypeptide encompasses delivering a polypeptide or composition of the invention as described herein, or a prodrug or other pharmaceutically acceptable derivative thereof, to a subject using any suitable formulation or route of administration (e.g., as described herein).

[0089] As used herein, the term "and / or" when used in a list of two or more items means that any one of the listed items may be taken alone or any combination of two or more of the listed items may be taken.

[0090] As used herein, "cancer" refers to a tumor or tumor caused by the abnormal and uncontrolled growth of cells. Cancer may also be referred to as a cell proliferative disease. Cancer can include different histological types, cell types, and different stages of cancer, such as primary tumors or metastatic growths. Cancer can include, for example, breast cancer, cholangiocarcinoma, colorectal cancer, endometriosis, esophageal cancer, gastric cancer, diffuse gastric cancer, pancreatic cancer, kidney cancer, soft tissue tumors, testicular cancer, heart: sarcomas (angiosarcomas, fibrosarcomas, rhabdomyosarcomas, liposarcoma), myxoma, rhabdomyomas, fibromas, lipomas, and teratomas; lung: bronchial cancer (squamous cell carcinoma, undifferentiated small cell carcinoma, undifferentiated large cell carcinoma, adenocarcinoma), alveolar (bronchiolar) cancer, bronchial adenomas, sarcomas, lymphomas, chondromatous hamartomas (chondromatous carcinomas). Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, vipoma), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyomas, hemangiomas, lipomas, neurofibromas, fibromas), large intestine (adenocarcinoma, tubular adenomas, villous adenomas, hamartomas, leiomyomas); genitourinary: kidney (adenocarcinoma, Wilm's tumor, lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, =Adenocarcinoma), prostate (adenocarcinoma, sarcoma), testicle (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatoid tumor, lipoma); liver: hepatocellular carcinoma, bile duct carcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticular cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (osteochronfroma), benign enchondroma, chondroblastoma, chondromyxofibrosarcoma, osteoid osteoma, and giant cell tumor; nervous system: skull (osteomas, hemangiomas, granulomas, xanthomas, osteodegenerative osteomas, and osteopetrosis); defornians), meninges (meningioma, meningeal sarcoma, glioma), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor [pinealoma], glioblastoma, glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal neurofibromas, meningiomas, gliomas, sarcomas);Gynecology: Uterus (endometrial cancer), cervix (cervical cancer, precancerous cervical atypical hyperplasia), ovary (ovarian cancer, ovarian cancer carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa cell tumor, sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma)), fallopian tube (carcinoma); hematologic system: blood (myeloid leukemias [acute and chronic], acute lymphocytic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndrome), Hodgkin disease, non-Hodgkin lymphoma [malignant lymphoma], CML; skin: melanoma, malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi sarcoma, nevus, dysplastic nevus, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and adrenal gland: neuroblastoma.

[0091] In some embodiments, the cancer comprises non-small cell lung cancer (NSCLC). In some embodiments, the cancer is resistant to therapy. In some embodiments, the cancer is not resistant to a therapy.

[0092] The term "carrier" refers to a vehicle used in the formulation of a composition and may be composed of a variety of excipients.

[0093] As used herein, the terms "combination" or "co-administration" are used interchangeably to refer to the use of more than one therapy (e.g., one or more prophylactic and / or therapeutic agents). The use of these terms does not limit the order in which the therapies (e.g., prophylactic and / or therapeutic agents) are administered to a subject.

[0094] As used herein, the term "excipient" refers to any pharmacologically inactive natural or synthetic component or substance, which is formulated together with (for example, accompanying) or after the active ingredient of the present invention. In some embodiments, the excipient can be any additive, adjuvant, adhesive, expander, carrier, coating agent, diluent, disintegrant, filler, glidant, lubricant, preservative, vehicle or a combination thereof, which can be used together with the recombinant polypeptide of the present invention, and or can be used to prepare the composition of the present invention. Excipients include any such substances known in the art that are nontoxic and do not interact with the other components of the composition. In some embodiments, when preparing the composition, in order to make the combination weight gain, the excipient can be formulated together with the recombinant polypeptide (therefore commonly referred to as expander, filler or diluent). In other embodiments, excipients can be used to impart enhancement to the active ingredient in the final dosage form, such as promoting absorption and / or solubility. In other embodiments, excipients can be used to provide stability or prevent contamination (for example, microbial contamination). In other embodiments, excipients can be used to impart physical properties to the composition (e.g., a composition in the physical form of dry granules or a flowable dry powder). Reference to an excipient includes both one and more than one such excipient. Suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences by E.W. Martin, the disclosure of which is incorporated herein by reference in its entirety.

[0095] The term "inhibitor" refers to a compound that inhibits or reduces the activity of a polypeptide. The inhibitor can bind to the polypeptide indirectly or directly and inhibit its activity, including binding activity or catalytic activity. For example, an inhibitor can prevent the expression of a polypeptide, or inhibit the ability of a polypeptide to mediate binding of a polypeptide to a ligand. An "allosteric inhibitor" refers to a compound that binds to a polypeptide at a secondary site that is different from the primary ligand binding site and inhibits or reduces the activity of the polypeptide. The term "inhibit" means that in the presence of an inhibitor, the activity is reduced or prevented relative to the absence of the inhibitor. The term "inhibit" refers to the reduction or downregulation of a process, or the elimination of the stimulus for a process, which results in the absence or minimization of the expression or activity of a biomolecule or polypeptide. Inhibition can be direct or indirect. Inhibition can be specific, that is, the inhibitor inhibits a certain biomolecule or polypeptide but not others.

[0096] As used herein, "EGFP × NRP1 bispecific antibody" or "anti-EGFP × NRP1 bispecific antibody" (used as a targeted protein degradation agent) is a bispecific binding molecule that comprises two different antigen-binding domains, one of which specifically binds to the antigen EGFR1 and one of which specifically binds to NRP1. Similar nomenclature is used throughout for other bispecific binding molecules that bind to targets other than EGFR, such as "HER2 × NRP1 bispecific antibody" or "cMET × NRP1 bispecific antibody" to describe bispecific binding molecules that comprise one antigen-binding domain that specifically binds to HER2 or cMET, respectively, and another antigen-binding domain that specifically binds to the antigen NRP1.

[0097] As used herein, the term "receptor tyrosine kinase" or "receptor tyrosine kinase" or "RTK" refers to a protein that acts as a receptor (ie, binds a ligand) and phosphorylates tyrosine residues.

[0098] As used herein, the term "non-receptor tyrosine kinase" or "non-receptor tyrosine kinase" or "non-RTK" refers to a protein that is not a RTK, ie, a protein that is not a receptor and / or does not phosphorylate tyrosine residues.

[0099] As used herein, "treatment" or "treating" are used interchangeably herein and refer to a method of obtaining a therapeutic benefit. Therapeutic benefit is determined by whether the tumor shrinks, remains the same size, or progression-free survival is prolonged compared to placebo. As used herein, "parenteral" includes, but is not limited to, subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques.

[0100] The term "pharmaceutically acceptable salts" is meant to include salts of the active bispecific antibodies that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the bispecific antibodies described herein.

[0101] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. "Polynucleotide" as used herein can be single-stranded or double-stranded, or can comprise portions of double-stranded and single-stranded sequences. Polynucleotides can be natural or synthetic nucleic acids, DNA, genomic DNA, cDNA, RNA or hybrids, wherein the polynucleotides can comprise a combination of deoxyribonucleotides and ribonucleotides, and a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine. Polynucleotides can be obtained by chemical synthesis or recombinant methods. The term "reduce" or other forms of the word, such as "reducing" or "reduction", generally refers to a reduction event or feature (e.g., one or more symptoms, or the combination of a protein with another protein). It should be understood that this is generally associated with some standard or expected value, in other words, it is relative, but does not always require a reference standard or relative value.

[0102] When used to refer to, for example, a cell, nucleic acid, polynucleotide, protein or vector, "recombinant" means that the cell, nucleic acid, polynucleotide, protein or vector has been modified by introducing a heterologous nucleic acid or protein or by changing a native polynucleotide or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not present in the native (non-recombinant) form of the cell, or expresses native genes that are otherwise abnormally expressed, underexpressed or not expressed at all. For example, the term "recombinant DNA molecule" as used herein refers to a DNA molecule comprising DNA segments that are linked together by molecular biology techniques. The term "recombinant protein" or "recombinant polypeptide" as used herein refers to a protein molecule expressed by a recombinant DNA molecule or a recombinant polynucleotide.

[0103] "Specific binding" generally means that an agent or polypeptide binds to a target when it binds to that target more readily than it binds to a random, unrelated target.

[0104] As used herein, " subject " can refer to any animal having cancer, for example, mammal, such as laboratory animal, farm animal, pet etc. In some embodiments, animal is primate, preferably people. As used herein, term " subject " is used interchangeably.Term " subject " refers to animal (for example, bird, such as chicken, quail or turkey, or mammal), particularly " mammal ", comprises non-primate (for example, cattle, pig, horse, sheep, rabbit, guinea pig, rat, cat, dog and mouse) and primate (for example, monkey, chimpanzee and people), and more particularly people. In one embodiment, subject is non-human animal, such as farm animal (for example, horse, cattle, pig or sheep) or pet (for example, dog, cat, guinea pig or rabbit). In a preferred embodiment, subject is " people ".

[0105] As used herein, the term "synergistic" refers to a combination of a polypeptide of the invention and another therapy (eg, a prophylactic or therapeutic agent) that is more effective than the additive effects of the individual therapies.

[0106] Unless otherwise defined, "therapeutically effective amount" or "effective dose" or "effective amount" are used interchangeably herein and refer to the dosage of an effective agent or drug over the time required to achieve the desired therapeutic effect. The effective dose can be determined by those skilled in the art and can vary according to factors such as the disease state, age, sex and weight of the individual, and the ability of the drug to elicit the desired response in the individual. As used herein, the term can also refer to an amount that effectively causes the desired in vivo effect in a subject. A therapeutically effective amount can be administered in one or more administrations (e.g., a composition can be given as a preventive treatment, or therapeutically administered at any stage of disease progression, before or after symptoms, etc.), applications, or dosages, and is not intended to be limited to a specific formulation, combination, or route of administration. It is within the scope of the present disclosure that the drug can be administered at different times during the course of treating the subject. The time of administration and the dosage used will depend on several factors, such as the therapeutic goal (e.g., treatment or prevention), the condition of the subject, etc., and can be easily determined by those skilled in the art. A therapeutically effective amount is also the amount in which the therapeutically beneficial effects of a substance outweigh its toxic or deleterious effects. A "prophylactically effective amount" refers to an amount that effectively achieves the desired preventive result within the necessary dosage and time period. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0107] As used herein, the term "therapeutic" or "treatment" refers to a treatment in which the goal is to slow down (mitigate) an undesirable physiological condition, disorder or disease, or to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to: alleviation of symptoms; alleviation of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the condition, disorder or disease (i.e., not worsening); delaying the onset of the condition, disorder or disease or slowing its progression; improving the state of the condition, disorder or disease; and alleviation (whether partial or complete) (whether detectable or undetectable) or improvement or improvement of the extent of the condition, disorder or disease. Treatment also includes a prolonged survival compared to the expected survival in the absence of treatment. The term "treatment" or "therapeutic" may include preventing, inhibiting, suppressing, improving or completely eliminating a disease. Preventing a disease may include administering a composition of the present invention to a subject before the onset of the disease. Suppressing a disease may include administering a composition of the present invention to a subject after the disease has been induced but before its clinical manifestation. Suppressing or ameliorating a disease may include administering a composition of the present invention to a subject after the clinical manifestation of the disease.

[0108] The term "variant" as used herein with respect to a polynucleotide refers to (i) a portion or fragment of a reference nucleotide sequence; (ii) the complement of a reference nucleotide sequence or a portion thereof; (iii) a polynucleotide that is substantially identical to a reference polynucleotide or its complement; or (iv) a polynucleotide that hybridizes under stringent conditions to a reference polynucleotide, its complement, or a sequence substantially identical thereto.

[0109] As used herein, the term "percent identity" between two sequences (e.g., amino acid or nucleotide sequences) refers to the percentage (out of a possible 100%) of positions that are identical (with appropriate insertions or deletions for optimal alignment) when optimally aligned and compared. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below. Methods and algorithms for determining % homology between two protein sequences are well established in the art.

[0110] For example, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol. (48): 444-453) algorithm, which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In addition, a protein amino acid sequence can be used as a "query sequence" to search against public databases, for example, to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215: 403-10. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0111] All patent applications, patents, and printed publications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. All patent applications, patents, and printed publications cited herein are incorporated by reference in their entirety, except for any definitions, subject matter disclaimers, or disclaimers, and to the extent the incorporated material is inconsistent with the explicit disclosure herein, in which case the language of the present disclosure controls.

[0112] The scope of the present invention is not limited to the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the above description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. It should be further understood that all values are approximate and are provided for illustrative purposes.

[0113] 2. Target protein degraders and EGFR degraders

[0114] The present invention further provides a target protein degradation agent. The target protein degradation agent comprises a target protein binding domain and a neuropilin binding domain. The target protein binding domain and the neuropilin binding domain of the target protein degradation agent specifically bind to the target protein and NRP1, and the target protein that is subsequently internalized is degraded via the lysosome. The target protein degradation agent comprises any heavy chain polypeptide of SEQ ID NO: 2 to 11 and a light chain polypeptide of SEQ ID NO: 12. Also provided are sequences shown in SEQ ID NO: 15 to 24 of nucleotides encoding the corresponding heavy chain polypeptides of SEQ ID NO: 2 to 11, and sequences shown in SEQ ID NO: 25 of nucleotides encoding the light chain polypeptide of SEQ ID NO: 12. The sequences of all polypeptides, polynucleotides and linkers disclosed in U.S. Provisional Application No. 63 / 325,312 are incorporated herein.

[0115] For example, an EGFR degrader is a bispecific antibody (EGFP×NRP1 Ab) that specifically binds to EGFR and NRP1. Binding of the EGFR degrader to EGFR and NRP1 results in degradation of EGFR. The EGFR degrader mediates EGFR degradation via the lysosomal degradation pathway. The EGFR degrader comprises the polypeptide sequences shown in SEQ ID NOs: 11 and 12. The polynucleotide sequences encoding the polypeptides of SEQ ID NOs: 11 and 12 are shown in SEQ ID NOs: 15 and 25. See Tables 1 and 2.

[0116] Table 1. Amino acid sequences of EGFR degraders

[0117]

[0118]

[0119] Table 2. Nucleotide sequences encoding EGFR degradation polypeptides

[0120]

[0121]

[0122] Additional exemplary bispecific antibody heavy chain polypeptide sequences for use in the protein degraders of the present disclosure are shown in Table 3 and in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 39, and polynucleotide sequences encoding heavy chain antibody polypeptide sequences of the bispecific antibodies are shown in Table 3 and in SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 40. In some aspects, the bispecific antibody further comprises a light chain polypeptide sequence comprising a variable light chain and a constant light chain, which is paired with the heavy chain polypeptide and is shown in SEQ ID NO: 12. The polynucleotide sequence encoding the light chain polypeptide of SEQ ID NO: 12 is shown in SEQ ID NO: 25. In some aspects, the present disclosure provides polynucleotide sequences encoding GGGGS subunits, and these sequences are shown in Table 3 and in SEQ ID NOs: 26-38.

[0123] In some aspects, the protein degrader is a bispecific antibody comprising an anti-EGFR binding arm comprising (or consisting of) a VH amino acid sequence as set forth in SEQ ID NO: 69 and a VL amino acid sequence as set forth in SEQ ID NO: 70. In some aspects, the bispecific antibody comprises an anti-EGFR binding arm comprising a heavy chain CDR1, CDR2, and CDR3 region comprising (or consisting of) the amino acid sequences set forth in SEQ ID NO: 71, 72, and 73, respectively. In some aspects, the bispecific antibody comprises an anti-EGFR binding arm comprising a heavy chain CDR3 region comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 73. In some aspects, the bispecific antibody comprises an anti-EGFR binding arm comprising a light chain CDR1, CDR2, and CDR3 region comprising (or consisting of) the amino acid sequence set forth in SEQ ID NOs: 74, 75, and 76, respectively. In some aspects, the bispecific antibody comprises an anti-EGFR binding arm comprising a light chain CDR3 region comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 76. In some embodiments, the anti-EGFR binding arm comprises (or consists of) one or more sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.8% identity to any of the above-described VH, VL, HCDR, or LCDR sequences. These sequences are also shown in Table 3.

[0124] In some aspects, the protein degrader is a bispecific antibody comprising an anti-NRP1 binding arm comprising (or consisting of) a VH amino acid sequence as set forth in SEQ ID NO: 77 and a VL amino acid sequence as set forth in SEQ ID NO: 78. In some aspects, the bispecific antibody comprises an anti-NRP1 binding arm comprising a heavy chain CDR1, CDR2, and CDR3 region comprising (or consisting of) the amino acid sequences set forth in SEQ ID NOs: 79, 80, and 81, respectively. In some aspects, the bispecific antibody comprises an anti-NRP1 binding arm comprising a heavy chain CDR3 region comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 81. In some aspects, the bispecific antibody comprises an anti-NRP1 binding arm comprising a light chain CDR1, CDR2, and CDR3 region comprising (or consisting of) the amino acid sequence set forth in SEQ ID NOs: 82, 83, and 84, respectively. In some aspects, the bispecific antibody comprises an anti-NRP1 binding arm comprising a light chain CDR3 region comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the anti-NRP1 binding arm comprises (or consists of) one or more sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.8% identity to any of the above-described VH, VL, HCDR, or LCDR sequences. These sequences are also shown in Table 3.

[0125] EGFR degraders can reduce or prevent cancer resistance to tyrosine kinase inhibitors. Anti-EGFR degraders can be, for example, antibodies, polynucleotides, small molecules, or combinations thereof. EGFR degraders can lead to degradation of the target protein, reducing or preventing cancer resistance to tyrosine kinase inhibitors. Following administration of an EGFR degrader, acquired resistance to tyrosine kinase inhibitors can be reduced or prevented. In some embodiments, tyrosine kinase inhibitor resistance can be reduced by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold. In some embodiments, tyrosine kinase inhibitor resistance is reduced by 2 to 10-fold. The activity of a tyrosine kinase inhibitor can be increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold. In some embodiments, the activity of a tyrosine kinase inhibitor is increased by at least 2-fold to at least 10-fold. In some embodiments, administering an amount of a target degrader to a patient with a cancer resistant to a therapeutic agent can increase the therapeutic activity of the therapeutic agent by at least 2-fold. In some embodiments, administering an amount of a target degrader to a patient with a cancer resistant to a therapeutic agent can increase the therapeutic activity of the therapeutic agent by at least 3-fold. In some embodiments, administering an amount of a target degrader to a patient with a cancer resistant to a therapeutic agent can increase the therapeutic activity of the therapeutic agent by at least 4-fold. In some embodiments, administering an amount of a target degrader to a patient with a cancer resistant to a therapeutic agent increases the therapeutic activity of the therapeutic agent by at least 5-fold. In some embodiments, administering an amount of a target degrader to a patient with a cancer resistant to a therapeutic agent increases the therapeutic activity of the therapeutic agent by at least 6-fold. In some embodiments, administering an amount of a target degrader to a patient with a cancer resistant to a therapeutic agent increases the therapeutic activity of the therapeutic agent by at least 7-fold. In some embodiments, administering an amount of a target degrader to a patient with a cancer resistant to a therapeutic agent increases the therapeutic activity of the therapeutic agent by at least 8-fold. In some embodiments, administering an amount of a target degrader to a patient with a cancer resistant to a therapeutic agent increases the therapeutic activity of the therapeutic agent by at least 9-fold. In some embodiments, administering an amount of a target degrader to a patient increases the therapeutic activity of the therapeutic agent by at least 10-fold.

[0126] a. Neuropilin

[0127] There are two forms of neuropilins: NRP-1 and NRP-2. Neuropilins are transmembrane glycoproteins composed of four domains: A (ala2), B (blb2), C (MAM), and a cytoplasmic domain. They are known to regulate neurogenesis and angiogenesis by complexing with plexin receptors / class 3 semaphorin ligands and vascular endothelial growth factor (VEGF) receptors / VEGF ligands, respectively. Neuropilins primarily function as coreceptors because their cytoplasmic domains are very small and they therefore rely on other cell surface receptors to transduce their signals across the cell membrane (Pellet et al., 2008 and Schwarz, 2010).

[0128] Recent studies have shown that neuropilins are multifunctional and can cooperate with a variety of transmembrane receptors. Neuropilins are closely associated with multiple signaling pathways, including those activated by epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), and transforming growth factor β (TGFI3) (Kofler, 2016 and Roy et al., 2017). Although neuropilins are usually found on the cell surface, they have also been reported to be localized in mitochondria and the nucleus (Issitt et al., 2019 and Mehta et al., 2018). Both neuropilin family members can also exist in soluble forms formed by alternative splicing or by shedding of the extracellular domain from the cell surface (Rossgnol et al., 2000 and Werneberg et al., 2016).

[0129] Neuropilins bind to many receptors including receptor tyrosine kinases (eg, EGFR), receptor serine / threonine kinases (eg, TGFPR), integrins, GPCRs (G protein coupled receptors), ion channels, but are not limited to CXCR.

[0130] The pleiotropic effects of NRP receptors lead to their involvement in cellular processes such as axon guidance and angiogenesis, immune responses, and remyelination (Mecollari et al., 2014). Consequently, dysregulation of NRP activity has been implicated in numerous pathological conditions, including many types of cancer and cardiovascular disease (Niland et al., 2019; Kofler, 2016; Pellet-Many et al., 2019; and Harma et al., 2020).

[0131] The NRP1 binding domain of the protein degrader used in the methods of the present disclosure includes an antibody or an NRP1 binding fragment thereof. For example, all or a portion of an NRP1 antibody known in the art or an anti-NRP1 antibody provided herein can be used. Non-limiting examples of anti-NRP1 mAb heavy chain polypeptide sequences are shown in SEQ ID NOs: 41-47, which comprise an N-terminal NPR1 binding domain. In some embodiments, the monoclonal antibody heavy chain polypeptide comprises an N-terminal NRP1 binding domain comprising a variable heavy chain (VH) and a constant heavy chain 1 (CH1), and an Fc domain comprising a constant heavy chain 2 (CH2) and a constant heavy chain 3 (CH3). In some embodiments, the monoclonal antibody comprises a light chain polypeptide sequence as shown in any one of SEQ ID NOs: 48-54. Thus, the heavy chains of SEQ ID NOs: 41-47 can be paired with the light chains of SEQ ID NOs: 48-54, respectively. The polynucleotide sequences encoding the heavy and light chain polypeptides are shown in SEQ ID NOs: 55-61 and 62-68, respectively. These sequences are also shown in Table 3.

[0132] b.EGFR

[0133] Epidermal growth factor receptor (EGFR) is one of the most frequently altered oncogenes in solid tumors. Enhanced EGFR signaling drives proliferation and cell survival in many cancer types. Mutations that affect EGFR expression or activity can lead to cancer. For example, mutations that lead to EGFR overexpression are associated with the development of many cancers. Resistance eventually develops with all known EGFR inhibitors, as well as with all kinase inhibitors that target oncogenic receptor tyrosine kinases and serine / threonine kinases. Figure 1 Existing EGFR blockers are unable to achieve sustained responses due to the lack of EGFR degradation ( Figure 2 ).

[0134] c. Additional protein degraders

[0135] cMET×NRP1 bispecific construct

[0136] In one embodiment, the protein degradation agent is a cMET×NRP1 bispecific construct. In one embodiment, the cMET×NRP1 bispecific antibody comprises an anti-NRP1 binding arm, comprising a heavy chain CDR1, CDR2, and CDR3 region, wherein the heavy chain CDR1, CDR2, and CDR3 region comprise (or consist of) the amino acid sequences set forth in SEQ ID NOs: 79, 80, and 81, respectively. In one embodiment, the bispecific antibody comprises an anti-NRP1 binding arm, comprising a heavy chain CDR3 region, wherein the heavy chain CDR3 region comprises (or consists of) the amino acid sequence set forth in SEQ ID NO: 81. In one embodiment, the bispecific antibody comprises an anti-NRP1 binding arm, comprising a light chain CDR1, CDR2, and CDR3 region, wherein the light chain CDR1, CDR2, and CDR3 region comprise (or consist of) the amino acid sequences set forth in SEQ ID NOs: 82, 83, and 84, respectively. In one embodiment, the bispecific antibody comprises an anti-NRPl binding arm comprising a light chain CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NO:84.

[0137] In one embodiment, the cMET binding arm comprises the sequence of an anti-cMET monoclonal antibody (mAb) known and available in the art, such as the heavy and light chain CDRs (1-3) of the mAb or the VH / VL polypeptide of the mAb. In one embodiment, the anti-cMET mAb is imatinib (also known in the art as LY2875358). In one embodiment, the anti-cMET mAb is onatuzumab. In one embodiment, the anti-cMET mAb is terituzumab.

[0138] HER2×NRP1 bispecific construct

[0139] In one embodiment, the protein degrader is a HER2×NRP1 bispecific antibody construct. In an embodiment, the HER2×NRP1 bispecific antibody comprises an anti-NRP1 binding arm comprising a heavy chain CDR1, CDR2, and CDR3 region, and the heavy chain CDR1, CDR2, and CDR3 region comprise the amino acid sequences shown in SEQ ID NOs: 79, 80, and 81, respectively (or consist of them). In an embodiment, the bispecific antibody comprises an anti-NRP1 binding arm comprising a heavy chain CDR3 region comprising the amino acid sequence shown in SEQ ID NO: 81 (or consist of them). In an embodiment, the bispecific antibody comprises an anti-NRP1 binding arm comprising a light chain CDR1, CDR2, and CDR3 region, and the light chain CDR1, CDR2, and CDR3 region comprise the amino acid sequences shown in SEQ ID NOs: 82, 83, and 84, respectively (or consist of them). In one embodiment, the bispecific antibody comprises an anti-NRPl binding arm comprising a light chain CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NO:84.

[0140] In an embodiment, the HER2 binding arm comprises the sequence of an anti-HER2 monoclonal antibody (mAb) known and available in the art, such as the heavy and light chain CDRs (1-3) of the mAb or the VH / VL polypeptides of the mAb. In one embodiment, the anti-HER2 mAb is trastuzumab. In one embodiment, the anti-HER2 mAb is pertuzumab.

[0141] IGF1R×NRP1 bispecific construct

[0142] In one embodiment, the protein degradation agent is an IGF1R×NRP1 bispecific antibody construct. In an embodiment, the IGF1R×NRP1 bispecific antibody comprises an anti-NRP1 binding arm, wherein the anti-NRP1 binding arm comprises a heavy chain CDR1, CDR2, and CDR3 region, and the heavy chain CDR1, CDR2, and CDR3 region comprise the amino acid sequences shown in SEQ ID NOs: 79, 80, and 81, respectively (or consist of them). In one embodiment, the bispecific antibody comprises an anti-NRP1 binding arm, wherein the anti-NRP1 binding arm comprises a heavy chain CDR3 region, and the heavy chain CDR3 region comprises the amino acid sequence shown in SEQ ID NO: 81 (or consist of them). In an embodiment, the bispecific antibody comprises an anti-NRP1 binding arm, wherein the anti-NRP1 binding arm comprises a light chain CDR1, CDR2, and CDR3 region, and the light chain CDR1, CDR2, and CDR3 region comprise the amino acid sequences shown in SEQ ID NOs: 82, 83, and 84, respectively (or consist of them). In one embodiment, the bispecific antibody comprises an anti-NRPl binding arm comprising a light chain CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NO:84.

[0143] In an embodiment, the IGF1R binding arm comprises the sequence of an anti-IGF1R monoclonal antibody (mAb) known and available in the art, e.g., the heavy and light chain CDRs (1-3) of the mAb or the VH / VL polypeptides of the mAb. In one embodiment, the anti-IGF1R mAb is ganituzumab (also known in the art as AMG 479). In one embodiment, the anti-IGF1R mAb is fentolumab. In one embodiment, the anti-IGF1R mAb is cixitumumab. In one embodiment, the anti-IGF1R mAb is darotozumab.

[0144] 3. Therapeutic agents including tyrosine kinase inhibitors

[0145] a. Tyrosine kinase

[0146] Tyrosine kinases are enzymes that transfer a gamma (terminal) phosphate group from ATP to polypeptides in cells. The phosphate group is attached to the amino acid tyrosine on the polypeptide. Tyrosine kinases can phosphorylate one or more tyrosine residues on a polypeptide. Phosphorylation of tyrosine residues on a polypeptide can result in altered polypeptide function. Tyrosine kinases are a subgroup of the larger class of protein kinases, which attach phosphate groups to other amino acids, such as serine and threonine. Phosphorylation of proteins by kinases is a mechanism for transmitting signals within cells (signal transduction) and regulating cellular activities, such as cell division.

[0147] b. Tyrosine kinase inhibitors

[0148] Tyrosine kinase inhibitors (TKIs) inhibit the activity or expression of tyrosine kinases. Tyrosine kinase inhibitors may include antibodies, polynucleotides (such as interfering RNA), small molecules, or combinations thereof. In some embodiments, tyrosine kinase inhibitors include antibodies. In some embodiments, the antibodies are monoclonal antibodies. In some embodiments, tyrosine kinase inhibitors include small molecules. Tyrosine kinase inhibitors can be specific for a particular tyrosine kinase, or for a particular tyrosine kinase family or subfamily. In some embodiments, tyrosine kinase inhibitors are specific for EGFR. Such inhibitors may be referred to as "EGFR inhibitors." Antibody EGFR inhibitors include, for example, cetuximab and panitumumab. Small molecule EGFR inhibitors include, for example, lapatinib (a mixed EGFR and ERBB2 inhibitor), myritinib (osimertinib), gefitinib, icotinib, erlotinib, afatinib, and brigatinib. EGFR inhibitors can bind to and inhibit the kinase domain of EGFR. Without kinase activity, EGFR may be unable to activate itself or induce downstream activation and signaling. In some embodiments, EGFR inhibitors include osimertinib.

[0149] All of the following, but not limited to, EGFR inhibitors, which target and inhibit certain oncogenic EGFRs and ultimately produce drug resistance, which is achieved by the individual including the present invention (Examples 1 and Figure 3 The disease is overcome by treatment with any EGFR degrader (compound 1) as well as any EGFR inhibitor combination that leads to drug resistance.

[0150] EGFR inhibitors include lazertinib, osimertinib (AZD9291), WZ4002, cyasterone, erlotinib (OSI-774) hydrochloride, gefitinib (ZD1839), lapatinib (GW-572016) ditosylate, afatinib (BIBW2992), sacatinib (AZD0530), vandetanib (ZD6474), neratinib (HKI-272), canertinib (CI-1033), lapatinib (GW-572016), AG-490 (tyrosine phosphorylation inhibitor B42), CP-724714, dacomitinib ( PF-00299804), sabutinib (AZD8931), CUDC-101, AG-1478 (tyrosine phosphorylation inhibitor AG-1478), PD153035 hydrochloride, pelitinib (EKB-569), AC480 (BMS-599626), AEE788 (NVP-AEE788), AP26113-analog (ALK-IN-1), OSI-420, WZ3146, HER2-inhibitor-1, WZ8040, alitinib tosylate, rociletinib (CO-1686), genistein (NPI 031L), valitinib, TQB33804 (EGFR-IN-7), icotinib (BPI-2009H), TAK-285, daphnetin, tyrosine phosphorylation inhibitor 9, AG-18, AG 555, AZ5104, CL-387785 (EKI-785), tyrosine phosphorylation inhibitor AG-258, AG-556, tucatinib, erlotinib (OSI-774), gefitinib-based PROTAC 3, zolitinib (AZD 3759), ErbB2, AV-412 free base, AST-1306, JND3229, BI-4020, cilitinib (HMPL-309), BDTX-189, rifirafenib (BGB-283), pyrotinib (SHR-1258), O-desmethyl gefitinib, epatinib hydrochloride, SU5214, avitinib (AC0010), AG 494, and without limitation, poziotinib (HM781-36B).

[0151] All of the following, but not limited to, ERBB2 (HER2), ERBB3 (HER3), and ERBB4 (HER4) inhibitors, which target and inhibit certain oncogenic ERBBs and ultimately lead to drug resistance, which is overcome by treatment with any ERBB degrader alone and the present invention, as well as in combination with any ERBB inhibitor that leads to drug resistance.

[0152] ERBB inhibitors include tucatinib, HER2 inhibitor 1, afatinib (BIBW2992), neratinib (HKI-272), CP-724714, muritinib (TAK 165), AC480 (BMS-599626), AEE778, TAK-285, tyrosine phosphoinhibitor AG 879, tyrosine phosphoinhibitor AG-528, SU5204, poziotinib (HM781-36B), TAS0728, BDTX-189, pyrotinib, and epatinib hydrochloride.

[0153] All of the following, but not limited to, cMET inhibitors, which target and inhibit certain oncogenic cMETs and ultimately lead to drug resistance, which is overcome by treatment with any cMET degrader alone and the present invention, as well as in combination with any cMET inhibitor that leads to drug resistance.

[0154] cMET inhibitors include crizotinib, cabozantinib, foretinib, PHA-665752, SU11274, SGX-523, BMS-777607, tivatinib, JNJ-38877605, PF-04217903, amvatinib (MP-470), MGCD-265 analogs, capmatinib, BMS-754807, BMS-794833, AMG-208, MK-2461, govatinib, AMG-458, NVP-BVU972, AMG 337, meritinib, JNJ-38877618, crizotinib hydrochloride, ningetinib, AMG-1, UNC2025, pamufetinib, atelatinib, NPS-1304, and savotinib.

[0155] All of the following, but not limited to, PDGFR and FGFR inhibitors, which target and inhibit certain oncogenic PDGFRs and FGFRs and ultimately develop drug resistance, which is overcome by any PDGFR degrader and any FGFR degrader alone and the present invention, as well as treatment in combination with any PDGFR or any FGFR inhibitor that leads to drug resistance.

[0156] PDGFR inhibitors and FGFR inhibitors are ponatinib (AP24534), infigratinib (BGJ398), nintedanib (BIBF 1120), pazopanib hydrochloride (GW786034HC1), pazopanib, AZD4547, tyrosine phosphorylation inhibitor AG 1296, SSR128129E, LY2874455, derazentinib (ARQ-087), SU5402, ODM-203, pemigatinib (INCB054828), derazentinib (E3810) hydrochloride, ferulic acid, masitinib mesylate, fisorinib (BLU-554), PRN1371, ON123300, FIIN-3, robitinib (FGF401), fobatinib (TAS-120), FIIN-2, zoligratinib (Debio-1347), nintedanib essylate, BLU9931, and surufatinib.

[0157] All of the following, but not limited to, cKIT inhibitors, which target and inhibit certain oncogenic cKIT and ultimately develop drug resistance that is overcome by treatment with any of the cKIT degraders and the present invention alone and in combination with each cKIT inhibitor that leads to drug resistance. cKIT inhibitors are dasatinib (BMS-354825), sorafenib (BAY 43-9006) mesylate, imatinib (STI571) mesylate, sunitinib (SU11248) malate, ponatinib (AP24534), axitinib (AG 013736), imatinib (STI571), nintedanib (BIBF 1120), regorafenib (BAY 73-4506), pazopanib hydrochloride (GW786034HC1), linifanib (ABT-869), clelanib (CP-868596), masitinib (AB1010), amovatinib (MP-470), olantinib (SU6668), CP-673451, telatinib, PP121, pazopanib, and tyrosine phosphorylation inhibitors (TPSs) such as AG-1. 1296, tyrosine phosphoinhibitor 9, SU14813, regorafenib hydrochloride, tyrosine phosphoinhibitor AG1433, sunitinib (SU11248), ripretinib (DCC-2618), masitinib mesylate, AZD3229, ON123300, afatinib (BLU-285), seraticinib (GB002), AZD2932, JNJ-10198409, nintedanib essylate, flumatinib (HI-I-GV-678), and regorafenib (BAY-734506) monohydrate.

[0158] All of the following, but not limited to, FTL3 inhibitors, which target and inhibit certain oncogenic FTL3s and ultimately lead to drug resistance that is overcome by treatment with any FTL3 degrader alone and the present invention and in combination with any FTL3 inhibitor that leads to drug resistance. FTL3 inhibitors are paclitinib (SB1518), TCS 359, linifanib (ABT-869), zotirapir, cediranib (AZD2171), dovitinib (TKI258) lactate, UNC2025 HC1, SU5614, FLT3-IN-2, FLT3-IN-4, FF-10101, meritinib (LY2801653), emavusertib (CA-4948), tandutinib (MLN518), R406 (free base), 5'-fluorobis(2-isobutylketone) oxime, tauzertinib, quizartinib (AC220), R406, AST-487 (NVP-AST487), sorafenib (BAY 43-9006) mesylate, BMS-794833, sorafenib (BAY 43-9006), 4SC-203, dovitinib (TKI-258), isoguanosine, TAK-659, ATH686, SGI-1776 free base, MK-2461, fostamatinib (R788) disodium, MRX-2843, brigatinib (AP26113), GW2580, rebastinib (DCC-2036), BMS-754807, UNC2025, fidartinib (TG101348), FLT3-IN-3, G-749, cleralinib (CP-868596), BPR1K871, PHA-680632, entuximab (GS-9973), HPK1-IN-2, SP600125, SKLB477 1(FLT3-IN-1), KW-2449, gilteritinib (ASP2215), CCT241736, PRT062607(P505-15)HC1, ENMD-2076, FN-1501, ceritinib (LDK378), cimisatinib (CX-4945), AZD2932, fostamatinib (R788), tivozanib (AV-951), PF-477736, BPR1J-097, pegidatinib (PLX3397), Go6976, HM43239, OSI-930, TG101209, PLX5622, amvatinib (MP-470), GNF-2, midostaurin (PKC412), AMG 925 and ENMD-2076 L-(+)-tartaric acid.

[0159] All of the following, but not limited to, VEGFR1, VEGFR2, VEGFR3, and VEGFR4 inhibitors, which target and inhibit any of oncogenic VEGFR1, VEGFR2, VEGFR3, and VEGFR4, and ultimately develop drug resistance, which is overcome by treatment with any of the VEGFR1, VEGFR2, VEGFR3, or VEGFR4 degraders alone and the present invention, as well as in combination with any of the VEGFR1, VEGFR2, VEGFR3, or VEGFR4 inhibitors that lead to drug resistance.

[0160] The VEGFR1, VEGFR2, VEGFR3, and VEGFR4 inhibitors are sorafenib (BAY 43-9006) mesylate, sunitinib (SU11248) malate, lenalidomide (CC-5013), cabozantinib (BMS-907351), ponatinib (AP24534), axitinib (AG 013736), forlatinib (GSK1363089), vandetanib (ZD6474), nintedanib (BIBF 1120), regorafenib (BAY 43-9006) mesylate, and stivarga (BAY 43-9006) malate. 73-4506), pazopanib hydrochloride (GW786034HC1), cediranib (AZD2171), PD173074, dovitinib (TKI-258), linifanib (ABT-869), vatalanib (PTK787) 2HC1, RAF265 (CHIR-265), tivozanib (AV-951), motesanib diphosphate (AMG-706), lenvatinib (E7080), brivanib (BMS-540215), MGCD-265 analog, AEE788 (NVP-AEE788), ENMD-2076, OSI-930, CYC116, Ki8751, telatinib, PP121, pazopanib, KRN 633, SAR131675, BMS-794833, apatinib (YN968D1) mesylate, sorafenib (BAY 43-9006), cabozantinib malate, brivanib alanine salt (BMS-582664), govatinib (E7050), semaxanib (SU5416), ZM 306416, ZM 323881HC1, ENMD-2076L-(+)-tartrate, LY2874455, BAW2881 (NVP-BAW2881), WHI-P180, SU14813, ZD-4190, SU1498, SU5402, PDGFR inhibitor 1, Ki20227, dovitinib (TKI258) lactate, tocinib phosphate, cedirimib Nibuvir maleate, apatinib, BFH772, lenvatinib (E7080) mesylate, SU5614, regorafenib hydrochloride, SU5204, SU5208, fruquintinib (HMPL-013), hVEGF-IN-1, ODM-203, erdafitinib (JNJ-42756493), tyrosine phosphorylation inhibitor AG1433, MAZ51, SKLB 610, sunitinib (SU11248), 4SC-203, sitrutinib (MGCD516), R1530, donafenib (sorafenib D3), envodostat (PTC299), AG-13958, SKLB1002, motesanib (AMG-706), 4,4'-Bis(4-aminophenoxy)biphenyl, deritinib (E3810) hydrochloride, ogifanil, sioroni, ningetinib, X-82 (voronib), Pamufetinib (TAS-115), Cassia seed extract, CS-2660 (JNJ-38158471), WAY-340935, (20R)-protopanaxadiol, litchi seed extract, atelatinib, vitamin E, SU5408, AZD2932, anlotinib (AL3818) dihydrochloride, nintedanib essylate, chebulic acid, SU5205, SU5214, regorafenib (BAY-734506) monohydrate, taxifolin (dihydroquercetin), surufatinib, and XL092.

[0161] All of the following, but not limited to, TGFβ1R and TGFβ2R inhibitors, which target and inhibit certain oncogenic TGFβ1R and TGFβ2R and ultimately develop drug resistance, which is overcome by treatment with any TGFβ1R and / or TGFβ2R degrader and the present invention alone and in combination with any of the TGFβ1R and TGFβ2R inhibitors that cause drug resistance.

[0162] TGFβ1R and TGFβ2R inhibitors include SD-208, GW788388, A-83-01, desistatin (P144), SRI-011381, TP0427736 HC1, LY2109761, ophiopogonin D, SB505124, SIS3 HC1, BIBF-0775, LY3200882, LSKL, thrombospondin (TSP-1) inhibitor, galunisertib (LY2157299), ginsenoside Rh4, LDN-193189, A77-01, LDN-193189 2HC1, vactosertib (TEW-7197), halofuginone hydrobromide, halofuginone, sulfasalazine (NSC 667219), BMS-986260, XAV-939, LY364947, oxymatrine, pirfenidone (S-7701), hypoaconitine, SB525334, ITD-1, and bufastatin, TA-02, and PD 169316. All of the following, but not limited to, integrin inhibitors target and inhibit certain oncogenic integrins and ultimately lead to drug resistance, which is overcome by any integrin degraders and treatments of the present invention, alone and in combination with any integrin inhibitor that leads to drug resistance. The integrin inhibitors were cilengitide trifluoroacetate, RGD (Arg-Gly-Asp) peptide, A-205804, SB273005, cilengitide, RGD peptide (GRGDNP), OSU-T315, ILK-IN-3, Cyclo (-RGDfK), A286982, Cyclo (RGDyK), and A286982.

[0163] All of the following, but not limited to, IGF1R and IR inhibitors, which target and inhibit certain oncogenic IGF1R and IR, and ultimately develop drug resistance, which is overcome by treatment with any IGF1R and IR degrader alone and in combination with any IGF1R or IR inhibitor that leads to drug resistance.

[0164] The IGF1R and IR inhibitors were lumispivir (NVP-AUY922), linsitinib (OSI-906), NVP-AEW541, GSK1904529A, NVP-ADW742, BMS-536924, ceritinib (LDK378), AG-1024, GSK1838705A, BMS-754807, PQ 401, ZD3463, nordihydroguaiaretic acid (NDGA), NT157, insulin (human), ceritinib dihydrochloride, Rhizoma Dioscoreae extract, MID-1, brigatinib (AP26113), picropodophyllin (PPP), MSDC-0160, degludec, chromium picolinate, SBI-477, and XL228.

[0165] 4. Additional target proteins

[0166] Neuropilins are closely associated with many drug-resistant tumors. Neuropilins and their degradation are linked to receptor tyrosine kinases, receptor serine / threonine kinases, G protein-coupled receptors, ion channels, CXCRs, and immune checkpoint regulators, which serve numerous biological functions. Therefore, co-degradation of neuropilins with other receptors has potential therapeutic applications in treating neuropilin-associated drug resistance.

[0167] All of the following, but not limited to, PARP inhibitors, which develop resistance that is overcome by treatment with any NRP degrading compound alone and in combination with any PARP1 inhibitor that leads to resistance.

[0168] PARP inhibitors are PJ34 HC1, AZD2461, olaparib (AZD2281), veliparib (ABT-888), XAV-939, rucaparib (AG-014699) phosphate, iniparib (BSI-201), talazoparib (BMN 673), AG-14361, 3-aminobenzamide, A-966492, niraparib (MK-4827), UPF 1069, ME0328, licochalcone D, DR2313, MN 64, 4',5,7-trimethoxyflavone, rucaparib, M2912, GeA-69, BYK204165, BGP-15 2HC1, atamparib (RBN-2397), vinadaparib (IDX-1197), niraparib (MK-4827) mesylate, NU1025, rucaparib camphorsulfonate, berberine hydrochloride (NSC 646666), pamiparib (BGB-290), fluzopanib (SHR-3162), G007-LK, NVP-TNKS656, berberine hydrochloride hydrate, HI-TOPK-032, stenoparib (E7449), 4-hydroxyquinazoline, NMS-P118, WIKI4, RBN012759, AZD5305, AZD-9574, RK-287107, benzamide, JW55, and picolinamide.

[0169] All of the following, but not limited to, RAF inhibitors, which develop resistance that is overcome by treatment with any neuropilin-degrading compound alone and in combination with any RAF inhibitor that leads to resistance.

[0170] The RAF inhibitors were vemurafenib (PLX4032), B-Raf inhibitor 1 (Compound 13) dihydrochloride, Raf inhibitor 1, Raf inhibitor 2, sorafenib (BAY 43-9006) mesylate, PLX-4720, dabrafenib (GSK2118436), regorafenib (BAY 73-4506), damanimide (BIRB 796), GDC-0879, RAF265 (CHIR-265), AZ 628, NVP-BHG712, SB590885, ZM 336372, sorafenib (BAY 43-9006), GW5074, TAK-632, agrafenib (RXDX-105), conagrafenib (LGX818), BAW2881 (NVP-BAW2881), PLX8394, TBAP-001, regorafenib hydrochloride, MCP110, naborafenib (LXH254), B-Raf IN 1. Donafenib (sorafenib D3), CCT196969, RAF709, rifolafenib (BGB-283), L-779450, PLX7904, LY3009120, dabrafenib mesylate, RO5126766 (CH5126766), AZ304, Belvarafenib (HM95573), regorafenib (BAY-734506) monohydrate, and tovorafenib (MLN2480).

[0171] All of the following, but not limited to, autophagy activators or autophagy inhibitors, which produce drug resistance that is overcome by treatment with any neuropilin-degrading compound alone and in combination with any autophagy activator or any inhibitor that causes drug resistance.

[0172] The autophagy activators are enzalutamide (MDV3100), obaclava mesylate (GX15-070), SRT1720 HC1, fulvestrant (ICI-182780), bicalutamide (ICI-176334), resveratrol (SRT501), forskolin, rosiglitazone (BRL-49653) maleate, rosiglitazone (BRL 49653), mifepristone (RU486), purmorphamine, clemastine (HS-592) fumarate, GW4064, chloroquine diphosphate, ivermectin (MK-933), loperamide hydrochloride, melatonin (NSC113928), methylprednisolone (NSC-19987), clonidine hydrochloride, flubendazole, fenofibrate (NSC-281319), montelukast sodium, LYN-1604 , EN6, Eprenetapopt (APR-246), 3BDO, MHY1485, methylprednisolone acetate, QX77, anisomycin, β-elemene, spermidine trihydrochloride, troglitazone (CS-045), conosine, obeticholic acid, SMER28, BC1618, monomethyl fumarate, PCNA-II, CA77.1, spermidine, xylitol, isorhynchophylline and MPP+iodide.

[0173] The autophagy inhibitors were MK-2206 2HC1, bortezomib (PS-341), olaparib (AZD2281), vemurafenib (PLX4032), vorinostat (SAHA), ABT-737, Y 276322HC1, daclis (BEZ235), sorafenib (BAY43-9006) mesylate, dasatinib (BMS-354825), rapamycin (AY-22989), crizotinib (PF-02341066), erlotinib (OSI-774) hydrochloride, everolimus (RAD001), gefitinib (ZD1839), veliparib (ABT-888), entinostat (MS-275), and BI. 2536, Pitilipsin (GDC-0941), Laduviglusib (CHIR-99021) HC1, LY294002, Trametinib (GSK1120212), Ruxolitinib (INCB018424), Panobinostat (LBH589), Imatinib (STI571) mesylate, KU-55933 (ATM kinase inhibitor), Alecetin (MLN8237), Afatinib (BIBW2992), U0126-EtOH, Idelalisib, Tauzacertib, AZD8055, Sacatinib (AZD0530), Paclitaxel (NSC 125973), SP600125, ponatinib (AP24534), tanespiramycin (17-AAG), YM155 (sapantrine bromide), DAPT (GSI-IX), cisplatin (NSC 119875), imatinib (STI571), nilotinib (AMN-107), temsirolimus (CCI-779), PI-103, lumispivir (NVP-AUY922), vandetanib (ZD6474), gemcitabine (LY-188011) hydrochloride, moxistat (MGCD0103), regorafenib (BAY 73-4506), SRT1720 HC1, pazopanib hydrochloride (GW786034HC1), bosutinib (SKI-606), cediranib (AZD2171), belinostat (PXD101), GSK690693, SB202190 (FHPI), carfilzomib (PR-171), fulvestrant (ICI-182780), SB216763, SU11274, linifanib (ABT-869), pemetrexed (LY-231514) disodium, tocotinib (PP242), etoposide (VP-16), wortmannin (KY12420), LY2109761, daruselutib (PHA-739358), cimisatinib (CX-4945), venetoclax (ABT-199), flavopiridol (L86-8275), SGI-1776 free base, lapatinib (GW-572016), vincristine (NSC-67574) sulfate, temozolomide (CCRG 81045), tacrolimus (FK506), metformin hydrochloride, fasudil (HA-1077) hydrochloride, oxaliplatin (NSC 266046), rabusertib (LY2603618), 3-methyladenine (3-MA), flavopiridol (L86-8275) hydrochloride, (+)-JQ1, zoledronic acid (ZOL 446), momelotinib (CYT387), ixazomib citrate (MLN9708) analog, tamoxifen (ICI 46474) citrate, letrozole (CGS20267), topotecan (NSC609699) hydrochloride, Torin 1, omelisabet (GSK2126458), Degrasyn (WP1130), 2-methoxyestradiol (2-MeOE2), azacitidine (5-azacytidine), BX-795, OSI-027, ixazomib (MLN2238), TWS119, apellisib (GDC-0980), BI-D1870, resveratrol (SRT501), dexamethasone (MK-125), cytarabine (U-19920A), simvastatin (MK 733), Vistusertib (AZD2014), CCT128930, Idarubicin hydrochloride, Gemcitabine (LY-188011), Verteporfin (CL 318952), PF-4708671, Torin 2, Streptozotocin (STZ), H 89 2HC1, Brefeldin A, Geldanamycin (NSC 122750), MK-5108 (VX-689), Forskolin, Valproic acid (NSC 93819) sodium salt, pitavastatin (NK-104) calcium, doxormorphine (Compound C) dihydrochloride, lovastatin (MK-803), rosiglitazone (BRL-49653) maleate, necrostatin-1, pazopanib, nocodazole (R17934), dapoline, clofarabine, cabazitaxel (XRP6258), atorvastatin calcium, (R)-(-)-gossypol acetic acid, pifitarin-a (PFTa) hydrobromide, SN-38, GSK343, BIX 01294, nutrin-3a, tigecycline (GAR-936), STF-62247, binitinib (MEK162), itraconazole (R 51211), mircini (PHA-848125), sorafenib (BAY43-9006), 10058-F4, YM201636, C646, hydroxyurea (NSC-32065), bardoxolone methyl, sodium butyrate, PR-619, linagliptin (BI-1356), niclosamide (BAY2353), nitazoxanide (NSC 697855), UNC1999, heparin sodium, Dynasore, curcumin, celastrol (NSC 70931), GSK2606414, honokiol (NSC 293100), laduviglusib (CHIR-99021), GANT61, omeprazole, amiodarone (NSC 85442) hydrochloride, dexamethasone sodium phosphate, aspirin (NSC 27223), AZD3463, GSK2656157, carbamazepine, bafilomycin Al (Baf-A1), azithromycin (CP-62993), nimodipine, IU1, sulfasalazine (NSC 667219), pifetaline-μ, PF-543 hydrochloride, tubastatin A, nitrendipine, DC661, KB-R7943 mesylate, tubastatin A TFA, PFK15, nordihydroguaiaretic acid (NDGA), nilotinib hydrochloride monohydrate, thalidomide, neferine, emetine hydrochloride, SBI-0206965, apatinib, EAD1, lanatoside C, ruxolitinib phosphate, lopimod (GW856553X), STO-609, IITZ-01, Vacuolin-1, entrectinib (RXDX-101), sunitinib (SU11248), valproic acid (VPA), URMC-099, spautin-1, erlotinib (OSI-774), pemetrexed disodium hydrate, crizotinib hydrochloride, atorvastatin, berberine hydrochloride (NSC 646666), quercetin (NSC 9221), VLX600, afatinib (BIBW2992) dimaleate, FL-411, pemetrexed, Autophinib, cryptotanshinone, nortriptyline hydrochloride, dihydroartemisinin (DHA), KN-93 phosphate, tiraginostat (CB-839), purvalanol A, brevicornulin A, chloroquine (NSC-187208), PFK158, sulfacetamide sodium hydrate, OTS964, sinomenine hydrochloride, MRT67307 HC1, Lys05, sulfacetamide sodium, resatovir (TAK-242), DMH1, hydroxychloroquine sulfate (NSC 4375), MRT68921HC1, PHY34, vinorelbine ditartrate (KW-2307), CA-5f, VPS34-IN1, DMOG, ICCB-19 hydrochloride, AS1842856, SAR405, PIK-III, ULK-101, vinblastine (NSC-49842) sulfate, SP2509, ROC-325, Codonopsis pilosula extract, ABTL-0812, ML-9HC1, AZD1208, lucanthone, leonurine, VER155008, RA-190LY3009120, 4-phenylbutyric acid (4-PBA), NSC 185058, E260, daurisoline, doxormorphine (Compound C), tamoxifen (ICI 46474), deferoxamine mesylate (Ba 33112), pepstatin A, trametinib DMSO solvate, and concanavalin A.

[0174] All of the following, but not limited to, mTOR inhibitors, which develop resistance that is overcome by treatment with any neuropilin-degrading compound alone and in combination with any mTOR inhibitor that leads to resistance.

[0175] mTOR inhibitors are mTOR inhibitor-1, everolimus (RAD001), KU-0063794, daclis (BEZ235), rapamycin (AY-22989), AZD8055, temsirolimus (CCI-779), PI-103, NU7441 (KU-57788), tocotinib (PP242), lidaformolimus (rapamycin, MK-8669), sapacerti (MLN0128), voxtalisib (XL765) analogs, Torin 1, omelisib (GSK2126458), OSI-027, PF-04691502, apellisib (GDC-0980), GSK1059615, gidalisib (PKI-587), WYE-354, vistusertib (AZD2014), Torin 2, WYE-125132 (WYE-132), BGT226 (NVP-BGT226) maleate, Palomid 529 (P529), PP121, WYE-687, Nitazoxanide (NSC 697855), WAY-600, ETP-46464, GDC-0349, XL388, 4EGI-1, JR-AB2-011, ferric acid, lanatoside C, compound 401, astragaloside IV, ginkgolide K, CC-115, zotarolimus (ABT-578), Paxalisib (GDC-0084), CZ415, SF2523, bimilisib (PQR309), voxtalisib (XL765), rhubarb acid, onatasertib (CC 223), 3-hydroxyanthranilic acid, Samotolisib (LY3023414), MTI-31, ABTL-0812, PQR620, MHY-1685, GNE-477, and GNE-493.

[0176] All of the following, but not limited to, PI3K activators or PIK3 inhibitors that produce drug resistance that is overcome by treatment with any neuropilin-degrading compound alone and in combination with any PI3K activator or any PI3K inhibitor that causes drug resistance.

[0177] The PI3K activators are higenamine, cinobufagin, resibufogenin, 740YP (PDGFR 740Y-P), erucic acid, picrogentioside, and YS-49.

[0178] PI3K inhibitors are PI-103, XL147 analogs, 3-methyladenine (3-MA), apellisib (GDC-0980), TG100713, taselisib (GDC 0032), daclis (BEZ235), pitilisib (GDC-0941), LY294002, idelalisib, buparisib (BKM120), NU7441 (KU-57788), TGX-221, IC-87114, wortmannin (KY12420), ZSTK474, apellisib (BYL719), AS-605240, PIK-75HC1, reglucosidase (ON-01910), A66, and voxtalisib (XL765). Analogs, Omilide (GSK2126458), PIK-90, AZD6482, PF-04691502, GSK1059615, Duvilice (IPI-145), Gidalice (PKI-587), TG100-115, AS-252424, NU7026, BGT226 (NVP-BGT226) maleate, Feminostat (CUDC-907), PIK-294, AS-604850, GSK2636771, Kupanisi (BAY 80-6946), YM201636, CH5132799, CAY10505, PIK-293, PKI-402, VS-5584 (SB2343), KU-0060648, CZC24832, demethylcoclaurine, oroxylin B, homosalate, AMG319, cinobufagin, lipobufogenin, homoplanin, GSK2292767, lanatoside C, zeaxanthin, desitatin (P144), cafestol, Paxalisib (GDC-0084), SKI-V, MTX-211, selelisib (UCB-5857), Trichosanthes peel extract, Rhizoma Dioscoreae extract, GDC-0326, 740YP (PDGFR 740Y-P), PIK-108, Pasalisib (INCB050465) hydrochloride, HS-173, SF2523, Lenilixib (CDZ 173), Lupetin, Cerelisib (TAK-117), Eganelisib (IPI-549), Quercetin (NSC9221), Bimilisib (PQR309), VPS34 inhibitor 1 (Compound 19), IHMT-PI3K6-372, Voxtalisib (XL765), Autophinib, GNE-317, (E)-Akt inhibitor-IV, Panax notoginseng saponin R1, Tenalisib (RP6530), Australosolamine, Alizarin Violet, Cirsin, Inalisib (GDC-0077), SRX3207, α-linolenic acid, Erbulisib (TGR-1202), acalisib (GS-9820), ME-401, 3-hydroxyanthranilic acid, Nemilisib, Samotolisib (LY3023414), VPS34-IN1, Ailanthone, Tripterygium wilfordii extract, IPI-3063, SAR405, PIK-III, IPI-3063, Pasalisib (INCB050465), Quercetin dihydrate, Pilaralisib (XL147), SPP-86, AZD8835, Trigonelline, Deguelin, Selective PI3K6 Inhibitor 1 (Compound 7n), Loureirin A, PF-4989216, AZD8186, GNE-477, and GNE-493.

[0179] All of the following, but not limited to, proteasome inhibitors, which develop resistance that is overcome by treatment with any neuropilin-degrading compound alone and in combination with any proteasome inhibitor that leads to resistance.

[0180] The proteasome inhibitors are salinosporamide A (NPI-0052), bortezomib (PS-341), MG132, carfilzomib (PR-171), ixazomib citrate (MLN9708), ixazomib (MLN2238), ONX-0914 (PR-957), oprozomib (ONX 0912), delanzomib (CEP-18770), celastrol (NSC 70931), epoxomicin (BU-4061T), shikonin (CI 75535), VR23, isoginkgetin, RA-190, and PI-1840.

[0181] All of the following, but not limited to, JNK inhibitors, which develop resistance that is overcome by treatment with any neuropilin-degrading compound alone and in combination with any JNK inhibitor that leads to resistance.

[0182] The JNK inhibitors are JNK inhibitor VIII, JNK inhibitor IX, JNK-IN-8, BI-78D3, SP600125, damarimod (BIRB 796), metformin hydrochloride, DB07268, 3'-hydroxypterostilbene, KB-R7943 mesylate, JNK-IN-7, lucerne B, astragaloside IV, cucurbitacin IIb, trans-zeatin, etanerstat, IQ 3, berberine hydrochloride (NSC 646666), SU3327, bemamod (AS602801), indirubin-3'-oxime, falcarindiol, tanzisetti (CC-930), NDMC101, morulin A, C-401 hydrochloride, RPI-1, ginsenoside Re, IQ-1S, and urolithin B.

[0183] All of the following, but not limited to, NF-кB inhibitors, which develop resistance, are overcome by treatment with any neuropilin-degrading compound alone and in combination with any NF-кB inhibitor that leads to resistance.

[0184] The NF-кB inhibitors are rabbit recombinant monoclonal antibody against phosphorylated IKBα (S32), ophiopogon saponin D, cornus officinalis glycosides, rubiacein 1-methyl ether, SM-7368, NF-кB-IN-1, chitosan oligosaccharides, Cynanchi Atrat extract, IAXO-102, Adjudin, CBL0137, IQ 3, erythrine, apigenin isoliquiritin, cyanidin, bortezomib (PS-341), urolithin B, sulfasalazine (NSC 667219), Acetylcysteine (N-Acetylcysteine), Erdosteine, Curcumin, Andrographolide, Dihydroartemisinin (DHA), Indole-3-Carbinol, Magnolia Bark, (-)-Parthenolide, Evodiamine, Chondroitin Sulfate, TPCA-1, Sodium Salicylate, Methylthiouracil, Articaine Hydrochloride, L-Quasilanol, UCB-9260, Zeaxanthin, Eriophorin, Myristyl Glycol, Gardenia Botanicum, Caulophylline (N-Methylcytisine), Demethinylberberine, 3-Hydroxyanthranilic Acid, (E / Z)-IT-603, Triptolide (PG490), Smilaxone, Berberine Hydrochloride, Pyrrolidine Dithiocarbamate Ammonium formate, stachydrine hydrochloride, sodium aescinate, stachydrine, tyrosol, mangiferin, baicalin, ginsenoside Re, dehydroevodiamine, (E)-cardamomine, musk ketone, guaiacol, curcumenol, schisandrin A, hederagenin, astragaloside IV, ginsenoside Rgl, ginsenoside Rbl, ginsenoside Rd, 4'-methoxyresveratrol, (+)-α-lipoic acid, sodium 4-aminosalicylate, IMM-H007, diethyl maleate, 4-hydroxychalcone, benfotiamine, QNZ(EVP4593), 4'-hydroxychalcone, neferine, vanillic acid, hyperoside, chelidonic acid, caffeic acid ethyl ester, sulforaphane, (R)-(-)-ibuprofen, SN50, C25 140, INH14, licorice chalcone D, SC75741, JSH-23, caffeic acid phenethyl ester, melinamide, APX-3330, DTP3, omasolone (RTA-408), bardoxolone methyl, shikonin (CI 75535), TAK-243 (MLN7243), CBL0137 HC1, withaferin A, NIK SMI1, arobuse (GS-5829), anthatin, maslinic acid, morusin A, eleutheroside E, berbamine, chrysanthemoside, aristolochic acid A, ginsenoside Rb3, 8-O-acetyl shanzhiside methyl ester, dauricine, 2',5'-dihydroxyacetophenone, (+)-peucedanin A, homoplanin, madecassic acid, and BTYNB.

[0185] All of the following, but not limited to, HSP90 inhibitors, which develop resistance, are overcome by treatment with any neuropilin-degrading compound alone and in combination with any HSP90 inhibitor that leads to resistance.

[0186] HSP90 inhibitors include lumispib (NVP-AUY922), tanespimycin (17-AAG), aspiramycin (17-DMAG) hydrochloride, ganetespib (STA-9090), ilisimol (STA-4783), BIIB021, tamoxifen (ICI46474) citrate, onaspib (AT13387), NVP-BEP800, geldanamycin (NSC 122750), SNX-2112 (PF-04928473), PF-04929113 (SNX-5422), KW-2478, XL888, Pifitrine-μ, NMS-E973, Zelavespib (PU-H71), Teprenone, Dimethylenastron, Apolazole, KRIBB11, Tebuconazole, TRC051384, DTHIB, Methamide, KNK437, VER-49009, HA15, Pimetinib (TAS-116), CH5138303, VER-50589, YUM70, Triptolide (PG490), VER155008, JG98, Tamoxifen (ICI 46474), NPX800, and HSP990 (NVP-HSP990).

[0187] All of the following, but not limited to, E3 ligase inhibitors that generate resistance that is overcome by treatment with any neuropilin-degrading compound alone and in combination with any E3 ligase inhibitor that leads to resistance.

[0188] E3 ligase inhibitors are lenalidomide (CC-5013), pomalidomide (CC-4047), thalidomide (K17), NSC207895, TAME, PRT4165, CC-885, dCBP-1, iberdomide (CC220), BC-1215, CC-90009, avadomide (CC-122), VL285, (S,R,S)-AHPC (MDK7526), Smurfl-IN-A01, (S,R,S)-AHPC-PEG4-NH2 hydrochloride, SZL P1-41, VH298, thalidomide-OH, MuRF1-IN-1, Skp2 inhibitor Cl (SKPin C1), GMB-475, mezigmid (CC-92480), isotype-PROTAC Cereblon degrader 1, THAL-SNS-032, NSC232003, Apcin, and thalidomide-O-COOH (Cereblon ligand 3).

[0189] All of the following, but not limited to, KRAS inhibitors that develop resistance that is overcome by treatment with any neuropilin-degrading compound alone and in combination with any KRAS ligase inhibitor that leads to resistance.

[0190] KRAS inhibitors include MRTX1133, sotolacib (AMG510), adagracib (MRTX849), LC-2, Deltarasin, BAY-293, ARS-1620, BI-2852, ARS-853 (ARS853), BI-3406, ASP2453, sotolacib (AMG510) racemate, Pan-RAS-IN-1, MRTX-1257, zoledronic acid (ZOL 446), lonafarnib (SCH66336), K-Ras(G12C) inhibitor 9, salirexetine, Alamandine, (Rac)-antineoplastin A10, K-Ras-IN-1, MCP110, 6H05, K-Ras(G12C) inhibitor 12, Kobe0065, K-Ras(G12C) inhibitor 6, BQU57, Kobe2602, NAV-2729, antineoplastin A10, fendiline hydrochloride, KRpep-2d, perillyl alcohol, RBC8, KY1220, CID-1067700, and zoledronic acid monohydrate.

[0191] 5. Pharmaceutical Compositions

[0192] The polypeptides described herein can be formulated into pharmaceutical compositions, which further comprise a pharmaceutically acceptable carrier, diluent, adjuvant, or vehicle. In one embodiment, the present disclosure provides a pharmaceutical composition comprising the disclosed polypeptides and a pharmaceutically acceptable carrier, diluent, adjuvant, or vehicle. In one embodiment, the present invention is a pharmaceutical composition comprising an effective amount of the disclosed bispecific antibody or pharmaceutically acceptable carrier, diluent, adjuvant, or vehicle.

[0193] Pharmaceutically acceptable carriers or excipients may contain inert ingredients that do not excessively inhibit the biological activity of the polypeptide. Pharmaceutically acceptable carriers should be biocompatible, for example, non-toxic, non-inflammatory, non-immunogenic, or have no other undesirable reactions or side effects after administration to a subject. Standard pharmaceutical formulation techniques can be used.

[0194] As used herein, pharmaceutically acceptable carriers, adjuvants or vehicles include any and all solvents, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., as suitable for the specific dosage form required. Remington's Pharmaceutical Sciences, sixteenth edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers for preparing pharmaceutically acceptable compositions and known preparation techniques thereof. Unless any conventional carrier medium is incompatible with the polypeptides described herein, such as producing any undesirable biological effect or otherwise interacting with any other component of the pharmaceutically acceptable composition in a harmful manner, its use is contemplated within the scope of the present invention. As used herein, the phrase "side effect" covers the undesirable and adverse effects of therapy.

[0195] Materials that can serve as pharmaceutically acceptable carriers for antibodies increase conformational stability, reduce protein dynamics, inhibit aggregation, and protect proteins adsorbed to liquid-air interfaces, and include, but are not limited to, cyclodextrin hydrogels, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as twin 80, phosphates, glycine, sorbic acid, or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts), colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, methylcellulose, hydroxypropyl methylcellulose, lanolin, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered Gum tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol and phosphate buffered solutions and other nontoxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives, and antioxidants.

[0196] In some embodiments, the compositions of the present invention comprise pharmaceutically acceptable salts. When the polypeptides of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such polypeptides with a sufficient amount of the desired base (neat or in a suitable inert solvent). Examples of pharmaceutically acceptable base addition salts include sodium salts, potassium salts, calcium salts, ammonium salts, organic amino salts or magnesium salts, or similar salts. When the polypeptides of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such polypeptides with a sufficient amount of the desired acid (neat or in a suitable inert solvent). Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid, or phosphorous acid, and the like, as well as salts derived from relatively nontoxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, oxalic acid, methanesulfonic acid, and the like. Also included are salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid or galacturonic acid, etc. (see Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific polypeptides disclosed herein contain both basic and acidic functional groups, allowing the polypeptides to be converted into base addition salts or acid addition salts.

[0197] Thus, the disclosed polypeptides may exist as salts, such as salts with pharmaceutically acceptable acids. The present invention includes such salts. Non-limiting examples of such salts include hydrochlorides, hydrobromides, phosphates, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, propionates, tartrates (e.g., (+)-tartrate, (-)-tartrate, or mixtures thereof, including racemic mixtures), succinates, benzoates, and salts with amino acids (such as glutamic acid) and quaternary ammonium salts (e.g., methyl iodide, ethyl iodide, etc.). These salts can be prepared by methods known to those skilled in the art.

[0198] The neutral forms of the polypeptide are preferably regenerated by contacting the salt with a base or acid and isolating the parent polypeptide in a conventional manner. The parent form of the polypeptide may differ from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0199] Certain polypeptides of the present invention may exist in unsolvated forms as well as solvated forms (including hydrate forms). In general, solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain polypeptides of the present invention may exist in polycrystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0200] In some embodiments, subcutaneous formulations may contain recombinant human PH20 hyaluronidase (rHuPH20) to facilitate dispersion of the antibody from the injection site.

[0201] 6. Combination therapy

[0202] In some embodiments, the compositions and methods described in detail herein can be used in combination with other cancer therapies. These additional cancer therapies can be administered to a subject simultaneously with, before, or after an anti-resistance agent or tyrosine kinase inhibitor. Additional cancer therapies can include, for example, chemotherapy, radiotherapy, and surgery.

[0203] Chemotherapy uses one or more chemotherapeutics to kill highly proliferative cells. Chemotherapeutics include compounds or compositions used in the treatment of cancer or other highly proliferative diseases. Chemotherapeutics can be classified according to their activity patterns in the cell, for example, whether and at which stage they affect the cell cycle. Alternatively, chemotherapeutics can be characterized based on their ability to directly crosslink DNA, insert into DNA, or induce chromosome and mitotic aberrations by affecting nucleic acid synthesis. The category of chemotherapeutics includes, for example, alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas. Special chemotherapeutics can include, for example, cyclophosphamide, doxorubicin, daunorubicin, vinblastine, vincristine, bleomycin, etoposide, topotecan, irinotecan, taxotere, paclitaxel, 5-fluorouracil, methotrexate, gemcitabine, cisplatin, carboplatin, and chlorambucil, as well as agonists of any of the above compounds.

[0204] Radiation therapy (also known as radiotherapy) is the use of ionizing radiation to treat cancer and other diseases. Ionizing radiation deposits energy that damages or destroys cells in the treated area by damaging their genetic material, rendering them unable to continue growing. Although radiation damages both cancer cells and normal cells, normal cells are able to repair themselves and function normally. Radiation therapy can be used to treat localized solid tumors, such as cancer of the skin, tongue, throat, brain, breast, or cervix. It can also be used to treat leukemias and lymphomas (cancers of the blood-forming cells and lymphatic system, respectively).

[0205] Immunotherapy can include monoclonal antibodies that recognize cancer cells and target them for destruction by the immune system. Such antibodies include rituximab (targeting CD-20), trastuzumab (targeting HER-2), and cetuximab (targeting EGFR). In some embodiments, the anti-cancer antibody is selected from rituximab, trastuzumab, and cetuximab.

[0206] Surgery can include total or partial removal of a tumor or cancerous tissue. Surgery can also include removal of any surrounding tissue. Surgery can include removal of any tissue that is considered to be at risk for cancer, such as from the spread or metastasis of cancer to that tissue.

[0207] 7. Application

[0208] The composition of the present invention can be administered to a subject in need of cancer treatment. The term "administering" refers to the act of providing a composition of the present invention (eg, a polypeptide or a pharmaceutically acceptable salt thereof) to a subject in need of cancer treatment.

[0209] As used herein, "intermittent administration" includes administering the agent for a period of time (which may be considered a "first administration period"), then discontinuing or taking the composition at a lower maintenance dose for a period of time (which may be considered a "drug-free period"), and then administering the composition for a period of time (which may be considered a "second administration period"). Typically, during the second administration period, the dosage level of the agent will match the dosage level administered during the first administration period, but may be increased or decreased as medically necessary.

[0210] As described in detail herein, the anti-resistance agent and / or tyrosine kinase inhibitor, or a pharmaceutical composition comprising the same, can be administered to a subject. As described in detail herein, the anti-resistance agent and / or tyrosine kinase inhibitor can be formulated into a composition and administered using dosages and techniques known to those skilled in the medical field, taking into account factors such as the age, sex, weight, and condition of the particular subject and the route of administration.

[0211] Anti-resistance agents and / or tyrosine kinase inhibitors can be administered prophylactically or therapeutically. In prophylactic administration, the anti-resistance agent and / or tyrosine kinase inhibitor can be administered in an amount sufficient to induce a response. In therapeutic administration, the anti-resistance agent and / or tyrosine kinase inhibitor is administered to a subject in need thereof in an amount sufficient to induce a therapeutic effect. The anti-resistance agent and / or tyrosine kinase inhibitor can be administered in a therapeutically effective amount.

[0212] For example, a therapeutically effective amount of an anti-resistance agent and / or a tyrosine kinase inhibitor, or a pharmaceutically acceptable salt thereof, can be from about 1 mg / kg to about 1000 mg / kg, from about 5 mg / kg to about 950 mg / kg, from about 10 mg / kg to about 900 mg / kg, from about 15 mg / kg to about 850 mg / kg, from about 20 mg / kg to about 800 mg / kg, from about 25 mg / kg to about 750 mg / kg, from about 30 mg / kg to about 700 mg / kg, from about 35 mg / kg to about 650 mg / kg, from about 40 mg / kg to about 500 mg / kg, or from about 50 mg / kg to about 600 mg / kg. 600 mg / kg, about 45 mg / kg to about 550 mg / kg, about 50 mg / kg to about 500 mg / kg, about 55 mg / kg to about 450 mg / kg, about 60 mg / kg to about 400 mg / kg, about 65 mg / kg to about 350 mg / kg, about 70 mg / kg to about 300 mg / kg, about 75 mg / kg to about 250 mg / kg, about 80 mg / kg to about 200 mg / kg, about 85 mg / kg to about 150 mg / kg, and about 90 mg / kg to about 100 mg / kg.

[0213] Anti-resistance agents and / or tyrosine kinase inhibitors can be administered by methods known in the art, such as those described in Donnelly et al. (Ann. Rev. Immunol. 1997, 15, 617-648); Feigner et al. (U.S. Pat. No. 5,580,859, issued December 3, 1996); Feigner et al. (U.S. Pat. No. 5,703,055, issued December 30, 1997); and Carson et al. (U.S. Pat. No. 5,679,647, issued October 21, 1997), the contents of all of which are incorporated herein by reference in their entirety. Anti-resistance agents and / or tyrosine kinase inhibitors can be complexed with particles or beads that can be administered to an individual using, for example, a vaccine gun. It will be appreciated by those skilled in the art that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration.

[0214] Anti-resistance agents and / or tyrosine kinase inhibitors can be delivered by a variety of routes. Typical delivery routes include parenteral administration, such as intradermal, intramuscular, or subcutaneous delivery. Other routes include oral administration, intranasal, intravaginal, transdermal, intravenous, intraarterial, intratumoral, intraperitoneal, and epidermal routes. In some embodiments, the anti-resistance agent and / or tyrosine kinase inhibitor is administered intravenously, intraarterially, or intraperitoneally to the subject.

[0215] The anti-resistance agent and / or tyrosine kinase inhibitor can be a liquid formulation, such as a suspension, syrup or elixir. The anti-resistance agent and / or tyrosine kinase inhibitor can be incorporated into liposomes, microspheres or other polymer matrices (e.g., by Feigner et al., U.S. Patent No. 5,703,055; Gregoriadis, Liposome Technology, Volumes I to III (2nd Edition, 1993), the contents of which are incorporated herein by reference in their entirety). Liposomes can be composed of phospholipids or other lipids and can be non-toxic, physiologically acceptable and metabolizable carriers that are relatively simple to prepare and administer.

[0216] Anti-resistance agents and / or tyrosine kinase inhibitors can be used as vaccines. Vaccines can be administered by electroporation, such as by the method described in U.S. Patent No. 7,664,545, the contents of which are incorporated herein by reference. Electroporation can be performed by the methods and / or devices described in U.S. Patent Nos. 6,302,874, 5,676,646, 6,241,701, 6,233,482, 6,216,034, 6,208,893, 6,192,270, 6,181,964, 6,150,148, 6,120,493, 6,096,020, 6,068,650, and 5,702,359, the contents of which are incorporated herein by reference in their entirety. Electroporation can be performed by minimally invasive devices.

[0217] In some embodiments, the anti-resistance agent and / or tyrosine kinase inhibitor is administered in a controlled release formulation. For example, the anti-resistance agent and / or tyrosine kinase inhibitor can be released into the blood circulation. In some embodiments, the anti-resistance agent and / or tyrosine kinase inhibitor can be released over a period of at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 1 week, at least about 1.5 weeks, at least about 2 weeks, at least about 2.5 weeks, at least about 3.5 weeks, at least about 4 weeks, or at least about 1 month.

[0218] Sterile injectable forms of the compositions described herein may be aqueous or oily suspensions. These suspensions can be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. Sterile injectable preparations may also be sterile injectable solutions or suspensions in a nontoxic, parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile, fixed oils are commonly used as solvents or suspending media. For this purpose, any bland, fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids (such as oleic acid and its glyceride derivatives) may be used to prepare injectables, as may natural pharmaceutically acceptable oils (such as olive oil or castor oil, particularly their polyoxyethylated forms). These oily solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans, and other emulsifying agents or bioavailability enhancers that are commonly used in preparing pharmaceutically acceptable dosage forms may also be used for formulation purposes.

[0219] In some embodiments, the formulation includes agents such as excipients, buffers, isotonic agents, preservatives, surfactants, and preferably zinc. The formulation may also include excipients or agents for stabilizing the polypeptide, such as buffers, reducing agents, bulk proteins, or carbohydrates. Bulk proteins that can be used to formulate at least one polypeptide composition include albumin, protamine, and the like. Typical carbohydrates that can be used to formulate at least one polypeptide include sucrose, mannitol, lactose, trehalose, glucose, and the like. The bispecific antibody formulation may also include a surfactant, which can reduce or prevent surface-induced aggregation of at least one polypeptide caused by atomization of the solution when forming an aerosol. Various conventional surfactants can be used, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitan fatty acid esters. The amount will generally range from about 0.001% to 4% by weight of the formulation. For the purposes of the present invention, particularly preferred surfactants are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, and the like. Other reagents known in the art for preparing polypeptides (such as antibody proteins) may also be included in the formulation.

[0220] Example

[0221] The examples in this specification are not intended to, and should not be used to, limit the present invention; they are provided merely to illustrate the present invention.

[0222] Example 1. Treatment with an EGFR degrader in combination with osimertinib enhances the survival of osimertinib-resistant cells. Antitumor activity in mouse xenografts

[0223] The EGFR degrader bispecific EGFP×NRP1 antibody has been found to degrade the oncogenic receptor EGFR through lysosomes and reduce cell viability in vitro. It was also found that the EGFR degrader significantly inhibited tumor growth in xenograft mouse models that were sensitive to osimertinib (H1975), resistant to osimertinib (H1975-OR), and refractory to osimertinib (H1975-HGF), respectively. All mouse xenografts had EGFR double mutations (T970M / L858R) (unpublished data). The xenograft mice (H1975-OR) resistant to osimertinib used in this article did not show anti-tumor activity at a dose of 3 mg / kg of osimertinib.

[0224] To investigate whether co-treatment with an EGFR degrader and a therapeutic enhances the therapeutic efficacy in cancers resistant to the therapeutic, an osimertinib-resistant mouse xenograft model (H1975-OR) was treated with panitumumab (10 mg / kg, ip, QD) plus osimertinib (10 mg / kg, ip, QD), an EGFR degrader (13.74 mg / kg, ip, QD) plus osimertinib (10 mg / kg), osimertinib alone, or the IgG1 isotype (5 mg / kg, ip, QD). Panitumumab is a fully humanized anti-EGFR monoclonal antibody that binds to EGFR and blocks EGFR signaling. Osimertinib is an EGFR tyrosine kinase inhibitor.

[0225] like Figure 3 As shown in , no synergistic effect was found in osimertinib-resistant tumors obtained from mice treated with panitumumab and osimertinib. This result suggests that co-treatment with osimertinib and panitumumab inhibits EGFR signaling in osimertinib-resistant tumors, while resistance to osimertinib persists. In contrast, osimertinib-resistant mice treated with an EGFR degrader and osimertinib showed more tumor regression. This result suggests that EGFR degraders degrade EGFR through lysosomes. T790M / L858R , and subsequent removal of EGFR T790M / L858R A highly effective synergistic effect was produced against osimertinib-resistant tumors. Combination therapy with a therapeutic agent and a target protein degrader enhanced the therapeutic effect of the therapeutic agent in mice resistant to the therapeutic agent. In summary, the combination therapy disclosed herein will become a new approach to overcome drug resistance and enhance the efficacy of treatment in cancer. A schematic diagram showing that combination therapy enhances the therapeutic efficacy of a therapeutic agent in patients with cancer resistant to a therapeutic agent is shown in Figure 4 middle.

[0226] Example 2. Treatment with EGFR degraders in combination with osimertinib enhances the survival of NRP1-overexpressing mouse xenografts. Antitumor activity in transplant models

[0227] In this example, a modified version of the H1975 xenograft model was used in which tumor cells were engineered to overexpress NRP1. In this xenograft model (referred to as H1975-NRP1OE), the tumor cells were EGFR L858R / T790M 、NRP1 +++ , EGFR + For the latter, treatment of the parental H1975 model with 3 mg / kg osimertinib showed strong and sustained tumor regression, whereas treatment of the H1975-NRP1 OE model with the same dose of osimertinib resulted in significant tumor progression, suggesting that NRP1 overexpression induces at least some resistance to osimertinib treatment.

[0228] To investigate whether co-treatment with EGFR degraders and osimertinib enhances the therapeutic efficacy of osimertinib in the H1975-NRP1OE xenograft model, mice were treated with panitumumab (10 mg / kg, ip, QW), EGFR degraders (13.74 mg / kg, ip, QW), osimertinib (3 mg / kg, ip, QD), EGFR degraders plus osimertinib, or IgG1 isotype control (10 mg / kg, ip, QW). The results of tumor volume over time are shown in Figure 5A The results of survival probability over time are shown in Figure 5B middle.

[0229] The results demonstrated that in this model, treatment with an EGFR degrader alone resulted in significantly superior tumor growth inhibition than treatment with panitumumab alone. Furthermore, the combination of an EGFR degrader and osimertinib produced a strong synergistic effect, leading to tumor regression. While not intended to be limited to mechanism, this suggests that removal of EGFR mutants through degradation mediated by EGFR degraders is necessary to achieve the optimal combined effect of tumor growth inhibition. Treatment with an EGFR degrader alone also resulted in an increase in median survival compared to treatment with panitumumab or osimertinib alone; and similarly, the combination of an EGFR degrader and osimertinib produced a strong synergistic effect, leading to a significant increase in mouse survival.

[0230] Thus, in summary, similar to the results observed in Example 1, EGFR degraders combined with osimertinib were the most effective treatments in inhibiting tumor growth and improving median survival in this NRP1 overexpression xenograft model.

[0231] Example 3. Treatment with EGFR degraders in combination with therapeutic agents enhances the survival of HGF-overexpressing mouse xenograft models. Antitumor activity in

[0232] In this example, a modified version of the H1975 xenograft model was used in which tumor cells were engineered to overexpress hepatocyte growth factor (HGF), a ligand for cMET. The HGF / cMET pathway was demonstrated to be active in H1975 cells by inhibiting it with either the small molecule inhibitor crizotinib (which blocks cMET pathway signaling) or the EGFR×cMET bispecific antibody construct ervantumab (which blocks cMET pathway signaling and leads to cMET degradation) (data not shown). The H1975-HGF cell line was prepared by recombinantly engineering the H1975 cell line to express HGF using standard methods. Therefore, the H1975-HGF xenograft model represents a dual activation model in which both the EGFR and cMET pathways are constitutively activated.

[0233] In the first set of experiments, to investigate whether co-treatment with an EGFR degrader and osimertinib enhances the therapeutic efficacy of osimertinib in the H1975-HGF xenograft model, mice were treated with panitumumab (5 mg / kg, ip, BIW), an EGFR degrader (6.87 mg / kg, ip, BIW), ervantumab (6.87 mg / kg, ip, BIW), osimertinib (3 mg / kg, ip, QD), an EGFR degrader plus osimertinib, or an IgG1 isotype control (5 mg / kg, ip, BIW). Tumor volume over time is shown in Figure 6A The results showed that the H1975-HGF model was resistant to both panitumumab monotherapy and low-dose osimertinib monotherapy, while EGFR degrader monotherapy and ervantumab monotherapy showed similar tumor growth inhibition. However, the combination of EGFR degrader plus osimertinib produced the strongest tumor growth inhibition over time.

[0234] In the second set of experiments, the effect of co-treatment with the EGFR degrader, osimertinib, and the cMET inhibitor crizotinib was investigated. Mice were treated with osimertinib (10 mg / kg, ip, QD), crizotinib (20 mg / kg, ip, QD), EGFR degrader (15 mg / kg, ip, QW), EGFR degrader plus osimertinib, EGFR degrader plus crizotinib, EGFR degrader plus osimertinib and crizotinib or IgG1 isotype control (5 mg / kg, ip, BIW). The results of tumor volume over time are shown in Figure 6BResults showed that in the H1975-HGF model, crizotinib monotherapy had limited efficacy, and high-dose osimertinib monotherapy failed to halt tumor growth over time. EGFR degrader monotherapy demonstrated tumor growth inhibition, while the combination of an EGFR degrader with crizotinib or osimertinib showed enhanced tumor growth inhibition compared to either agent alone. The triple combination of an EGFR degrader, osimertinib, and crizotinib demonstrated the best efficacy over time.

[0235] Thus, in summary, similar to the results observed in Examples 1 and 2, EGFR degraders combined with osimertinib or crizotinib, or with both agents, significantly improved tumor growth inhibition in this dual EGFR / cMET pathway dually activated HGF-overexpressing xenograft model.

[0236] Example 4. Treatment with an EGFR degrader in combination with a KRAS inhibitor in a mutant KRAS mouse xenograft model Enhanced anti-tumor activity

[0237] In this example, a mutant KRAS mouse xenograft model was used to examine the effects of EGFR degraders, alone or in combination with KRAS inhibitors, on tumor growth in the mutant KRAS model H358. G12C 、NRP1 + , EGFR + And responded to the irreversible KRAS G12C selective inhibitor sotolacizumab.

[0238] Mice were treated with sotolacib (5 mg / kg, ip, QD, 5x / wk), EGFR degrader (5 mg / kg, ip, QW), EGFR degrader plus sotolacib, or IgG1 isotype control (3.66 mg / kg, ip, QW). The results of tumor volume over time are shown in Figure 7 The results demonstrated that both sotolacib monotherapy and EGFR degrader monotherapy exhibited robust antitumor activity of approximately equal magnitude, while the combination of EGFR degrader and sotolacib demonstrated synergy, leading to the greatest tumor regression. These results further confirm the effects observed and reported in Examples 1-3, demonstrating the effectiveness of combining EGFR degraders with therapeutic agents that target tumor cell pathways to enhance the antitumor activity of both agents.

[0239] The description of the foregoing specific aspects will fully reveal the general nature of the present invention so that others can easily modify and / or adjust these specific aspects for various applications without departing from the general concept of the present disclosure by applying the knowledge of those skilled in the art without excessive experimentation. Therefore, based on the teachings and guidance provided herein, such adjustments and modifications are intended to fall within the meaning and scope of the equivalents of the disclosed aspects. It should be understood that the wording or terminology herein is for descriptive purposes only and not for limitation, so that those skilled in the art understand the terms or wording of this specification in accordance with the teachings and guidance.

[0240] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary aspects, but should be defined only in accordance with the following claims and their equivalents.

[0241] All publications, patents, patent applications and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application and / or other document were individually indicated to be incorporated by reference for all purposes.

[0242] Table 3: Summary of sequence listing

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

Claims

1. A method of enhancing the effect of a therapeutic agent in a subject having a cancer that is resistant or refractory to the therapeutic agent, the method comprising: administering to the subject (i) the therapeutic agent; and (ii) a target protein degrader, such that the effect of the therapeutic agent is enhanced compared to administration of the therapeutic agent alone; The target protein degrader comprises a bispecific binding molecule, wherein the bispecific binding molecule comprises: (a) a target protein binding domain that specifically binds to a target protein on a cancer cell in the subject; and (b) An NRP1 binding domain that binds to neuropilin-1 (NRP1), comprising an antibody or an NRP-1 binding fragment thereof.

2. The method of claim 1, wherein the target protein is a receptor tyrosine kinase (RTK).

3. The method of claim 2, wherein the receptor tyrosine kinase is selected from the group consisting of epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), Met receptor tyrosine kinase (MET) and vascular endothelial growth factor (VEGFR).

4. The method of claim 2, wherein the receptor tyrosine kinase is EGFR.

5. The method of claim 2, wherein the receptor tyrosine kinase is cMET.

6. The method according to claim 2, wherein the receptor tyrosine kinase is HER2.

7. The method of claim 2, wherein the receptor tyrosine kinase is IGF1R.

8. The method of claim 1, wherein the target cell is a cancer cell.

9. The method of claim 8, wherein the cancer cells are selected from the group consisting of lung cancer, breast cancer, colon and rectal cancer, head and neck cancer, esophagogastric cancer, liver cancer, glioblastoma, prostate cancer, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, and pancreatic cancer.

10. The method of claim 8, wherein the cancer cells are non-small cell lung cancer (NSCLC) cells.

11. The method of claim 1 , wherein the target protein binding domain and the NRP1 binding domain are each independently selected from IgG, half antibodies, single domain antibodies, nanobodies, Fab, monospecific Fab2, Fc, scFv, minibodies, IgNAR, V-NAR, hcIgG, VHH domains, camelid antibodies, and antibody-polypeptide conjugates.

12. The method of claim 1, wherein the NRP1 binding domain comprises: (i) an antibody heavy chain variable (VH) domain comprising CDR1, CDR2, and CDR3 regions (HCDR1, HCDR2, and HCDR3, respectively), wherein HCDR1 consists of the sequence set forth in SEQ ID NO: 79, HCDR2 consists of the sequence set forth in SEQ ID NO: 80, and HCDR3 consists of the sequence set forth in any one of SEQ ID NOs: 81-84; and (ii) an antibody light chain variable (VL) domain comprising CDR1, CDR2, and CDR3 regions (LCDR1, LCDR2, and LCDR3, respectively), wherein LCDR1 consists of the sequence set forth in any one of SEQ ID NOs: 85-87, LCDR2 consists of the sequence set forth in SEQ ID NO: 88, and LCDR3 consists of the sequence set forth in SEQ ID NO:

89.

13. The method according to claim 12, wherein: (iii) HCDR1 consists of the sequence shown in SEQ ID NO: 79, HCDR2 consists of the sequence shown in SEQ ID NO: 80, and HCDR3 consists of the sequence shown in any one of SEQ ID NO: 84; and (iv) LCDR1 consists of the sequence shown in SEQ ID NO: 85, LCDR2 consists of the sequence shown in SEQ ID NO: 88, and LCDR3 consists of the sequence shown in SEQ ID NO:

89.

14. The method of claim 1, wherein the enhanced effect comprises increased tumor growth inhibition.

15. The method of claim 1, wherein the enhanced effect comprises an increased median survival time.

16. The method of claim 1, wherein the therapeutic agent targets the EGFR pathway.

17. The method of claim 16, wherein the therapeutic agent is selected from the group consisting of lazertinib, osimertinib (AZD9291), WZ4002, cyasterone, erlotinib (OSI-774) hydrochloride, gefitinib (ZD1839), lapatinib (GW-572016) ditosylate, afatinib (BIBW2992), sacatinib (AZD0530), vandetanib (ZD6474), neratinib (HKI-272), canertinib (CI-1033), lapatinib (GW-572016), AG-490 (tyrosine phosphorylation inhibitor B42), CP-724 714, dacomitinib (PF-00299804), sabutinib (AZD8931), CUDC-101, AG-1478 (tyrosine phosphorylation inhibitor AG-1478), PD153035 hydrochloride, pelitinib (EKB-569), AC480 (BMS-599626), AEE788 (NVP-AEE788), AP26113-analog (ALK-IN-1), OSI-420, WZ3146, HER2-inhibitor-1, WZ8040, alitinib tosylate, rociletinib (CO-1686), genistein (NPI 031L), valitinib, TQB33804 (EGFR-IN-7), icotinib (BPI-2009H), TAK-285, daphnetin, tyrosine phosphorylation inhibitor 9, AG-18, AG 555, AZ5104, CL-387785 (EKI-785), tyrosine phosphorylation inhibitor AG-258, AG-556, tucatinib, erlotinib (OSI-774), gefitinib-based PROTAC 3, zolitinib (AZD 3759), ErbB2, AV-412 free base, AST-1306, JND3229, BI-4020, cilitinib (HMPL-309), BDTX-189, rifurafenib (BGB-283), pyrotinib (SHR-1258), O-desmethyl gefitinib, epatinib hydrochloride, SU5214, avitinib (AC0010), AG 494, and poziotinib (HM781-36B).

18. The method of claim 16, wherein the therapeutic agent is osimertinib.

19. The method of claim 1, wherein the therapeutic agent targets the cMET pathway.

20. The method of claim 19, wherein the therapeutic agent is selected from crizotinib, cabozantinib, foretinib, PHA-665752, SU11274, SGX-523, BMS-777607, tivatinib, JNJ-38877605, PF-04217903, amvatinib (MP-470), MGCD-265 analogs, capmatinib, BMS-754807, BMS-794833, AMG-208, MK-2461, govatinib, AMG-458, NVP-BVU972, AMG 337, Meritinib, JNJ-38877618, Crizotinib Hydrochloride, Ningetinib, AMG-1, UNC2025, Pamufetinib, Atelatinib, NPS-1304, and Savotinib.

21. The method of claim 19, wherein the therapeutic agent is crizotinib.

22. The method of claim 1, wherein the therapeutic agent targets the KRAS protein.

23. The method of claim 22, wherein the therapeutic agent is selected from MRTX1133, Sotolacib (AMG510), Adalasib (MRTX849), LC-2, Deltarasin, BAY-293, ARS-1620, BI-2852, ARS-853 (ARS853), BI-3406, ASP2453, Sotolacib (AMG510) racemate, Pan-RAS-IN-1, MRTX-1257, Zoledronic acid (ZOL 446), lonafarnib (SCH66336), K-Ras(G12C) inhibitor 9, salirexetine, Alamandine, (Rac)-antineoplastin A10, K-Ras-IN-1, MCP110, 6H05, K-Ras(G12C) inhibitor 12, Kobe0065, K-Ras(G12C) inhibitor 6, BQU57, Kobe2602, NAV-2729, antineoplastin A10, fendiline hydrochloride, KRpep-2d, perillyl alcohol, RBC8, KY1220, CID-1067700, and zoledronic acid monohydrate.

24. The method of claim 23, wherein the therapeutic agent is sotolacib.

25. The method of claim 1, wherein the therapeutic agent targets a protein selected from the group consisting of HER2, IGF1R, ALK, Braf, VEGF, and PDGF.

26. The method of claim 1, further comprising administering to the subject a second therapeutic agent, wherein the protein target degrader enhances the effect of the second therapeutic agent compared to administration of the second therapeutic agent alone.

27. The method of claim 26, wherein the first therapeutic agent targets EGFR and the second therapeutic agent targets cMET.

28. The method of claim 27, wherein the therapeutic agent is osimertinib and the second therapeutic agent is sotolacib.

29. A method of enhancing the effect of a receptor tyrosine kinase (RTK) inhibitor in a subject having a cancer that is resistant or refractory to the RTK inhibitor, the method comprising: administering to the subject (i) the RTK inhibitor; and (ii) a target protein degrader, such that the effect of the RTK inhibitor is enhanced compared to administration of the RTK inhibitor alone; The target protein degrader comprises a bispecific binding molecule, wherein the bispecific binding molecule comprises: (a) a target protein binding domain that specifically binds to a RTK on a cancer cell in the subject; and (b) An NRP1 binding domain that binds to neuropilin-1 (NRP1), comprising an antibody or an NRP-1 binding fragment thereof.

30. A method of enhancing the effect of an epidermal growth factor receptor (EGFR) inhibitor in a subject having a cancer that is resistant or refractory to the EGFR inhibitor, the method comprising: administering to the subject (i) the EGFR inhibitor; and (ii) a target protein degrader, such that the effect of the EGFR inhibitor is enhanced compared to administration of the EGFR inhibitor alone; The target protein degrader comprises a bispecific binding molecule, wherein the bispecific binding molecule comprises: (a) a target protein binding domain that specifically binds to EGFR on a cancer cell in the subject; and (b) An NRP1 binding domain that binds to neuropilin-1 (NRP1), comprising an antibody or an NRP-1 binding fragment thereof.

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