Methods and materials for treating cancer

Abstract

This document provides methods and materials involved in treating cancer. For example, methods and materials for using (a) one or more antibody-drug conjugates (ADCs) and (b) one or more proteolysis-targeting chimera (PROTAC) compounds to treat a mammal (e.g., a human) having cancer are provided.

Description

[0001] METHODS AND MATERIALS FOR TREATING CANCER

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 560,270, filed on March 1, 2024. The entire contents of which are hereby incorporated by reference.

[0004] TECHNICAL FIELD

[0005] This document relates to methods and materials involved in treating cancer. For example, this document provides methods and materials for using (a) one or more antibodydrug conjugates (ADCs) and (b) one or more proteolysis-targeting chimera (PROTAC) compounds for treating a mammal (e.g., a human) having cancer.

[0006] BACKGROUND

[0007] Cancer is a leading cause of death worldwide. By 2040, the estimated number of cancer related deaths is expected to increase to 16.4 million (National Cancer Institute, Cancer Statistics, Published September 25, 2020). ADCs are a group of cancer treatments that deliver drugs to tumor cells that express specific tumor antigens. Despite the success of ADCs such as trastuzumab deruxtecan, off-target toxicity remains a major challenge. Thus, there is a need to optimize the therapeutic modality of ADCs to improve its efficacy. See, e g., Dumontet et al., Nat. Rev. Drug Di sc w, 22(8) 641-661 (2023); Tsuchikama et al., Nat. Rev. Clin. Oncol., 21(3)1203-223 (2024); Tarantino et al., Nat. Rev. Clin. Oncol., 20(8): 558- 576 (2023); Drago et al., Nat. Rev. Clin. Oncol., 18(6)1327-344 (2021); and Fu et al., Signal Transduct. Target Ther., 7(1)193 (2022).

[0008] SUMMARY

[0009] This document provides methods and materials involved in treating cancer. For example, this document provides methods and materials for using (a) one or more ADCs and (b) one or more PROTAC compounds for treating a mammal (e.g., a human) having cancer. PROTAC compounds are bifunctional molecules that include a targeting moiety and an E3 ligase ligand (and, optionally, a linker connecting the targeting moiety and the E3 ligase ligand) that can recruit an E3 ligase polypeptide to a polypeptide targeted (e.g., targeted and bound) by the targeting moiety resulting in ubiquitination of the target polypeptide (e.g., to mark the target polypeptide for degradation through polyubiquitylation). As demonstrated herein, PROTAC compounds that can target (e.g., can target and mark for degradation) a particular surface antigen (e.g., a particular surface polypeptide) expressed by a cell can increase cellular internalization of ADCs including ADCs that target that same surface antigen. For example, when a mammal (e.g., a human such as a human having cancer) is administered (a) one or more ADCs and (b) one or more PROTAC compounds where the ADC(s) and the PROTAC compound(s) can bind the same target polypeptide on a cell, the PROTAC compound can mark the target polypeptide for degradation and the cell can internalize the target polypeptide together with the bound ADC(s). Having the ability to improve cellular internalization of ADCs as described herein (e.g., by administering the ADCs together with one or more PROTAC compounds that can target (e.g., target and mark for degradation) the same target polypeptide as the ADC) provides a unique and unrealized opportunity to improve the cytotoxicity of ADCs (e.g., thereby improving the efficacy of treatment with ADCs).

[0010] In general, one aspect of this document features methods for treating a mammal having cancer. The methods can include, or consist essentially of, (a) administering an antibody-drug conjugate (ADC) to a mammal having cancer, and (b) administering a proteolysis-targeting chimera (PROTAC) compound to the mammal, where the ADC and the PROTAC target a surface antigen expressed by a cancer cell in the mammal. The mammal can be a human. The cancer can be a pancreatic cancer, an ovarian cancer, a cervical cancer, a myeloma, a breast cancer, a melanoma, a lung cancer, a mesothelioma, or a sarcoma. The surface antigen can be a HER-2 polypeptide, an EGFR polypeptide, a MET polypeptide, an estrogen receptor polypeptide, an androgen receptor polypeptide, a RET polypeptide, an ALK polypeptide, a TROP-2 polypeptide, a CLDN18.2 polypeptide, a HER3 polypeptide, a nectin-4 polypeptide, a CD276 polypeptide, a folate receptor polypeptide (e.g., a folate receptor alpha polypeptide), a PD-L1 polypeptide, a PD-1 polypeptide, a CD19 polypeptide, a BCMA polypeptide, a R0R1 polypeptide, a CD30 polypeptide, a CD22 polypeptide, a PSMA polypeptide, a mesothelin polypeptide, a B7-H3 polypeptide, a B7-H4 polypeptide, a CD70 polypeptide, a CLDN6 polypeptide, an EphA2 polypeptide, a MUCl polypeptide, a CD20 polypeptide, a CD33 polypeptide, a CD37 polypeptide, a CD79b polypeptide, a tissue factor (TF) polypeptide, an AXL polypeptide, a R0R2 polypeptide, an ALCAM polypeptide, a CD 123 polypeptide, a CD25 polypeptide, a CEACAM5 polypeptide, a NaPi2b polypeptide, or a STING polypeptide. The ADC can include an antigen-binding domain that binds the surface antigen. The antigen-binding domain can be trastuzumab, cetuximab, atezolizumab, amivantamab, telisotuzumab, anetumab, tarlatamab, enfortumab, sacituzumab, datopotamab, mirvetuximab, tisotumab, disitamab, loncastiuximab, belantamab, polatuzumab, inotuzumab, brentuximab, gemtuzumab, patritumab, depatuxizumab, ifmatamab, tusamitamab, upifitamab, vobramitamab, zilovertamab, camidanlumab, coltuximab, farletuzumab, glembatumumab, indatuximab, labetuzumab, luveltamab, mecbotamab, naratuximab, ozuriftamab, praluzatamab, epratuzumab, pivekimab, aprutumab, bivatuzumab, cofetuzumab, ispectamab, losatuxizumab, or lupartumab. The ADC can include an anti-cancer drug selected from the group consisting of deruxtecan, emtansine (DM1), ravtansine (DM4), vedotin, ozogamicin, govitecan mafodotin, pasudotox, tesirine, soravtansine, sarotalocan, mafodotin, govitecan, ozogamicin, tirumotecan, botidotin, exatecan, camptothecin, 7- aminom ethyl- 10, 11 -methylenedi oxy camptothecin (AMDCPT), indenoisoquinoline, dibenzonapthyridinone, fluoroindenoisoquinoline, belotecan, auristatin, epothilone, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), calicheamicin, SN- 38, and Dxd. The ADC can be trastuzumab deruxtecan, trastuzumab emtansine, telisotuzumab vedotin, anetumab ravtansine, datopotamab deruxtecan, gemtuzumab ozogamicin, brentuximab vedotin, inotuzumab ozogamicin, polatuzumab vedotin, enfortumab vedotin, sacituzumab govitecan, loncastuximab tesirine, tisotumab vedotin, mirvetuximab soravtansine, or belantamab mafodotin. The PROTAC compound can include a targeting moiety that can bind the surface antigen. The targeting moiety can be lapatinib, capmatinib, erlotinib, afatinib, alectinib, brigatinib, lorlatinib, tepotinib, crizotinib, or ceritinib. The PROTAC compound can include an E3 ligase ligand selected from the group consisting of a von Hippel-Lindau (VHL) polypeptide, a cereblon (CRBN) polypeptide, an AHR polypeptide, a RNF4 polypeptide, a DCAF11 polypeptide, a DCAF15 polypeptide, a DCAF16 polypeptide, a MDM2 polypeptide, and a XIAP polypeptide. The targeting moiety and the E3 ligase ligand can be connected by a linker. The PROTAC compound can be SJF- 1528, 48-284, CFT8634, CFT8919, CG001419, CFT1946, KT-253, AC0176, CC-94676, HP518, GT20029, AC0682, HSK29116, BGB-16673, RNK05047, AC682, ARV-766, DT2216, FHD-609, KT-474, KT-413, KT-333, NX-2127, NX-5948, CC-99282, CFT7455, DKY709, ARV-110, ARV-471, CC-220, CC-92480, or CC-90009.

[0011] In another aspect, this document features methods for enhancing cellular internalization of an ADC. The methods can include, or consist essentially of, (a) contacting a cell with an ADC; and (b) contacting the cell with a PROTAC compound, where the ADC and the PROTAC compound can target a surface antigen expressed by the cell, and where the amount of the ADC that enters the cell is more than the amount that enters a comparable cell not contacted with the PROTAC compound. The cell can be within a mammal. The contacting can include administering the ADC and the PROTAC compound to the mammal. The mammal can be a human. The mammal can have cancer. The cancer can be a pancreatic cancer, an ovarian cancer, a cervical cancer, a myeloma, a breast cancer, a melanoma, a lung cancer, a mesothelioma, or a sarcoma. The surface antigen can be a HER-2 polypeptide, an EGFR polypeptide, a MET polypeptide, an estrogen receptor polypeptide, an androgen receptor polypeptide, a RET polypeptide, an ALK polypeptide, a TROP-2 polypeptide, a CLDN18.2 polypeptide, a HER3 polypeptide, a nectin-4 polypeptide, a CD276 polypeptide, a folate receptor polypeptide (e.g., a folate receptor alpha polypeptide), a PD-L1 polypeptide, a PD-1 polypeptide, a CD19 polypeptide, a BCMA polypeptide, a RORl polypeptide, a CD30 polypeptide, a CD22 polypeptide, a PSMA polypeptide, a mesothelin polypeptide, a B7-H3 polypeptide, a B7-H4 polypeptide, a CD70 polypeptide, a CLDN6 polypeptide, an EphA2 polypeptide, a MUC1 polypeptide, a CD20 polypeptide, a CD33 polypeptide, a CD37 polypeptide, a CD79b polypeptide, a tissue factor (TF) polypeptide, an AXL polypeptide, a R0R2 polypeptide, an ALCAM polypeptide, a CD123 polypeptide, a CD25 polypeptide, a CEACAM5 polypeptide, a NaPi2b polypeptide, or a STING polypeptide. The ADC can include an antigen-binding domain that can bind the surface antigen. The antigen-binding domain can be trastuzumab, cetuximab, atezolizumab, amivantamab, telisotuzumab, anetumab, tarlatamab, enfortumab, sacituzumab, datopotamab, mirvetuximab, tisotumab, disitamab, loncastiuximab, belantamab, polatuzumab, inotuzumab, brentuximab, gemtuzumab, patritumab, depatuxizumab, ifinatamab, tusamitamab, upifitamab, vobramitamab, zilovertamab, camidanlumab, coltuximab, farletuzumab, glembatumumab, indatuximab, labetuzumab, luveltamab, mecbotamab, naratuximab, ozuriftamab, praluzatamab, epratuzumab, pivekimab, aprutumab, bivatuzumab, cofetuzumab, ispectamab, losatuxizumab, or lupartumab. The ADC can include an anti-cancer drug selected from the group consisting of deruxtecan, emtansine (DM1), ravtansine (DM4), vedotin, ozogamicin, govitecan mafodotin, pasudotox, tesirine, soravtansine, sarotalocan, mafodotin, govitecan, ozogamicin, tirumotecan, botidotin, exatecan, camptothecin, AMDCPT, indenoisoquinoline, dibenzonapthyridinone, fluoroindenoisoquinoline, belotecan, auristatin, epothilone, MMAE, MMAF, calicheamicin, SN-38, and Dxd. The ADC can be trastuzumab deruxtecan, trastuzumab emtansine, telisotuzumab vedotin, anetumab ravtansine, datopotamab deruxtecan, gemtuzumab ozogamicin, brentuximab vedotin, inotuzumab ozogamicin, polatuzumab vedotin, enfortumab vedotin, sacituzumab govitecan, loncastuximab tesirine, tisotumab vedotin, mirvetuximab soravtansine, or belantamab mafodotin. The PROTAC compound includes a targeting moiety that binds the surface antigen. The targeting moiety can be lapatinib, capmatinib, erlotinib, afatinib, alectinib, brigatinib, lorlatinib, tepotinib, crizotinib, or ceritinib. The PROTAC compound can include an E3 ligase ligand selected from the group consisting of a VHL polypeptide, a CRBN polypeptide, an AHR polypeptide, a RNF4 polypeptide, a DCAFll polypeptide, a DCAF15 polypeptide, a DCAF16 polypeptide, a MDM2 polypeptide, and a XIAP polypeptide. The targeting moiety and the E3 ligase ligand can be connect by a linker. The PROTAC compound can be SJF-1528, 48-284, CFT8634, CFT8919, CG001419, CFT1946, KT-253, AC0176, CC-94676, HP518, GT20029, AC0682, HSK29116, BGB-16673, RNK05047, AC682, ARV-766, DT2216, FHD-609, KT- 474, KT-413, KT-333, NX-2127, NX-5948, CC-99282, CFT7455, DKY709, ARV-110, ARV- 471, CC-220, CC-92480, or CC-90009.

[0012] 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 invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figures 1A-1C. PROTACs improved internalization of antibodies targeting oncogenic tyrosine kinase receptors. Cellular internalization of labeled antibodies was evaluated by in vitro cell-live imaging and was plotted as the area under the curve (AUC) of integrated intensity over the percentage of cell confluence as (red calibrated unit (RCU) x pM2 / well / %). Figure 1A) Cellular internalization of antibodies by breast cancer cell lines expressing HER2 (BT-474 and SKBR-3) and treated with PROTAC SJF-1528 100 nM + trastuzumab (Tmab) at 4 pg / mL compared against DMSO + Tmab at 4 pg / mL as a control condition. Figure IB) Cellular internalization of antibodies by lung cancer cells (A549) and pancreatic (BxPC3) cancer cells expressing an epidermal growth factor receptor (EGFR) polypeptide and treated with PROTAC SJF-1528 100 nM + cetuximab (Cmab) at 4 pg / mL and 2 pg / mL, respectively. Figure 1C) Cellular internalization of antibodies by mesenchymal -epithelial transition factor (Met) cell models treated with PROTAC 48-284 500 nM + anti-Met antibody at 4 pg / mL for HCC827 GR6 cells and Hek293T cells treated with PROTAC 48-284 500 nM + anti-Met antibody or human IgG control at 4 pg / mL. Data shown here are representative experiments, every condition was done in triplicate and lines and error bars represent the medians and S.E.M. Statistical significance was evaluated with GraphPad Prism 10 by unpaired t test and two-tailed p value. ***P<0.001; ****7’<0.0001.

[0015] Figures 2A-2B. Antibody internalization was antibody and PROTAC concentration dependent. Internalization of Tmab and Cmab was evaluated by in vitro cell-live imaging and was plotted as AUC (RCU x pM2 / Well / %) as described for Figure 1. Figure 2A) Cellular internalization of antibodies by SKBR-3 cells treated with PROTAC SJF1528 100 nM + Tmab (4 pg / mL, 2 pg / mL, or 1 pg / mL) and BxPC3 cells treated with PROTAC SJF1528 100 nM + Cmab (4 pg / mL, 2 pg / mL, or 1 pg / mL). Control conditions included antibodies without PROTAC. Figure 2B) Cellular internalization of antibodies by SKBR-3 cells treated with Tmab 2 pg / mL + PROTAC SJF1528 at (200 nM, 100 nM, or 50 nM) and BT-474 cells treated with Tmab 2 pg / mL + PROTAC SJF1528 at (200 nM or 100 nM). Figures 3A-3D. Dyngo4a disrupted PROTAC induced-antibody internalization and MG132 blocked intemalized-antibody degradation. Antibody internalization was evaluated in the presence of endocytosis inhibitors (Dyngo4a and Pitstop2) and the proteosome inhibitor MG132. Figure 3A) BT-474 and SKBR-3 cells were assayed by in vitro cell-live imaging for internalization of antibodies when treated with Tmab + PROTAC SJF-1528 in presence of the dynamin mediated endocytosis inhibitor Dyngo4a (30 pM or 15 pM). Figure 3B) BT-474 and SKBR-3 cells were also assayed for internalization of antibodies when treated with Tmab + PROTAC SJF-1528 in presence of the clathrin inhibitor Pitstop2 which inhibits clathrin-mediated endocytosis and clathrin-coated vesicle transport. Figure 3C) Time course evaluation of internalization of antibodies by BT-474 cells treated with or without SJF-1528 200 nM + Tmab 2 pg / mL and SJF-1528 200 nM + Tmab 2 pg / mL + MG132 5 pM. Internalized Tmab was evaluated by western blotting with antibodies specific for human IgG light chains, human IgG heavy chains, and HER2. Figure 3D) Representative images of immunofluorescence staining of BT-474 cells treated overnight with SJF1528 200 nM + labeled Tmab 2 pg / mL or SJF1528 200 nM + Tmab 2 pg / mL + MG132 5 pM and a FITC conjugated secondary antibody specific for human IgG.

[0016] Figures 4A-4C. PROTAC enhanced the cytotoxic activity of ADCs. Cell viability was assayed on BT-474 and SKBR-3 cells treated with PROTAC SJF-1528 at 200 nM or 100 nM and a range of concentrations of Tmab-DMl (TDM1, KADCYLA®) or Tmab-deruxtecan (ENHERTU®) for 24, 48, and 72 hours. The data are represented as a percentage of surviving cells as relative luminescence units (RLU), with or without PROTAC. Dose-response curves were generated with GraphPad Prism 10 and four-parameter fitting curves (inhibitor vs response variable slope) and log of KADCYLA® and ENHERTU® concentration use. Figure 4A) Representative graphs of independent experiments showing viability of BT-474 cells after 24, 48, and 72 hours of treatment with KADCYLA®. Figure 4B) Representative graphs of independent experiments showing viability of SKBR-3 cells after 24, 48, and 72 hours of treatment with ENHERTU®. Figure 4C) Representative images of BT-474 cells without treatment and treated with PROTAC SJF-1528 200 nM only, TDM1 1 pg / mL only, and the combination PROTAC SJF-1528 200 nM + TDM1 1 pg / mL at the indicated time points.

[0017] Figures 5A-5B. PROTAC increased internalization of antibodies directed to cell surface proteins. Representative phase and red fluorescent images captured during the in vitro cell imaging assay for antibody internalization experiments. Figure 5A) BT-474 cells assayed with PROTAC SJF1528 + Tmab or Tmab only after 6 and 12 hours of treatment. Figure 5B) HCC827 GR6 cells treated with PROTAC 48-284 + anti-MET antibody or anti- Met only after 6 and 12 hours of treatment.

[0018] Figures 6A-6C. Tmab internalization was affected by endocytosis inhibitors. Tmab internalization was evaluated as control in the presence of endocytosis inhibitors Dyngo4a and Pitstop. Both inhibitors affected the internalization of antibodies when evaluated by cell- live imaging for 48 hours. Figure 6A) BT-474 and SKBR-3 cells treated with Dyngo4a (30 pM and 15 pM) were assayed in vitro for Tmab internalization. Figure 6B) BT-474 and SKBR-3 cells were also assayed for Tmab internalization in the presence of Pitstop2 (30 pM and 15 pM). Figure 6C) Time course evaluation of internalization of antibodies by SKBR-3 cells treated with or without SJF1528 200 nM + Tmab 2 pg / mL and SJF1528 200 nM + Tmab 2 pg / mL + MG132 5 pM. Internalized Tmab was evaluated by western blotting with antibodies specific for human IgG light chain, human IgG heavy chain, and HER2.

[0019] Figures 7A-7C. PROTACs improved ADC-efficacy and reduced cell survival. Representative phase images of BT-474 cells treated with SJF1528 200 nM and various concentrations of Tmab-Dml 24 hours after treatment (Figure 7A), 48 hours after treatment (Figure 7B), and 72 hours after treatment (Figure 7C).

[0020] Figures 8A-8C. PROTACs improved internalization of antibodies targeting oncogenic tyrosine kinase receptors. Cellular internalization of labeled antibodies was evaluated by in vitro live-cell imaging and plotted as AUC of Red integrated intensity over percentage of cell confluence as (RCU x pM2 / Well / %). Figure 8A) Cellular internalization in breast cancer cell lines expressing HER2 (BT-474 and SKBR3) that were treated with PROTAC SJF1528 at 100 nM + Tmab at 4 pg / mL compared against DMSO + 4 pg / mL of Tmab control condition. Figure B) Cellular internalization in lung (A549) and pancreatic (BxPC-3) cancer cells expressing EGFR that were treated with PROTAC SJF1528 at lOOnM + Cmab at 4 pg / mL and 2 pg / mL, respectively. Figure 8C) Cellular internalization in MET cell models that were treated with PROTAC 48-284 at 500 nM + anti-MET antibody at 4 pg / mL for HCC827 GR6 (Lung) and HEK 293T cells treated with PROTAC 48-284 at 500 nM + anti-MET antibody at 4 pg / mL. Data shown here are representative experiments. Every condition was tested in triplicate, and lines and error bars represent the medians and S.E.M. Statistical significance was evaluated with GraphPad Prism 10 by unpaired t test and two- tailed p value. ***P<0.001 ****P<0.0001.

[0021] Figures 9A-9C. Antibody internalization was antibody and PROTAC concentration dependent and relied on degrader target specific activity. Internalization of Tmab, Cmab and anti-MET was evaluated by in vitro live-cell imaging and plotted as AUC (RCU x pM2 / Well / %) as described for Figure 8. Figure 9A) SKBR3 cells were treated with 100 nM of PROTAC SJF1528 + Tmab (4 pg / mL, 2 pg / mL, 1 pg / mL), and BxPC-3 cells were treated with Cmab (4 pg / mL, 2 pg / mL, 1 pg / mL). Control condition was DMSO without the PROTAC. Figure 9B) SKBR3 cells were treated with 2 pg / mL of Tmab + PROTAC SJF1528 (200 nM, 100 nM, or 50 nM), and BT-474 cells were treated with 2 pg / mL of Tmab + PROTAC SJF1528 (200 nM or 100 nM). Control condition was DMSO without the PROTAC. Figure 9C) SKBR-3 cells were treated with Tmab (2 pg / mL) or human IgG (2 pg / mL) + a PROTAC (either PROTAC SJF1528 (200 nM), off-target antigen specificity PROTAC SJF1521 (200 nM), or an inactive degrader SJF0661 (200 nM)). HCC827 GR6 cells were treated with anti-MET (2 pg / mL) or human IgG (2 pg / mL) + a PROTAC (either PROTAC 48-284 (500 nM) or inactive degrader 48-279 (500 nM)). Data shown here are representative experiments, every condition was tested in triplicate, and lines and error bars represent the medians and S.E.M.

[0022] Figures 10A-10D. Endocytosis and Ubal inhibitors disrupted PROTAC -induced antibody internalization, and MG132 blocked internalized-antibody degradation. Antibody internalization was evaluated in the presence of endocytosis inhibitors (Dyngo-4a and Pitstop2) and ubiquitin-proteosome system inhibitors (TAK-243, PYZD-4409, and MG132). Figure 10A) BT-474 and SKBR3 cells were assayed by in vitro live-cell imaging for Tmab internalization in the presence of PROTAC SJF1528 and the dynamin mediated endocytosis inhibitor Dyngo-4a (30 pM and 15 pM). Figure 10B) SKBR3 and HCC827 GR6 cells were assayed for Tmab or anti-MET internalization, respectively, in the presence of PROTAC SJF1528 or 48-284 and the Ubal inhibitors TAK-243 and PYZD-4409. Figure 10C) Time course evaluation of the internalized Tmab by western blots using antibodies specific against the light and heavy human IgG chains and HERZ expression on SKBR3 cells that were treated with or without SIF1528 (200 nM) + Tmab (2 pg / mL) or SIF1528 (200 nM) + Tmab (2 pg / mL) + MG132 (5 pM). Figure 10D) Representative images of immunofluorescence staining of BT-474 cells assayed overnight with SJF1528 (200 nM) + labeled Tmab (2 pg / mL) or SJF1528 200 nM + Tmab 2 pg / mL + MG132 5 pM + a secondary FITC- conjugated antibody for human IgG. Data shown here are representative experiments, every condition was tested in triplicate, and lines and error bars represent the medians and S.E.M. Statistical significance was evaluated with GraphPad Prism 10 by unpaired t test and two- tailed p value. **** <0.0001.

[0023] Figures 11A-11E. PROTACs enhanced the cytotoxic activity of ADCs. Viability and proliferation were assayed on HER2-positive cells (BT-474 and SKBR3) and breast cancer organoids (BJ11) treated with PROTAC SJF1528 (at 200 nM or 100 nM) and a range of concentrations of Tmab-DMl or Tmab-Deruxtecan. Also, lapatinib and lapatinib with Tmab- Deruxtecan combination were assayed in SKBR-3 cells and with similar ADC concentrations as described above. The data are represented as percentage of surviving cells (RLU) or relative area (pM2), normalized to a non-treated control condition (DMSO). Figure HA) Viability of BT-474 cells after 24, 48, and 72 hours of treatment. Figure 1 IB) Representative images of BT-474 cells without treatment (DMSO) and cells treated with PROTAC SJF1528 only (200 nM), Tmab-DMl only (1 pg / mL), or a combination of PROTAC SJF1528 (200 nM) + Tmab-DMl (1 pg / mL) at 72 hours. Figure 11C) Viability of SKBR-3 cells after 24, 48, and 72 hours of treatment. Figure 1 ID) Representative graphs of independent experiments with breast cancer organoids (BJ11) showing relative area as measurement of proliferation rate in a single organoid experimental setting and as the percentage of surviving cells (RLU) with multiple organoids / well after 72 hours of treatment. Figure 1 IE) Survival of SKBR-3 cells was assayed with lapatinib and the combinations of lapatinib + Tmab-

[0024] Deruxtecan, and SJF1528 + Tmab-Deruxtecan for comparison. - DMSO non-treated control; PROTAC (SJF1528) treatment; lapatinib at 100 nM and

[0025] > . . > . . > . . iapapnibat 200 nM, respectively. Data shown here are representative experiments, every condition was tested in triplicate, and lines and error bars represent the medians and S.E.M. Individual data points are shown.

[0026] Figures 12A-12B. PROTACs increased antibody internalization directed against cell surface proteins. Representative phase and red fluorescent images were captured during in vitro cell imaging assay for antibodies internalization experiments. Figure 12A) BT-474 cells were assayed with PROTAC SJF1528 + Tmab or Tmab only as a control after 6 and 12 hours of treatment. Figure 12B) HCC827 GR6 cells were treated with PROTAC 48-284 + anti- MET antibody or anti -MET only after 6 and 12 hours of treatment.

[0027] Figures 13A-13D. Tmab internalization was affected by endocytosis inhibitors. Tmab internalization was evaluated as control in the presence of endocytosis inhibitors Dyngo-4a and Pitstop2. Both inhibitors differentially affected antibody internalization in the presence of PROTAC SJF1528 or DMSO control as evaluated by live-cell imaging for 48 hours. Figure 13 A) BT-474 and SKBR3 cells treated with Dyngo-4a (30 pM and 15 pM) were assayed in vitro for Tmab internalization with DMSO control. Figure 13B) BT-474 and SKBR3 cells were also assayed for Tmab internalization in the presence of Pitstop2 (30 pM and 15 pM) and PROTAC SJF1528 at 200 nM. Figure 13C) BT-474 and SKBR3 cells were assayed for Tmab internalization in the presence of Pitstop2 (30 pM and 15 pM). Figure D) Time course evaluation of the internalized Tmab by western blots with antibodies specific against the light and heavy human IgG chains and HER2 in BT-474 cells treated with or without SJF1528 200nM + Tmab 2 pg / mL, and SJF1528 200nM + Tmab 2 pg / mL + MG132 5 pM.

[0028] Figures 14A-14C. PROTAC improved ADC efficacy and reduced cell survival. Representative phase images of BT-474 cells treated with SJF1528 200 nM and various concentrations of Tmab-DMl 24 hours after treatment (Figure 14A), 48 hours after treatment (Figure 14B), and 72 after treatment (Figure 14C).

[0029] DETAILED DESCRIPTION

[0030] This document provides methods and materials involved in treating cancer. For example, this document provides methods and materials for using (a) one or more ADCs and (b) one or more PROTAC compounds to treat a mammal (e.g., a human) having cancer. In some cases, a mammal (e.g., a human such as a human having cancer) can be administered (a) one or more ADCs and (b) one or more PROTAC compounds to increase the cellular internalization of the ADC(s), thereby enhancing the anti -turn or effects of the ADC(s). For example, one or more PROTAC compounds and one or more ADCs, where both the

[0031] PROTAC compound(s) and the ADC(s) target the same polypeptide (e.g. same cell surface polypeptide), can be administered to a mammal (e.g., a human) having cancer (e.g., a cancer including one or more cancer cells that express the polypeptide targeted by both the PROTAC compound(s) and the ADC(s)) such that the PROTAC compounds and the ADC(s) target (e.g., target and bind) cancer cells expressing the target polypeptide. The PROTAC compound(s) can mark the target polypeptide for degradation (e.g., via ubiquitination), and thereby be used to cause cells expressing the target polypeptide to internalize (e.g., via endocytosis) the target polypeptide together with the bound ADC(s).

[0032] In some cases, the methods and materials provided herein can be effective to enhance cellular internalization of one or more ADCs. For example, when (a) one or more ADCs and (b) one or more PROTAC compounds targeting the same polypeptide expressed by a cell (e.g., a cancer cell) are administered to a mammal in need thereof (e.g., a mammal having cancer such as a human having cancer) greater than 1-fold (e.g., greater than 1.1 fold, greater than 1.3-fold, greater than 1.4-fold, greater than 1.5-fold, greater than 1.6-fold, greater than 1.7-fold, greater than 18-fold, greater than 1.9-fold, or greater than 2-fold) more of the ADC(s) can be internalized by the cells within the mammal (e g., as compared to cells that are administered ADCs in the absence of PROTACs).

[0033] In some cases, the methods and materials provided herein can be used to reduce the size of cancer within a mammal. For example, a mammal in need thereof (e.g., a mammal having cancer such as a human having cancer) can be administered (a) one or more ADCs and (b) one or more PROTAC compounds targeting the same polypeptide expressed by a cell (e.g., a cancer cell) to reduce the size of the cancer in the mammal. In some cases, the methods and materials provided herein can be used as described herein to reduce the number of cancer cells in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, the methods and materials provided herein can be used as described herein to reduce the volume of one or more solid tumors in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

[0034] In some cases, the methods and materials provided herein can be used to improve survival of a mammal (e.g., a human) having cancer. For example, a mammal in need thereof (e.g., a mammal having cancer such as a human having cancer) can be administered (a) one or more ADCs and (b) one or more PROTAC compounds targeting the same polypeptide expressed by a cell (e.g., a cancer cell) to improve survival of the mammal. For example, the methods and materials described herein can be used to improve the survival of a mammal having cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, the methods and materials described herein can be used to improve the survival of a mammal having cancer by, for example, at least 6 months (e.g., about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, about 5 years, or more).

[0035] Any appropriate mammal having cancer can be treated as described herein (e.g., by administering (a) one or more ADCs and (b) one or more PROTAC compounds). Examples of mammals that can have cancer and can be treated as described herein include, without limitation, humans, non-human primates (e.g., monkeys), horses, bovine species, porcine species, dogs, cats, mice, and rats. In some cases, a human having cancer can be treated by administering (a) one or more ADCs and (b) one or more PROTAC compounds targeting the same polypeptide expressed by a cell (e.g., a cancer cell).

[0036] A mammal (e.g., a human) having any type of cancer can be treated as described herein (e.g., by administering (a) one or more ADCs and (b) one or more PROTAC compounds targeting the same polypeptide expressed by a cell (e.g., a cancer cell)). In some cases, a cancer that can be treated as described herein can include one or more solid tumors. In some cases, a cancer that can be treated as described herein can be a blood cancer. In some cases, a cancer treated as described herein can be a primary cancer. In some cases, a cancer treated as described herein can be a metastatic cancer. In some cases, a cancer treated as described herein can be a refractory cancer. Examples of cancers that can be treated as described herein include, without limitation, pancreatic cancers (e.g., pancreatic adenocarcinoma), ovarian cancers, cervical cancers, myelomas, breast cancers, melanomas, lung cancers, mesotheliomas, and sarcomas.

[0037] In some cases, one or more cancer cells in a cancer that can be treated as described herein (e.g., by administering (a) one or more ADCs and (b) one or more PROTAC compounds) can express one or more surface antigens (e.g., one or more surface polypeptides). In some cases, an antigen expressed by a cancer cell can be a tumor-specific antigen. Examples of antigens that can be expressed by a cancer cell (e.g., and can be targeted by one or more ADCs and / or one or more PROTAC compounds) include, without limitation, a HER-2 polypeptide, an EGFR polypeptide, a MET polypeptide, an estrogen receptor polypeptide, an androgen receptor polypeptide, a RET polypeptide, an ALK polypeptide, a TROP-2 polypeptide, a CLDN18.2 polypeptide, a HER3 polypeptide, a nectin-4 polypeptide, a CD276 polypeptide, a folate receptor polypeptide (e.g., a folate receptor alpha polypeptide), a PD-L1 polypeptide, a PD-1 polypeptide, a CD 19 polypeptide, a BCMA polypeptide, a R0R1 polypeptide, a CD30 polypeptide, a CD22 polypeptide, a PSMA polypeptide, a mesothelin polypeptide, a B7-H3 polypeptide, a B7-H4 polypeptide, a CD70 polypeptide, a CLDN6 polypeptide, an EphA2 polypeptide, a MUCl polypeptide, a CD20 polypeptide, a CD33 polypeptide, a CD37 polypeptide, a CD79b polypeptide, a tissue factor (TF) polypeptide, an AXL polypeptide, a R0R2 polypeptide, an ALCAM polypeptide, a CD 123 polypeptide, a CD25 polypeptide, a CEACAM5 polypeptide, a NaPi2b polypeptide, and a STING polypeptide.

[0038] In some cases, one or more ADCs and one or more PROTAC compounds that can be administered to a mammal (e.g., a human) having cancer as described herein can target different epitopes of the same polypeptide expressed by a cell (e.g., a cancer cell).

[0039] In some cases, one or more ADCs and one or more PROTAC compounds that can be administered to a mammal (e.g., a human) having cancer as described herein can target different polypeptides within the same polypeptide complex or cluster expressed by a cell (e g., a cancer cell).

[0040] In some cases, the methods described herein also can include identifying a mammal as having cancer. Examples of methods that can be used to identify a mammal as having cancer include, without limitation, physical examination, laboratory tests (e.g., blood and / or urine), biopsy, imaging tests (e.g., X-ray, PET / CT, MRI, and / or ultrasound), nuclear medicine scans (e.g., bone scans), endoscopy, and / or genetic tests.

[0041] Once identified as having cancer, a mammal can be treated as described herein (e.g., by administering (a) one or more ADCs and (b) one or more PROTAC compounds targeting the same polypeptide expressed by a cell (e.g., a cancer cell)).

[0042] A mammal (e.g., a human) having cancer can be administered, or can be instructed to self-administer, any appropriate ADC. An ADC is a molecule that includes an antigenbinding domain linked to a drug. An ADC can include any appropriate antigen-binding domain. In some cases, an antigen-binding domain that can be used to design an ADC can be an antibody. Examples of antigen-binding domains that can be used to design an ADC that can be used to treat a mammal (e.g., a human) having cancer as described herein include, without limitation, trastuzumab, cetuximab, atezolizumab, amivantamab, telisotuzumab, anetumab, tarlatamab, enfortumab, sacituzumab, datopotamab, mirvetuximab, tisotumab, disitamab, loncastiuximab, belantamab, polatuzumab, inotuzumab, brentuximab, gemtuzumab, patritumab, depatuxizumab, ifinatamab, tusamitamab, upifitamab, vobramitamab, zilovertamab, camidanlumab, coltuximab, farletuzumab, glembatumumab, indatuximab, labetuzumab, luveltamab, mecbotamab, naratuximab, ozuriftamab, praluzatamab, epratuzumab, pivekimab, aprutumab, bivatuzumab, cofetuzumab, ispectamab, losatuxizumab, and lupartumab.

[0043] An antigen-binding domain that can be used to design an ADC that can be used to treat a mammal (e.g., a human) having cancer as described herein can target (e.g., target and bind) any appropriate antigen. In some cases, an antigen-binding domain of an ADC can target (e.g., target and bind) a surface antigen (e.g., a surface polypeptide) expressed by a cancer cell (e.g., a tumor-specific antigen). Examples of antigens that can be expressed by a cancer cell and can be targeted by an antigen-binding domain of an ADC include, without limitation, HER-2 polypeptides, EGFR polypeptides, MET polypeptides, estrogen receptor polypeptides, androgen receptor polypeptides, RET polypeptides, ALK polypeptides, TROP- 2 polypeptides, CLDN18.2 polypeptides, HER3 polypeptides, nectin-4 polypeptides, CD276 polypeptides, folate receptor polypeptides (e.g., folate receptor alpha polypeptides), PD-L1 polypeptides, PD-1 polypeptides, CD19 polypeptides, BCMA polypeptides, R0R1 polypeptides, CD30 polypeptides, CD22 polypeptides, PSMA polypeptides, mesothelin polypeptides, B7-H3 polypeptides, B7-H4 polypeptides, CD70 polypeptides, CLDN6 polypeptides, EphA2 polypeptides, MUC1 polypeptides, CD20 polypeptides, CD33 polypeptides, CD37 polypeptides, CD79b polypeptides, tissue factor (TF) polypeptides, AXL polypeptides, R0R2 polypeptides, ALCAM polypeptides, CD 123 polypeptides, CD25 polypeptides, CEACAM5 polypeptides, NaPi2b polypeptides, and STING polypeptides.

[0044] In some cases, an antigen-binding domain that can be used to design an ADC that can be used to treat a mammal having cancer as described herein can be as described elsewhere (see, e.g., Hu et al., Nat., Cancer, 2(12): 1406-1422 (2021); Dumontet et al., Nat. Rev. Drug Discov., 22(8):641-661 (2023); Tsuchikama et al., Nat. Rev. Clin. Oncol., 21(3):203-223

[0045] (2024); Tarantino -576 (2023); Drago et al., Nat. Rev. Clin. Oncol., 18(6):327-344 (2021); and Fu et al., Signal Transduct. Target Ther., 7(1):93 (2022)).

[0046] An ADC provided herein can be designed to include any appropriate drug. In some cases, a drug that can be used to design an ADC provided herein can be a cytotoxic drug. In some cases, a drug that can be used to design an ADC provided herein can be an anti-cancer drug. Examples of drugs that can be used to design an ADC provided herein that can be used to treat a mammal (e.g., a human) having cancer as described herein include, without limitation, deruxtecan, emtansine (DM1), ravtansine (DM4), vedotin, ozogamicin, govitecan mafodotin, pasudotox, tesirine, soravtansine, sarotalocan, mafodotin, govitecan, ozogamicin, tirumotecan, botidotin, exatecan, camptothecin, 7-aminomethyl-10,l 1- methylenedioxycamptothecin (AMDCPT), indenoisoquinoline, dibenzonapthyridinone, fluoroindenoisoquinoline, belotecan, auristatin, epothilone, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), calicheamicin, SN-38, and Dxd.

[0047] In some cases, a drug (e.g., an anti -cancer drug) that can be used to design an ADC provided herein that can be used to treat a mammal having cancer as described herein can be as described elsewhere (see, e.g., Conilh et al., J. Hematol. Oncol., 16:3(2023)).

[0048] In some cases, an antigen-binding domain (e.g., an antibody) and a drug (e.g., an anticancer drug) can be directly conjugated to produce an ADC that can be used in the methods and materials provided herein.

[0049] In some cases, an antigen-binding domain (e.g., an antibody) and a drug (e.g., an anticancer drug) can be indirectly conjugated to produce an ADC that can be used in the methods and materials provided herein. For example, an antigen-binding domain and a drug can be connected via a linker such as a peptide linker to produce an ADC that can be used in the methods and materials provided herein. In some cases, a linker can be a cleavable linker such as an acid cleavable linker, a GSH cleavable linker, a cathepsin cleavable linker, a glycosidase cleavable linker, or a phosphatase cleavable linker. Examples of linkers that can be used to connect an antigen-binding domain and a drug of an ADC include, without limitation, those described in Su et al., Acta Pharmaceutica Sinica B, 11 (12):3889-3907 (2021).

[0050] Examples of ADCs that can be used in the methods and materials provided herein include, without limitation, trastuzumab deruxtecan (e.g., ENHERTU®), trastuzumab emtansine (trastuzumab-DMl; e.g., KADCYLA®), telisotuzumab vedotin, anetumab ravtansine, datopotamab deruxtecan, gemtuzumab ozogamicin (e.g., MYLOTARG®), brentuximab vedotin (e.g., ADCETRIS®), inotuzumab ozogamicin (e.g., BESPONSA®), polatuzumab vedotin (e.g., POLIVY®), enfortumab vedotin (e.g., PADCEV®), sacituzumab govitecan (e.g., TRODELVY®), loncastuximab tesirine (e.g., ZYNLONTA®), tisotumab vedotin (e.g., TIVDAK®), mirvetuximab soravtansine (e.g., ELAHERE®), and belantamab mafodotin (e.g., BLENREP®).

[0051] In some cases, an ADC that can be used to treat a mammal having cancer as described herein can be as described elsewhere (see, e.g., Baah et al., Molecules, 26(10): 2943 (2021); and Dumontet et al., Nat. Rev. Drug Discov., 22(8):641-661 (2023)).

[0052] A mammal (e.g., a human) having cancer can be administered, or can be instructed to self-administer, any appropriate PROTAC compound. A PROTAC compound is a bifunctional molecule including a targeting moiety and an E3 ligase ligand. A PROTAC compound can include any appropriate targeting moiety. In some cases, a targeting moiety can be an orthosteric ligand. In some cases, a targeting moiety can be an allosteric ligand. A targeting moiety can be any appropriate type of molecule. For example, a targeting moiety that can be used to design a PROTAC compound provided herein can be an oligonucleotide, a polypeptide, or a small molecule.

[0053] A targeting moiety that can be used to design a PROTAC compound provided herein that can be used in the methods and materials provided herein can be capable of targeting (e.g., targeting and binding) any appropriate antigen. In some cases, a targeting moiety of a PROTAC compound can target (e.g., target and bind) an antigen (e.g., a surface polypeptide) expressed by a cancer cell (e.g., a tumor-specific antigen). Examples of antigens that can be expressed by a cancer cell and can be targeted by a targeting moiety of a PROTAC compound include, without limitation, HER-2 polypeptides, EGFR polypeptides, MET polypeptides, estrogen receptor polypeptides, androgen receptor polypeptides, RET polypeptides, ALK polypeptides, TROP-2 polypeptides, CLDN18.2 polypeptides, HER3 polypeptides, nectin-4 polypeptides, CD276 polypeptides, folate receptor polypeptides (e.g., folate receptor alpha polypeptides), PD-L1 polypeptides, PD-1 polypeptides, CD 19 polypeptides, BCMA polypeptides, ROR1 polypeptides, CD30 polypeptides, CD22 polypeptides, PSMA polypeptides, mesothelin polypeptides, B7-H3 polypeptides, B7-H4 polypeptides, CD70 polypeptides, CLDN6 polypeptides, EphA2 polypeptides, MUC1 polypeptides, CD20 polypeptides, CD33 polypeptides, CD37 polypeptides, CD79b polypeptides, tissue factor (TF) polypeptides, AXL polypeptides, R0R2 polypeptides, ALCAM polypeptides, CD 123 polypeptides, CD25 polypeptides, CEACAM5 polypeptides, NaPi2b polypeptides, and STING polypeptides. Examples of targeting moieties that can be used to design a PROTAC compound provided herein that can be used to treat a mammal (e.g., a human) having cancer as described herein include, without limitation, lapatinib, capmatinib, erlotinib, afatinib, alectinib, brigatinib, lorlatinib, tepotinib, crizotinib, and ceritinib.

[0054] In some cases, a targeting moiety that can be used to design a PROTAC provided herein can be as described elsewhere (e.g., International Patent Application Publication No. WO 2022 / 183006, International Patent Application Publication No. WO 2023 / 249994, and Moon et al., Pharmaceutics, 15(2):411 (2023)).

[0055] A PROTAC compound can include any appropriate E3 ligase ligand. An E3 ligase ligand can be any appropriate molecule that can target (e.g., target and bind) to an E3 ligase polypeptide. An E3 ligase ligand can target (e.g., target and bind to) any appropriate E3 ligase polypeptide. Examples of E3 ligase polypeptides that can be targeted by an E3 ligase ligand of a PROTAC compound provided herein include, without limitation, von Hippel- Lindau (VHL) polypeptides, cereblon (CRBN) polypeptides, AHR polypeptides, RNF4 polypeptides, DCAF11 polypeptides, DCAF15 polypeptides, DCAF16 polypeptides, MDM2 polypeptides, and XIAP polypeptides.

[0056] In some cases, an E3 ligase ligand that can be used to design a PROTAC compound provided herein that can be used to treat a mammal (e.g., a human) having cancer as described herein can be as described elsewhere (see, e.g., International Patent Application Publication No. WO 2022 / 183006, International Patent Application Publication No. WO 2023 / 249994, and Liu et al., Nat. Commun., 14(l):6509 (2023)).

[0057] In some cases, a targeting moiety and an E3 ligase ligand can be directly conjugated to create a PROTAC compound provided herein that can be used in the methods and materials provided herein.

[0058] In some cases, a targeting moiety and an E3 ligase ligand can be indirectly conjugated to create a PROTAC compound provided herein that can be used in the methods and materials provided herein. For example, a targeting moiety and an E3 ligase ligand can be connected via a linker such as a peptide linker to create a PROTAC compound provided herein that can be used in the methods and materials provided herein. In some cases, a linker can be a cleavable linker such as an acid cleavable linker, a GSH cleavable linker, a cathepsin cleavable linker, a glycosidase cleavable linker, or a phosphatase cleavable linker.

[0059] Examples of PROTAC compounds that can be used in the methods and materials provided herein include, without limitation, SJF-1528, 48-284, CFT8634, CFT8919, CG001419, CFT1946, KT-253, AC0176, CC-94676, HP518, GT20029, AC0682, HSK29116, BGB-16673, RNK05047, AC682, ARV-766, DT2216, FHD-609, KT-474, KT- 413, KT-333, NX-2127, NX-5948, CC-99282, CFT7455, DKY709, ARV-110, ARV-471, CC-220, CC-92480, and CC-90009.

[0060] In some cases, a PROTAC compound that can be used to treat a mammal having cancer as described herein can be as described elsewhere (see, e.g., International Patent Application Publication No. WO 2022 / 183006, International Patent Application Publication No. WO 2023 / 249994, Moon et al., Pharmaceutics, 15(2):411 (2023); and Bekes et al., Nat. Rev. DrugDiscov., 21(3): 181-200 (2022)).

[0061] In some cases, a mammal (e.g., a human) having a cancer that includes one or more cancer cells that express a HER2 polypeptide can be administered (a) one or more ADCs that can target the HER2 polypeptide (e.g., trastuzumab deruxtecan and / or trastuzumab emtansine) and (b) one or more PROTAC compounds that can target the HER2 polypeptide (e.g., SJF-1528), such that both the ADC(s) and the PROTAC compound(s) can target and bind the HER2 polypeptide expressed by cancer cells within the mammal. The PROTAC compound(s) that can target the HER2 polypeptide (e.g., SJF-1528) can mark the HER2 polypeptide for degradation (e.g., via ubiquitination), thereby inducing the cell to internalize the HER2 polypeptide (e.g., via endocytosis) and also to internalize the one or more ADCs that targeted and bound to the HER2 polypeptide (e.g., trastuzumab deruxtecan and / or trastuzumab emtansine).

[0062] In some cases, a mammal (e.g., a human) having a cancer that includes one or more cancer cells that express a MET polypeptide can be administered (a) one or more ADCs that can target the MET polypeptide (e.g., telisotuzumab vedotin) and (b) one or more PROTAC compounds that can target the MET polypeptide (e.g., 48-284), such that both the ADC(s) and the PROTAC compound(s) can target and bind the MET polypeptide expressed by cancer cells within the mammal. The PROTAC compound(s) that can target the MET polypeptide (e.g., 48-284) can mark the MET polypeptide for degradation (e.g., via ubiquitination), thereby inducing the cell to internalize the MET polypeptide (e.g., via endocytosis) and also to internalize the one or more ADCs that targeted and bound to the MET polypeptide (e.g., telisotuzumab vedotin).

[0063] In some cases, one or more ADCs and one or more PROTAC compounds can be administered to a mammal at the same time (e.g., in a single composition).

[0064] One or more ADCs and / or one or more PROTAC compounds can be administered to a mammal by any appropriate route. For example, a composition including one or more ADCs and / or one or more PROTAC compounds can be administered locally or systemically. In some cases, a composition including one or more ADCs and / or one or more PROTAC compounds can be designed for parenteral (e.g., subcutaneous, intramuscular, intravenous, intraperitoneal, and intratumoral) administration. Compositions suitable for parenteral administration include, without limitation, aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

[0065] In some cases, a composition including one or more ADCs and / or one or more PROTAC compounds can be administered by intratumoral injection to one or more tumors present within a mammal (e.g., a human) having cancer.

[0066] In some cases, one or more ADCs and one or more PROTAC compounds can be administered a mammal (e.g., a human) separately. For example, one or more ADCs and one or more PROTAC compounds can be administered to a mammal at the same time (e.g., concurrently) as independent compositions. When one or more ADCs and one or more PROTAC compounds are administered concurrently, the composition including the one or more ADCs and the composition including one or more PROTAC compounds can be administered to a mammal within from about 1 second to about 15 minutes (e.g., about 2 seconds to about 15 minutes, about 5 seconds to about 15 minutes, about 10 seconds to about 15 minutes, about 15 seconds to about 15 minutes, about 1 second to about 10 minutes, about 1 second to about 5 minutes, or about 5 seconds to about 10 minutes) of each other. In some cases, a composition including one or more ADCs and a composition including one or more PROTAC compounds can be administered a mammal (e.g., a human) at different times. When a composition including one or more ADCs and a composition including one or more PROTAC compounds are administered at different times, the composition including the one or more ADCs and the composition including the one or more PROTAC compounds can be administered to a mammal with from about 1 hour to about 48 hours between each administration.

[0067] In some cases, a composition including one or more ADCs can be administered to a mammal (e.g., a human) first, and a composition including one or more PROTAC compounds can be administered to the mammal second.

[0068] When one or more ADCs and one or more PROTAC compounds are administered as separate compositions, each composition can be administered to a mammal by any appropriate route. In some cases, a composition including one or more ADCs and a composition including one or more PROTAC compounds can be administered by the same route. In some cases, a composition including one or more ADCs and a composition including one or more PROTAC compounds can be administered by different routes.

[0069] When a composition including one or more ADCs and a composition including one or more PROTAC compounds are administered as separate compositions, the composition including the one or more ADCs can be administered first, and the composition including the one or more PROTAC compounds administered second, or vice versa.

[0070] One or more ADCs can be administered to a mammal (e.g., a human) having cancer in any appropriate amount (e.g., any appropriate dose). In some cases, an effective dose of one or more ADCs can be a flat dose. In some cases, an effective dose of one or more ADCs can be based on the body of a mammal (e.g., a human) to be treated as described herein. In some cases, an effective amount of one or more ADCs can be from about 1 mg of ADC(s) per kg body weight of a mammal (mg / kg) to about 10 mg / kg (e.g., from about 1 mg / kg to about 8 mg / kg, from about 1 mg / kg to about 6 mg / kg, from about 1 mg / kg to about 5 mg / kg, from about 1 mg / kg to about 4 mg / kg, from about 1 mg / kg to about 3 mg / kg, from about 1 mg / kg to about 2 mg / kg, from about 2 mg / kg to about 10 mg / kg, from about 3 mg / kg to about 10 mg / kg, from about 5 mg / kg to about 10 mg / kg, from about 7 mg / kg to about 10 mg / kg, from about 2 mg / kg to about 8 mg / kg, from about 3 mg / kg to about 7 mg / kg, from about 4 mg / kg to about 6 mg / kg, from about 2 mg / kg to about 4 mg / kg, from about 3 mg / kg to about 5 mg / kg, from about 5 mg / kg to about 7 mg / kg, from about 6 mg / kg to about 8 mg / kg, or from about 7 mg / kg to about 9 mg / kg). For example, an effective amount of trastuzumab emtansine can be from about 2.4 mg / kg to about 3.6 mg / kg. For example, an effective amount of trastuzumab deruxtecan can be from about 3.2 mg / kg to about 6.4 mg / kg. The effective amount of one or more ADCs can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and / or severity of the cancer in the mammal being treated may require an increase or decrease in the actual effective amount administered.

[0071] One or more ADCs can be administered to a mammal (e.g., a human) having cancer at any appropriate frequency. The frequency of administration can be any frequency that can treat a mammal having cancer without producing significant toxicity to the mammal. For example, the frequency of administration can be from about once a day to about once a month, from about once a week to about once a month, or from about twice a month to about once a month. The frequency of administration can remain constant or can be variable during the duration of treatment. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, and / or route of administration may require an increase or decrease in administration frequency.

[0072] One or more ADCs can be administered to a mammal (e.g., a human) having cancer for any appropriate duration. An effective duration can be any duration that can treat a mammal having cancer without producing significant toxicity to the mammal. For example, the effective duration can vary from several weeks to several months, from several months to several years, or from several years to a lifetime. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, and / or route of administration.

[0073] One or more PROTAC compounds can be administered to a mammal (e.g., a human) having cancer in any appropriate amount (e.g., any appropriate dose). In some cases, an effective dose of one or more PROTAC compounds can be a flat dose. In some cases, as effective dose of one or more PROTAC compounds can be based on the body of a mammal (e.g., a human) to be treated as described herein. An effective amount of one or more PROTAC compounds can be any amount that can treat a mammal having cancer without producing significant toxicity to the mammal. In some cases, an effective amount of one or more PROTAC compounds can be from about 0.1 mg / kg to about 25 mg / kg (e.g., from about 0.1 mg / kg to about 20 mg / kg, from about 0.1 mg / kg to about 15 mg / kg, from about 0.1 mg / kg to about 12 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 8 mg / kg, from about 0.1 mg / kg to about 5 mg / kg, from about 0.1 mg / kg to about 2 mg / kg, from about 1 mg / kg to about 25 mg / kg, from about 5 mg / kg to about 25 mg / kg, from about 8 mg / kg to about 25 mg / kg, from about 10 mg / kg to about 25 mg / kg, from about 13 mg / kg to about 25 mg / kg, from about 15 mg / kg to about 25 mg / kg, from about 18 mg / kg to about 25 mg / kg, from about 20 mg / kg to about 25 mg / kg, from about 1 mg / kg to about 20 mg / kg, from about 3 mg / kg to about 15 mg / kg, from about 5 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 5 mg / kg, from about 3 mg / kg to about 8 mg / kg, from about 5 mg / kg to about 10 mg / kg, from about 8 mg / kg to about 12 mg / kg, from about 10 mg / kg to about 15 mg / kg, from about 13 mg / kg to about 18 mg / kg, from about 15 mg / kg to about 20 mg / kg, or from about 17 mg / kg to about 22 mg / kg). The effective amount of one or more PROTAC compounds can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and / or severity of the cancer in the mammal being treated may require an increase or decrease in the actual effective amount administered.

[0074] One or more PROTAC compounds can be administered to a mammal (e.g., a human) having cancer at any appropriate frequency. The frequency of administration can be any frequency that can treat a mammal having cancer without producing significant toxicity to the mammal. In some cases, a single dose of one or more PROTAC compounds can be administered to a mammal having cancer (e g., following each administration of one or more ADCs to the mammal). In some cases, multiple doses of one or more PROTAC compounds can be administered to a mammal having cancer. For example, the frequency of administration can be from about twice a day to about once every other day, from about once a day to about once a week, from about once a day to about once a month, from about once a week to about once a month, or from about twice a month to about once a month. The frequency of administration can remain constant or can be variable during the duration of treatment. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, and / or route of administration may require an increase or decrease in administration frequency.

[0075] One or more PROTAC compounds can be administered to a mammal (e.g., a human) having cancer for any appropriate duration. An effective duration can be any duration that can treat a mammal having cancer without producing significant toxicity to the mammal. For example, the effective duration can vary from several weeks to several months, from several months to several years, or from several years to a lifetime. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, and / or route of administration.

[0076] In some cases, one or more ADCs and one or more PROTAC compounds can be administered to a mammal (e g., a human) having cancer as the sole active agents to treat the cancer.

[0077] In some cases, methods for treating a mammal (e.g., a human) as described herein (e.g., by administering one or more ADCs and one or more PROTAC compounds) also can include administering to the mammal one or more (e.g., one, two, three, or more) additional agents used to treat cancer and / or performing one or more (e.g., one, two, three, or more) therapies used to treat cancer. For example, a combination therapy used to treat a mammal (e.g., a human) having cancer can include administering to the mammal (a) one or more ADCs and (b) one or more PROTAC compounds, and administering to the mammal one or more (e.g., one, two, three, or more) additional agents used to treat cancer. In some cases, an additional agent that can be administered to a mammal to treat cancer can be a chemotherapeutic agent. In some cases, an additional agent that can be administered to a mammal to treat cancer can be a cytotoxic agent. Examples of additional agents that can be administered to a mammal (e.g., a human) having cancer (e.g., a cancer including one or more solid tumors) to treat the mammal include, without limitation, sorafenib, regorafenib, ramucirumab, carboplatin, pemetrexed, paclitaxel, docetaxel, gemcitabine, cisplatin, nab- paclitaxel, bevacizumab, adriamycin, cyclophosphamide, and any combinations thereof.

[0078] In cases where one or more ADCs and one or more PROTAC compounds are used in combination with additional agents used to treat a mammal (e.g., a human) having cancer, the one or more additional agents can be administered at the same time (e.g., in a single composition containing one or more ADCs and one or more PROTAC compounds and containing the one or more additional agents) or independently. For example, a composition including one or more ADCs and one or more PROTAC compounds can be administered first, and the one or more additional agents administered second, or vice versa.

[0079] In some cases, a combination therapy used to treat a mammal (e.g., a human) having cancer can include administering to the mammal one or more ADCs and one or more PROTAC compounds, and can include performing one or more (e.g., one, two, three, or more) therapies used to treat cancer. Examples of additional therapies that can be used to treat a mammal (e.g., a human) having cancer include, without limitation, radiation therapies, surgeries, percutaneous ablation, and / or radiotherapeutics. In cases where one or more ADCs and one or more PROTAC compounds are used in combination with one or more therapies used to treat a mammal (e.g., a human) having cancer, the one or more additional therapies can be performed at the same time or independently of the administration of the one or more ADCs and one or more PROTAC compounds. For example, one or more ADCs and one or more PROTAC compounds can be administered before, during, or after the one or more additional therapies are performed.

[0080] In some cases, the methods and materials provided herein can include monitoring the mammal (e.g., the human) being treated as described herein (e.g., by administering (a) one or more ADCs and (b) one or more PROTAC compounds). For example, the size of the cancer (e g., the number of cancer cells and / or the volume of one or more tumors) present within a mammal can be monitored. Any appropriate method can be used to determine whether or not the size of the cancer present within a mammal is reduced. For example, imaging techniques can be used to assess the size of the cancer present within a mammal (e.g., a human).

[0081] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0082] Example 1: PROTAC compounds can improve the cytotoxicity of ADCs

[0083] The activity of ADCs is predicated upon cellular internalization to release the cytotoxic payloads intracellularly. The degree of ADC internalization is incomplete for many cell surface targets, thus limiting the activity of ADCs.

[0084] This Example demonstrates that PROTAC compounds targeting the same cell surface antigen as an ADC can be used to increase the cellular internalization of the ADC. For example, PROTAC compounds that target the same cell surface antigen as ADCs can improve the internalization and cytotoxicity of these ADCs by marking the targeted cell surface antigen for degradation such that the cell internalizes the cell surface antigen and the bound ADC (e.g., via endocytosis).

[0085] Materials and method

[0086] Cells and cell culture

[0087] Breast cancer cell lines (BT-474 and SK-BR-3), lung cancer cells (A549, HCC827 and HCC827 GR6), and pancreatic cancer cell line (BxPC3) were cultured in RPMI 1640 with L-glutamine (Corning 10-040-CV). HEK293T cells were cultured in MEM (Gibco 11095-080) supplemented with 10% fetal bovine serum (Gibco 10437-028) and penicillinstreptomycin (Gibco 15140-122) at 37 °C with 5% CO2 in a humidified incubator.

[0088] Live-cell imaging and antibodies internalization

[0089] All live-cell imaging experiments for antibodies internalization were performed with IncuCyte S3-C2 (Sartorius corporation). Cells were seeded at a density of 10.000 cell / well and 50 uL of culture medium in black-walled 96-well plate (Corning CLS3603) and incubated overnight before treatment. PROTAC reagents targeting mesenchymal epithelial transition factor receptor (MET) (48-284) or epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) (SJF1580 Tocris Bioscience 7262) were assayed in a range of concentration (from 50 nM to 500 nM). Human IgGl (Sino Biological HG1K), anti-Met (Creative Biolabs ABT-700), cetuximab (Med Chem Express HY-P9905) and trastuzumab (Med Chem Express HY-P9907) antibodies were used at a final concentration of 1 pg / mL, 2 pg / mL, and 4 pg / mL and were labeled with human FabFluor-pH red reagent (Sartorius corporation 4722). A total of 50 pL of antibody-dye mix was added to each well and four images per well were acquired every 30 minutes for at least 24 hours with the adequate light channel following the manufacturer’s recommendations. The quantitative data generated from these experiments were exported as excel files and used to generate the AUC and statistical analysis on GraphPad Prism 10.

[0090] Inhibition experiments

[0091] Antibody internalization was evaluated in the presence of endocytosis inhibitors. Breast tumor cell lines BT-474 and SK-BR-3 were assessed for live-cell imaging as described before. Clathrin inhibitor Pistop2 (Med Chem Express HY-115604), dynamin VII inhibitor Dyngo-4a (Med Chem Express HY-13863), and labeled antibody trastuzumab were added to the cells at the same time. Internalization of the antibodies was imaged with IncuCyte every 30 minutes for 24-48 hours. BT-474 and SK-BR-3 cell lines were treated with endocytosis inhibitors or proteosome inhibitor MG132 (Selleckchem S2619) and the internalized antibodies were tested by western blotting using primary antibodies that detect light and heavy chains (R&D Systems MAB 100502 and MAB1101, respectively) derived from human IgGs. Cells were seeded at a density of 400.000 cell / well in 1.5 mL of culture medium in 6-well plates (Corning CLS3603), and were incubated overnight at 37 °C before antibody or PROTAC + antibody treatment were added at different time points.

[0092] Western immunoblots

[0093] Cells seeded and treated in 6-well plates as described before were washed 2-3 times with PBS (Corning 21-031-CV) and lysed with home-made NETN lyses buffer plus protease inhibitors cocktail (Roche 11836170001). Cell lyses was cleared by centrifugation at 4 °C for 10 minutes at 15.000 x g and protein concentration was measured with Pierce BCA protein assay kit (Thermo scientific 23225) and Promega GloMax explorer plate reader. Equal amounts of proteins from cleared cell lysates were mixed with loading SDS buffer plus P- mercaptoethanol and were resolved in 4-20% precast mini-protean gels (Bio-Rad 4561096). The gels were then transferred to a nitrocellulose membrane and incubated with Pierce ECL western blotting substrate (Thermo scientific 32106). The membranes were imaged using chemiluminescence by ChemiDoc digital imaging system. Other primary antibodies used for immunoblotting were anti-HER2 (D8F12 Cell Signaling 4290), anti- -actin (BioLegend 643802), and anti-GAPDH (Santa Cruz Biotechnology sc-365062). Secondary antibody goat anti-rabbit (Santa Cruz Biotechnology sc-2030) and mouse IgG kappa binding protein (Santa Cruz Biotechnology sc-516102) were also used.

[0094] Immunofluorescence staining

[0095] Red Fabfluor-pH sensitive labeled Tmab was assayed by immunodetection after internalization overnight in BT-474 cells. A secondary anti human IgG (Fc specific)-FITC antibody (F9512 Millipore Sigma) was used for human IgG detection. Briefly, 40000 cells / well were seeded in the Millicell EZ Slide (C86024 Millipore Sigma) and incubated for a day. Then, SJF1528 + Red Fabfluor labeled Tmab 2 pg / mL or SJF1528 + Red Fabfluor labeled Tmab 2 + MG132 5 pM was added and incubate overnight. The cells were fixed with paraformaldehyde 4 % for 10 minutes, permeabilized with Triton 0.1% and blocked with 1% BSA and 22.5 mg / mL glycine in PBST for 1 hour. Later, the cells were incubated with antiHuman IgG-FITC antibody for 1 hour at 37 °C in a humidified chamber. The cells were washed 3 times for 10 minutes each and were mounted with Doulink in situ counter staining media with DAPI (DU082040 Sigma). Images were acquired with EVOS 5000 Imaging system and DAPI, GFP and Cy5 light filter cubes (ThermoFisher Scientific).

[0096] Cell viability assays

[0097] The effects of PROTAC and the ADC KADCYLA® (trastuzumab emtansine) or ADC ENHERTU® (trastuzumab deruxtecan) on the viability of BT-474 and SKBR-3 tumor cell lines was assessed with Cell Titer-Gio (Promega Corporation G7571). Cells were seed at a density of 10.000 cell / well incubated overnight in a 50 pL of culture medium in a blackwalled 96-well plate (Corning CLS3603). The cells were treated with different concentrations of ADC, PROTAC or the combination of PROTAC + ADC for 24 hours, 48 hours, and 72 hours. After the desired time, 100 pL of Cell Titer-Gio reagent was added to each well, incubated at room temperature for 10-15 minutes and luminescence was measured with Promega GloMax explorer. Dose-response curves were generated with GraphPad Prism 10 and four-parameter fitting curves (inhibitor vs response variable slope). Cells treated with PROTAC only were used as control to normalize the data. Results

[0098] Internalization of cell surface protein targeting antibodies

[0099] To determine whether antibodies targeting cell surface oncogenic proteins were internalized, cell lines that expressed EGFR, HER2, and MET were selected. Internalization of antibodies that target EGFR, HER2, and MET was observed in all cell lines (Figure 1, Figure 5). The lapatinib-based PROTAC SJF-1528 that degrades EGFR and HER2, and the capmatinib-based PROTAC 48-284 that degrades MET, were selected to test the effects of targeted degradation on antibody internalization. The addition of the PROTACs with antibodies to the cells that expressed the same cell surface proteins as the antibodies resulted in significantly increased internalization of the antibodies by 1.4 to 1.9 fold in all cell lines with strong target expression (Figure 1, Figure 5), and a 1.1 fold increase in internalization of EGFR in the A549 cell line with weaker EGFR target expression (Figure 1). Increasing concentrations of antibodies, with stable concentrations of PROTACs, resulted in greater internalization of these antibodies with or without PROTACs (Figure 2A), showing that the targets were not saturated at the tested concentrations. Increasing concentrations of the PROTAC SJF-1528, with stable concentrations of antibody, significantly increased the antibody internalization at each dose strongly for SKRB-3 and mildly for BT-474 (Figure 2B).

[0100] Dependence on endocytosis and proteolysis

[0101] To investigate which process mediates antibody internalization upon exposure to PROTACs, endocytosis inhibitors Dyngo4a and Pitstop2 were used. The dynamin inhibitor Dyngo4a delayed the internalization of trastuzumab with PROTAC SJF-1528 in the HER2- positive breast cancer cell line BT-474, and reduced antibody internalization in HER2- positive breast cancer cell line SKBR-3 to below basal levels (Figure 3A). Addition of the clathrin inhibitor Pitstop2 with PROTACs in the BT-474 and SKBR-3 cell lines showed no effect on antibody internalization (Figure 3B). In the absence of SJF-1528, inhibition of dynamin resulted in a similar delay and reduction in antibody internalization in BT-474 and SKBR-3, respectively (Figure 6A). In contrast, in the absence of SJF-1528, inhibition of clathrin by Pitstop2 in the BT-474 cell line resulted in a significant reduction in antibody internalization but not in the SKBR-3 cell line (Figure 6B). There was time-dependent degradation of the heavy and light chains of intracellular immunoglobulins upon treatment with PROTACs that was blocked by the addition of the proteasome inhibitor MG132 (Figure 3C-3D, Figure 6C). These results showed that antibody internalization with PROTACs was more dependent on dynamin rather than clathrin in these models, and that internalization results in degradation by the proteasome.

[0102] Enhanced cytotoxicity of antibody-drug conjugate with PROTAC

[0103] To determine the effects of PROTACs on the activity of ADCs, the HER2-positive breast cancer cell lines BT-474 and SKBR-3 were treated with the ADC trastuzumab-DMl (TDM1) and ADC trastuzumab-deruxtecan with and without the HER2 targeting PROTAC SJF-1528. The addition of the PROTAC reduced cell viability across multiple antibody concentrations at multiple time points (Figure 4, Figure 7). Typically, when the PROTAC SJF-1528 was added to trastuzumab deruxtecan or trastuzumab emtansine, there were 20- 30% fewer surviving cells than when the ADCs were used alone.

[0104] Example 2: PROTAC compounds can improve the cytotoxicity of ADCs

[0105] The results in this Example re-present and expand on at least some of the results provided in other Examples.

[0106] Materials and method

[0107] Cells and cell culture

[0108] Breast cancer cell lines (BT-474 and SKBR3), lung cancer cell lines (A549 [ATCC] and HCC827 GR6 [Sigma Aldrich]), and pancreatic cancer cell line (BxPC-3 [ATCC]) were cultured in RPMI 1640 with L-glutamine (Corning 10-040-CV). HEK 293T (ATCC) cells were cultured in MEM (Gibco 11095-080) supplemented with 10% fetal bovine serum (Gibco 10437-028), 100 I.U. / mL penicillin and 100 pg / mL streptomycin (Gibco 15140-122) at 37 °C with 5% CO2 in a humidified incubator.

[0109] PROTACs 48-284 and 48-279

[0110] To develop MET -targeting PROTACs, capmatinib was linked to thalidomide, lenalidomide and VHL binders, as described elsewhere (Mallareddy et al., bioRxiv, 2024:11 (2024)). The MET PROTAC 48-284 resulted in the strongest degradation of MET in the prior screen. In contrast, 48-279 had a shorter linker than 48-284 and was inactive.

[0111] 3D culture models

[0112] Organoids derived from PDX breast cancer models were generated. Tumor tissues were minced into small pieces of 1-3 mm, transferred into gentleMACS™ C tubes (Miltenyi Biotec 130-093-237) containing DMEM medium (Corning 10-013-CV) without fetal bovine serum and tumor dissociation enzymes (enzymes H, R and A) (Miltenyi Biotec 130-095- 929), and digested for 1 hour in gentleMACS™ dissociator (Miltenyi Biotec 130-093-235) according to the manufacturer’s recommendations. After dissociation, the cell suspensions were centrifuged, washed twice with DMEM medium, and filtered with 40 pm cell strainers (Falcon 352340) to remove undigested tissues. The single cell suspensions were then subjected to negative selection using a mouse cell depletion kit (Miltenyi Biotec 130-104- 694) for 20 minutes at 4°C with continuous rotation. To enrich and recover human breast tumor cells magnetic separation with LS Columns (Miltenyi Biotec 130-042-401) was performed. Between 10,000 to 15,000 cells / well were seeded in 96 well suspension culture plates (Greiner Bio-One 655185) containing DMEM medium supplemented with 10% fetal bovine serum (Gibco 10437-028), 1% glutamax (Gibco 35050-061), non-essential amino acids (Corning 25-025-CI), 100 I.U. / mL penicillin, 100 pg / mL streptomycin (Gibco 15 MO- 122), and 5 pM of ROCK inhibitor (Tocris Bioscience 1254), and incubated for 7-10 days at 37°C degrees with 5% CO2 in a humidified incubator.

[0113] Live-cell imaging and antibody internalization

[0114] All live-cell imaging experiments for antibody internalization were performed with IncuCyte S3-C2 (Sartorius corporation). Cells were seeded at a density of 10,000 cells / well with 50 pL of culture medium in black-walled 96-well plates (Corning CLS3603) and incubated overnight before the treatment. PROTAC reagents targeting HER2 and EGFR, SJF1528 (Tocris Bioscience 7262) and MET (48-284) respectively, were assayed in a range of concentrations (from 50 nM to 500 nM). Other PROTAC molecules such as SJF1521 (Tocris Bioscience 7261) that favor EGFR degradation over HER2, SJF0661 (Tocris Bioscience 7464) designed as negative control for a BRAF degrader, and 48-279 targeting MET with no degradation activity, were also assayed across a range of concentrations (from 200 nM to 500 nM). Trastuzumab (Tmab) (Med Chem Express HY-P9907), cetuximab (Cmab) (Med Chem Express HY-P9905), anti-MET (Creative Biolabs ABT-700), and Human IgGl (Sino Biological HG1K) antibodies were used at a final concentration of 1, 2 and 4 pg / mL and labeled with Human FabFluor-pH red reagent (Sartorius corporation 4722). A total of 50 pL of antibody-dye mix was added to each well. Four images per well were acquired every 30 minutes for at least 24 hours with the adequate light channel following the manufacturer’s recommendations. The quantitative data generated from these experiments were exported as Excel files and used to generate the AUC and statistical analysis on GraphPad Prism 10.

[0115] Inhibition experiments

[0116] Antibody internalization was evaluated in the presence of endocytosis and ubiquitin- proteosome system inhibitors. The breast cancer cell lines BT-474 and SKBR3 and the lung cancer cell line HCC827 GR6 were assessed for live-cell imaging as described above. Both the dynamin I / II or clathrin inhibitors Dyngo-4a and Pitstop2 (Med Chem Express HY-13863 and HY-115604, respectively), and Ubal inhibitors TAK-243 and PYZD-4409 (Med Chem Express HY-100487 and HY-13297, respectively) were added to the cells at the same time as the labeled antibodies Tmab and anti-MET. Internalization was evaluated by acquiring images with IncuCyte every 30 minutes for 24-48 hours.

[0117] Internalized antibodies were also tested by western blots using primary antibodies detecting light and heavy chains (R&D Systems MAB 100502 and MAB1101, respectively) derived from Human IgGs in BT-474 and SKBR3 cell lines treated with proteosome inhibitor MG132 (Selleckchem S2619). Cells were seeded at a density of 400,000 cell / well in 1.5 m of culture medium in 6-well plates (Coming CLS3603) and incubated overnight at 37°C before addition of antibody or PROTAC with antibody at different time points.

[0118] Western immunoblots

[0119] Cells seeded and treated in 6-well plates as described before were washed 2-3 times with PBS (Coming 21-031-CV) and lysed with home-made NETN lysis buffer containing protease inhibitors cocktail (Roche 11836170001). Cell lysates were cleared by centrifugation at 4°C for 10 minutes at 15,000 x g. Protein concentration was measured with Pierce BCA protein assay kit (Thermo scientific 23225) using the Promega GloMax Explorer plate reader. Equal amounts of proteins from cleared cell lysates were mixed with loading SDS buffer plus P-mercaptoethanol, resolved in 4-20% precast mini-protean gels (Bio-Rad 4561096), and transferred to PVDF membranes. Chemiluminescence was detected by the ChemiDoc digital imaging system after incubation with Pierce ECL western blotting substrate (Thermo scientific 32106). Other primary antibodies used for immunoblotting were anti-HER2 (D8F12 Cell Signaling 4290), anti-P-actin (BioLegend 643802), and anti-GAPDH (Santa Cruz Biotechnology sc-365062). Secondary antibodies used were goat anti-rabbit (Santa Cruz Biotechnology sc-2030) and mouse IgG kappa binding protein (Santa Cruz Biotechnology sc-516102).

[0120] Immunofluorescence staining

[0121] Red Fabfluor-pH sensitive labeled Tmab was assayed by immunodetection after internalization overnight in BT-474 cells. A secondary anti-human IgG (Fc specific)-FITC antibody (F9512 Millipore Sigma) was used for human IgG detection. Briefly, 40,000 cells / well were seeded in the Millicell EZ Slide (C86024 Millipore Sigma) and incubated for 1 day. Then, SJF1528 and Red Fabfluor labeled Tmab (2 pg / mL), or SJF1528 and Red Fabfluor labeled Tmab (2 pg / mL) and MG132 (5 pM) were added and incubated overnight. The cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature, permeabilized with Triton 0.1%, and blocked with 1% BSA and 22.5 mg / mL glycine in PBST for 1 hour. The cells were incubated with anti-human IgG-FITC antibody for 1 hour at 37°C in a humidified chamber. The cells were washed 3 times for 10 minutes each with PBS and mounted with Doulink® in situ counter staining media with DAPI (DU082040 Sigma). Images were acquired with EVOS 5000 Imaging system using DAPI, GFP, and Cy5 light filter cubes (ThermoFisher Scientific).

[0122] Cell lines viability assays

[0123] The effects of PROTAC and the ADC KADCYLA® (trastuzumab-emtansine (Tmab- DM1)) or ENHERTU® (trastuzumab-deruxtecan (Tmab-deruxtecan)) on the viability of BT- 474 and SKBR3 tumor cell lines were assessed with Cell Titer-Gio (Promega Corporation G7571). For live-cell imaging, cells were seed at a density of 10,000 cells / well in a 50 pL of culture medium in black-walled 96-well plates (Corning CLS3603) and incubated overnight. Cells were then treated with different ADCs concentrations, PROTAC or the combination of PROTAC and ADCs in separate experiments for 24, 48, and 72 hours. Also, the effects of lapatinib (Med Chem Express HY-50898) alone and the combination of lapatinib with Tmab- deruxtecan on cell survival in SKBR3 cells were evaluated for comparison against lapatinib- based PROTAC SJF1528 with Tmab-deruxtecan combination. After the desired time, 100 pL of Cell Titer-Gio reagent was added to each well, incubated at room temperature for 10-15 minutes, and luminescence was measured with Promega GloMax Explorer. Dose-response curves were generated with GraphPad Prism 10 and four-parameter fitting curves (inhibitor vs response variable slope). Cells treated with DMSO only were used as the control to normalize the data.

[0124] Organoids proliferation and viability

[0125] Organoids derived from PDX breast cancer models were evaluated for proliferation by measuring the relative area (pm2) after treatment with PROTAC, Tmab-deruxtecan, or the combination of PROTAC and Tmab-deruxtecan in a range of concentrations for 72 hours. Briefly, single organoid was transferred in 30 pL of culture medium into ultra-low attachment 96 well U-bottom plates (Coming 4520) and cultured overnight. Then, 30 pL of culture medium and reagents were added to achieve the desired concentration. Bright field images were taken at 10X for further analysis. All the images were calibrated using Fiji to convert pixels into Micras, and after setting the measurements the freehand selection tool was used to draw the perimeter of at least 3 organoids per experimental condition. The quantitative data generated from these experiments were exported into Excel files and used to generate the graphs on GraphPad Prism 10 as relative area (pM2). DMSO control condition was used to normalize the data for other treatments (Figure 1 ID).

[0126] Viability assays were also performed using multiple organoids per well. For these experiments, organoids were treated similarly to the cell line viability experiments described herein. Briefly, organoids were culture in 50 pL of culture medium. Then, 50 pL of different ADC concentrations, PROTAC, or the combination of PROTAC with ADC was added and treated for 72 hours. Then, 100 pL of Cell Titer-Gio reagent was added to each well, shaken for 5 minutes, and incubated at room temperature for 20 minutes. Luminescence was measured with Promega GloMax® Explorer. Dose-response curves were generated with GraphPad Prism 10 and four-parameter fitting curves (inhibitor vs response variable slope) as described above. Organoids treated with DMSO only were used as the control to normalize the data.

[0127] Statistics and reproducibility

[0128] The total area under the curves representing antibody internalization were determined using GraphPad from triplicates and compared between the experimental groups using a two- tailed unpaired t-test. The null hypothesis was that there would no differences in antibody internalization between the groups. There were no adjustments for multiple comparisons. To calculate fold changes, the areas under the curves of the experimental groups were divided by that of the control groups. The raw data obtained with IncuCyte®, and the summary statistics derived were organized by figure and models, to improve reproducibility of the analyses.

[0129] Results

[0130] Internalization of cell surface protein targeting antibodies

[0131] To determine whether antibodies targeting cell surface oncogenic proteins were internalized, cell lines expressing HER2, EGFR, and MET were selected. Internalization of antibodies specific to each of the cell surface targets, including trastuzumab for HER2, cetuximab for EGFR, and ABT-700 for MET, was observed in all cell lines (Figures 8A-8C and 12). The lapatinib-based PROTAC SJF1528 that degrades HER2 and EGFR, and the capmatinib-based PROTAC 48-284 that degrades MET, were selected to test the effects of targeted degradation on antibody internalization. Co-administration of PROTACs that target the same cell surface proteins as their respective antibodies significantly increased (1.4 to 1.9 fold) internalization of the antibodies in all cell lines with strong target expression, and mildly increased internalization of EGFR (1.1 fold) in the A549 cell line with weak EGFR target expression (Figures 8A-8C and 12).

[0132] Magnitude of internalization and antibody and PROTAC concentrations

[0133] Increasing concentrations of antibodies, while maintaining constant concentrations of their respective protein targeting PROTACs, resulted in greater internalization of these antibodies compared to the controls (Figure 9A), suggesting that the targets were not saturated by the antibodies at the tested concentrations. Increasing concentrations of the PROTAC SJF1528, while maintaining constant concentrations of the antibody significantly increased the antibody internalization at each dose, strongly for SKBR3 and mildly for BT- 474 (Figure 9B). To determine the role of degradation on internalization, a second lapatinib- based degrader molecule SJF1521, which favors EGFR degradation over HER2; SJF0661, an inactive PROTAC; and 48-279, an inactive PROTAC that targets MET were used to test the internalization of trastuzumab (Tmab) for HER2 and ABT-700 for MET. The PROTAC SJF1521 did not result in the same degree of trastuzumab internalization as observed for SJF1528 (Figure 9C). Similarly, the inactive MET-targeting PROTAC 48-279 did not result in the same degree of ABT-700 internalization as observed for 48-248 (Figure 9C). The inactive PROTAC SJF0661 also did not enhance antibody internalization compared to DMSO with antibody control conditions (Figure 9C). Furthermore, none of the PROTACs assayed enhanced internalization of non-specific human IgG controls in the HER2 and MET models (Figure 9C). These results demonstrated that the internalization rate was largely modulated by the activity of the degrader and the concentrations of the antibody and the PROTAC.

[0134] Role of endocytosis and proteolysis

[0135] To investigate which processes mediate antibody internalization upon exposure to PROTACs, endocytosis inhibitors Dyngo-4a and Pitstop2 were used. The dynamin inhibitor Dyngo-4a delayed internalization of Tmab with PROTAC SJF1528 in the HER2 -positive breast cancer cell line BT-474, and reduced antibody internalization in HER2 -positive breast cancer cell line SKBR3 compared to the treatment without Dyngo-4a (Figure 10A). Similar delay or reduction in antibody internalization was observed without SJF1528 when Dyngo-4a was administrated to the DMSO and Tmab control conditions (Figure 13 A). Pitstop2 had no effect on antibody internalization with PROTACs in these models (Figure 13B). In contrast, in the absence of SJF1528, there was a significant reduction in antibody internalization with the clathrin inhibitor Pitstop2 in the BT-474 cell line but not in the SKBR3 cell line (Figure 13C). These results demonstrated that some basal rates of antibody internalization were more sensitive to inhibition of clathrin-independent endocytosis, whereas PROTAC-mediated antibody internalization was more sensitive to clathrin-dependent endocytosis. In addition, inhibitors of the ubiquitin-like modifier-activating enzyme 1 (Ubal) TAK-243 and PYZD- 4409 were assessed for their effects on Tmab and anti-MET internalization in the presence of SJF1528 and 48-284 PROTACs. Both inhibitors showed significant reduction in antibody internalization suggesting that ubiquitylation was required for antibody internalization driven by PROTACs (Figure 10B). Furthermore, there was a time-dependent degradation of the heavy and light chains of intracellular immunoglobulins upon treatment with PROTACs that was reduced by the addition of the proteasome inhibitor MG132 (Figures 10C, 10D, and 13D). These results demonstrated that antibody internalization with PROTACs was more dependent on dynamin than clathrin in these models, and that internalization resulted in degradation of the antibody by the proteasome.

[0136] Enhanced cytotoxicity of ADC and PROTAC combinations

[0137] To determine the effects of PROTACs on the activity of ADCs, the HER2-positive breast cancer cell lines BT-474 and SKBR3 were treated with the ADC trastuzumab-DMl (Tmab-DMl) and Tmab-Deruxtecan, with and without the HER2 targeting PROTAC SJF1528. The addition of the PROTAC reduced cell viability across multiple antibody concentrations at multiple time points (Figures 11A-11C and 14A-14C). Also, organoids derived from HER2-positive patient-derived xenografts (PDX) were treated with Tmab- Deruxtecan with and without the HER2 targeting PROTAC SJF1528. The addition of SJF1528 improved the cytotoxicity of ADC compared to the ADC treatment alone (Figure 1 ID). The effects of lapatinib on cell survival was also evaluated, which showed that this inhibitor had a stronger cytotoxic effect than the lapatinib-based PROTAC SJF1528. However, unlike the observed improvement in cytotoxicity of the combination of SJF1528 and Tmab-Deruxtecan, the addition of lapatinib to Tmab-Deruxtecan did not impact the cytotoxicity (Figure 1 IE), suggesting that proteolysis had a role in the increased cytotoxicity of the PROTAC and ADC combination.

[0138] OTHER EMBODIMENTS

[0139] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for treating a mammal having cancer, wherein said method comprises:(a) administering an antibody-drug conjugate (ADC) to said mammal; and(b) administering a proteolysis-targeting chimera (PROTAC) compound to said mammal, wherein said ADC and said PROTAC compound target a surface antigen expressed by a cancer cell in said mammal.

2. The method of claim 1, wherein said mammal is a human.

3. The method of any one of claims 1-2, wherein said cancer is selected from the group consisting of a pancreatic cancer, an ovarian cancer, a cervical cancer, a myeloma, a breast cancer, a melanoma, a lung cancer, a mesothelioma, and a sarcoma.

4. The method of any one of claims 1-3, wherein said surface antigen is selected from the group consisting of a HER-2 polypeptide, an EGFR polypeptide, a MET polypeptide, an estrogen receptor polypeptide, an androgen receptor polypeptide, a RET polypeptide, an ALK polypeptide, a TROP-2 polypeptide, a CLDN18.2 polypeptide, a HER3 polypeptide, a nectin-4 polypeptide, a CD276 polypeptide, a folate receptor polypeptide (e.g., a folate receptor alpha polypeptide), a PD-L1 polypeptide, a PD-1 polypeptide, a CD19 polypeptide, a BCMA polypeptide, a R0R1 polypeptide, a CD30 polypeptide, a CD22 polypeptide, a PSMA polypeptide, a mesothelin polypeptide, a B7-H3 polypeptide, a B7-H4 polypeptide, a CD70 polypeptide, a CLDN6 polypeptide, an EphA2 polypeptide, a MUC1 polypeptide, a CD20 polypeptide, a CD33 polypeptide, a CD37 polypeptide, a CD79b polypeptide, a tissue factor (TF) polypeptide, an AXL polypeptide, a R0R2 polypeptide, an ALCAM polypeptide, a CD 123 polypeptide, a CD25 polypeptide, a CEACAM5 polypeptide, a NaPi2b polypeptide, and a STING polypeptide.

5. The method of any one of claim 1-4, wherein said ADC comprises an antigen-binding domain that binds said surface antigen.

6. The method of claim 5, wherein said antigen-binding domain is selected from the group consisting of trastuzumab, cetuximab, atezolizumab, amivantamab, telisotuzumab, anetumab, tarlatamab, enfortumab, sacituzumab, datopotamab, mirvetuximab, tisotumab, disitamab, loncastiuximab, belantamab, polatuzumab, inotuzumab, brentuximab, gemtuzumab, patritumab, depatuxizumab, ifinatamab, tusamitamab, upifitamab, vobramitamab, zilovertamab, camidanlumab, coltuximab, farletuzumab, glembatumumab, indatuximab, labetuzumab, luveltamab, mecbotamab, naratuximab, ozuriftamab, praluzatamab, epratuzumab, pivekimab, aprutumab, bivatuzumab, cofetuzumab, ispectamab, losatuxizumab, and lupartumab.

7. The method of any one of claim 1-6, wherein said ADC comprises an anti-cancer drug selected from the group consisting of deruxtecan, emtansine (DM1), ravtansine (DM4), vedotin, ozogamicin, govitecan mafodotin, pasudotox, tesirine, soravtansine, sarotalocan, mafodotin, govitecan, ozogamicin, tirumotecan, botidotin, exatecan, camptothecin, 7- aminomethyl-10,11 -methylenedi oxy camptothecin (AMDCPT), indenoisoquinoline, dibenzonapthyridinone, fluoroindenoisoquinoline, belotecan, auristatin, epothilone, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), calicheamicin, SN- 38, and Dxd.

8. The method of any one of claim 1-5, wherein said ADC is selected from the group consisting of trastuzumab deruxtecan, trastuzumab emtansine, telisotuzumab vedotin, anetumab ravtansine, datopotamab deruxtecan, gemtuzumab ozogamicin, brentuximab vedotin, inotuzumab ozogamicin, polatuzumab vedotin, enfortumab vedotin, sacituzumab govitecan, loncastuximab tesirine, tisotumab vedotin, mirvetuximab soravtansine, and belantamab mafodotin.

9. The method of any one of claims 1-8, wherein said PROTAC compound comprises a targeting moiety that binds said surface antigen.

10. The method of claim 9, wherein said targeting moiety is selected from the group consisting of lapatinib, capmatinib, erlotinib, afatinib, alectinib, brigatinib, lorlatinib, tepotinib, crizotinib, and ceritinib.

11. The method of any one of claims 1-10, wherein said PROTAC compound comprises an E3 ligase ligand selected from the group consisting of a von Hippel-Lindau (VHL) polypeptide, a cereblon (CRBN) polypeptide, an AHR polypeptide, a RNF4 polypeptide, a DCAF11 polypeptide, a DCAF15 polypeptide, a DCAF16 polypeptide, a MDM2 polypeptide, and a XIAP polypeptide.

12. The method of any one of claims 9-11, wherein said targeting moiety and said E3 ligase ligand are connected by a linker.

13. The method of any one of claim 1-8, wherein said PROTAC compound is selected from the group consisting of SJF-1528, 48-284, CFT8634, CFT8919, CG001419, CFT1946, KT-253, AC0176, CC-94676, HP518, GT20029, AC0682, HSK29116, BGB-16673, RNK05047, AC682, ARV-766, DT2216, FHD-609, KT-474, KT-413, KT-333, NX-2127, NX-5948, CC-99282, CFT7455, DKY709, ARV-110, ARV-471, CC-220, CC-92480, and CC-90009.

14. A method for enhancing cellular internalization of an ADC, wherein said method comprises:(a) contacting a cell with an antibody-drug conjugate (ADC); and(b) contacting said cell with a proteolysis-targeting chimera (PROTAC) compound, wherein said ADC and said PROTAC compound target a surface antigen expressed by said cell, and wherein the amount of said ADC that enters said cell is more than the amount that enters a comparable cell not contacted with said PROTAC compound.

15. The method of claim 14, wherein said cell is within a mammal.

16. The method of claim 15, wherein said contacting comprises administering said ADC and said PROTAC compound to said mammal.

17. The method of claim 15, wherein said mammal is a human.

18. The method of any one of claims 15-17, wherein said mammal has cancer.

19. The method of claim 18, wherein said cancer is selected from the group consisting of a pancreatic cancer, an ovarian cancer, a cervical cancer, a myeloma, a breast cancer, a melanoma, a lung cancer, a mesothelioma, and a sarcoma.

20. The method of any one of claims 14-19, wherein said surface antigen is selected from the group consisting of a HER-2 polypeptide, an EGFR polypeptide, a MET polypeptide, an estrogen receptor polypeptide, an androgen receptor polypeptide, a RET polypeptide, an ALK polypeptide, a TROP-2 polypeptide, a CLDN18.2 polypeptide, a HER3 polypeptide, a nectin-4 polypeptide, a CD276 polypeptide, a folate receptor polypeptide (e.g., a folate receptor alpha polypeptide), a PD-L1 polypeptide, a PD-1 polypeptide, a CD19 polypeptide, a BCMA polypeptide, a R0R1 polypeptide, a CD30 polypeptide, a CD22 polypeptide, a PSMA polypeptide, a mesothelin polypeptide, a B7-H3 polypeptide, a B7-H4 polypeptide, a CD70 polypeptide, a CLDN6 polypeptide, an EphA2 polypeptide, a MUCl polypeptide, a CD20 polypeptide, a CD33 polypeptide, a CD37 polypeptide, a CD79b polypeptide, a tissue factor (TF) polypeptide, an AXL polypeptide, a ROR2 polypeptide, an ALCAM polypeptide, a CD 123 polypeptide, a CD25 polypeptide, a CEACAM5 polypeptide, a NaPi2b polypeptide, and a STING polypeptide.

21. The method of any one of claim 14-20, wherein said ADC comprises an antigenbinding domain that binds said surface antigen.

22. The method of claim 21, wherein said antigen-binding domain is selected from the group consisting of trastuzumab, cetuximab, atezolizumab, amivantamab, telisotuzumab, anetumab, tarlatamab, enfortumab, sacituzumab, datopotamab, mirvetuximab, tisotumab,disitamab, loncastiuximab, belantamab, polatuzumab, inotuzumab, brentuximab, gemtuzumab, patritumab, depatuxizumab, ifinatamab, tusamitamab, upifitamab, vobramitamab, zilovertamab, camidanlumab, coltuximab, farletuzumab, glembatumumab, indatuximab, labetuzumab, luveltamab, mecbotamab, naratuximab, ozuriftamab, praluzatamab, epratuzumab, pivekimab, aprutumab, bivatuzumab, cofetuzumab, ispectamab, losatuxizumab, and lupartumab.

23. The method of any one of claim 14-20, wherein said ADC comprises an anti-cancer drug selected from the group consisting of deruxtecan, emtansine (DM1), ravtansine (DM4), vedotin, ozogamicin, govitecan mafodotin, pasudotox, tesirine, soravtansine, sarotalocan, mafodotin, govitecan, ozogamicin, tirumotecan, botidotin, exatecan, camptothecin, AMDCPT, indenoisoquinoline, dibenzonapthyridinone, fluoroindenoisoquinoline, belotecan, auristatin, epothilone, MMAE, MMAF, calicheamicin, SN-38, and Dxd.

24. The method of any one of claim 14-20, wherein said ADC is selected from the group consisting of trastuzumab deruxtecan, trastuzumab emtansine, telisotuzumab vedotin, anetumab ravtansine, datopotamab deruxtecan, gemtuzumab ozogamicin, brentuximab vedotin, inotuzumab ozogamicin, polatuzumab vedotin, enfortumab vedotin, sacituzumab govitecan, loncastuximab tesirine, tisotumab vedotin, mirvetuximab soravtansine, and belantamab mafodotin.

25. The method of any one of claims 14-24, wherein said PROTAC compound comprises a targeting moiety that binds said surface antigen.

26. The method of claim 25, wherein said targeting moiety is selected from the group consisting of lapatinib, capmatinib, erlotinib, afatinib, alectinib, brigatinib, lorlatinib, tepotinib, crizotinib, and ceritinib.

27. The method of any one of claims 14-24, wherein said PROTAC compound comprises an E3 ligase ligand selected from the group consisting of a VHL polypeptide, a CRBNpolypeptide, an AHR polypeptide, a RNF4 polypeptide, a DCAFll polypeptide, a DCAF15 polypeptide, a DCAF16 polypeptide, a MDM2 polypeptide, and a XIAP polypeptide.

28. The method of any one of claims 25-27, wherein said targeting moiety and said E3 ligase ligand are connect by a linker.

29. The method of any one of claim 14-24, wherein said PROTAC compound is selected from the group consisting of SJF-1528, 48-284, CFT8634, CFT8919, CG001419, CFT1946, KT-253, AC0176, CC-94676, HP518, GT20029, AC0682, HSK29116, BGB-16673, RNK05047, AC682, ARV-766, DT2216, FHD-609, KT-474, KT-413, KT-333, NX-2127, NX-5948, CC-99282, CFT7455, DKY709, ARV-110, ARV-471, CC-220, CC-92480, and CC-90009.

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