Degradation of EGFR using bispecific binding agents

Abstract

The present disclosure provides a method for degrading EGFR protein on a target cell. The present disclosure further discloses a bispecific binding agent that binds to EGFR protein and a membrane-bound internalizing protein. The method includes contacting an endogenous internalizing receptor and a target protein on the surface of the target cell with a binding agent, the binding agent comprising: (i) a first binding domain that specifically binds to an endogenous internalizing receptor selected from the group consisting of MUC1, ITGB6, CEACAM5, and CDH17; and (ii) a second binding domain that specifically binds to the target protein, where the target protein comprises EGFR.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 371,371, filed August 12, 2022, U.S. Provisional Application No. 63 / 384,877, filed November 23, 2022, and U.S. Provisional Application No. 63 / 479,497, filed January 11, 2023, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] background Targeted protein degradation is a promising novel therapeutic strategy compared to traditional inhibition-based therapies. Inhibitors rely on occupancy-driven pharmacology, which requires high-affinity binders capable of persistently disabling catalytic or binding functions. Inhibition of protein-protein interactions or scaffolding functions has been extremely challenging for standard, binding-based small molecules. In contrast, protein degraders are catalytic and utilize event-driven pharmacology, which alleviates the need for high-affinity binders, to simultaneously and permanently disable all protein functions. Therefore, degrader technologies such as proteolysis-targeting chimeras (PROTACs) have shown great success in targeting traditionally challenging proteins. Several PROTACs are currently in clinical trials.

[0003] Many degradation technologies, including PROTACs, utilize intracellular mechanisms of action and are thus primarily limited to targeting proteins with cytoplasmic domains. However, recent approaches, such as LYTACs, have been described to specifically degrade cell surface proteins. These utilize recycling glycan receptors, such as mannose-6-phosphate receptor (M6PR) or asialoglycoprotein receptor (ASGR), to target proteins for internalization and transport to lysosomes for degradation. These require complex glycans conjugated to antibodies or small molecules to effect membrane protein degradation.

[0004] We recently described antibody-based PROTACs (AbTACs) as a hybrid approach that is broadly applicable to many cell types. AbTACs utilize a standard IgG bispecific antibody format to bring cell surface E3 ligases (RNF43) into close proximity with membrane proteins of interest (POIs) and mediate their degradation via the lysosomal pathway. The traditional bispecific IgG scaffold on which AbTACs are constructed has favorable pharmacokinetic properties compared to LYTACs and other small molecule-based degraders. Furthermore, in contrast to other degradative modalities such as LYTACs and PROTACs, AbTACs are fully recombinant. However, there remains a need for targeted protein degraders that efficiently and selectively induce degradation of target proteins. Summary of the Invention [Means for solving the problem]

[0005] Abstract In one embodiment, a method for degrading a target protein on the surface of a target cell includes contacting an endogenous internalization receptor and the target protein on the surface of the target cell with a binding agent, wherein the binding agent comprises: (i) a first binding domain that specifically binds to an endogenous internalization receptor selected from the group consisting of MUC1, ITGB6, CEACAM5, and CDH17; and (ii) a second binding domain that specifically binds to the target protein, wherein the target protein comprises EGFR.

[0006] In some embodiments, the binding agent is a multispecific antibody, a bispecific diabody, a bispecific Fab2, a bispecific camelid antibody, a bispecific peptibody scFv-Fc, a bispecific IgG, a knob-and-hole bispecific IgG, an Fc-Fab, or a knob-and-hole bispecific Fc-Fab. In some embodiments, the first binding domain comprises a first binding domain variable heavy chain and a first binding domain variable light chain.

[0007] In some embodiments, the endogenous internalizing receptor is MUC1. In some embodiments, the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 71. In some embodiments, the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 71. In some embodiments, the first binding domain variable heavy chain comprises SEQ ID NO: 71. In some embodiments, the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO: 73.

[0008] In some embodiments, the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO: 73. In some embodiments, the first binding domain variable light chain comprises SEQ ID NO: 73. In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 71 and 73. In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 71 and 73. In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that does not include any amino acids from the epitope bound by an antibody comprising SEQ ID NOs: 71 and 73.

[0009] In some embodiments, the endogenous internalizing receptor is CDH17. In some embodiments, the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 47. In some embodiments, the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 47. In some embodiments, the first binding domain variable heavy chain comprises SEQ ID NO: 47. In some embodiments, the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO: 49. In some embodiments, the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO: 49. In some embodiments, the first binding domain variable light chain comprises SEQ ID NO: 49.

[0010] In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 47 and 49. In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 47 and 49. In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that does not comprise any amino acids from the epitope bound by an antibody comprising SEQ ID NOs: 47 and 49.

[0011] In some embodiments, the endogenous internalizing receptor is ITGB6. In some embodiments, the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 287. In some embodiments, the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 287. In some embodiments, the first binding domain variable heavy chain comprises SEQ ID NO: 287. In some embodiments, the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO: 289. In some embodiments, the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO: 289. In some embodiments, the first binding domain variable light chain comprises SEQ ID NO: 289.

[0012] In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 287 and 289. In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 287 and 289. In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that does not include any amino acids from the epitope bound by an antibody comprising SEQ ID NOs: 287 and 289.

[0013] In some embodiments, the endogenous internalizing receptor is CEACAM5. In some embodiments, the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 87. In some embodiments, the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 87. In some embodiments, the first binding domain variable heavy chain comprises SEQ ID NO: 87. In some embodiments, the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO: 89. In some embodiments, the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO: 89. In some embodiments, the first binding domain variable light chain comprises SEQ ID NO: 89.

[0014] In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 87 and 89. In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 87 and 89. In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that does not include any amino acids from the epitope bound by an antibody comprising SEQ ID NOs: 87 and 89.

[0015] In some embodiments, the second binding domain comprises a second binding domain variable heavy chain. In some embodiments, the second binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 651. In some embodiments, the second binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 651. In some embodiments, the second binding domain variable heavy chain comprises SEQ ID NO: 651.

[0016] In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 80% sequence identity to the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 90% sequence identity to the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that does not contain any amino acids from the epitope to which cetuximab binds.

[0017] In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 80% sequence identity to the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 90% sequence identity to the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that does not contain any amino acids from the epitope to which Mav2 binds.

[0018] In some embodiments, after contact, the EGFR is internalized into the target cell together with the endogenous internalized receptor, and the EGFR is degraded. In some embodiments, the endogenous internalized receptor is recycled to the target cell surface after internalization of the binding agent. In some embodiments, the endogenous internalized receptor is degraded.

[0019] In some embodiments, the target cell is a cancer cell, hi some embodiments, the cancer cell is selected from the group consisting of breast cancer cells, B-cell lymphoma cells, pancreatic cancer cells, Hodgkin's lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells, non-Hodgkin's B-cell lymphoma (B-NHL) cells, melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, glioma cells, glioblastoma cells, bladder cancer cells, colorectal cancer cells, and head and neck cancer cells.

[0020] In some embodiments, EGFR expression on the cancer cells is reduced after contact with the multispecific binding agent compared to control cancer cells not contacted with the binding agent. In some embodiments, EGFR expression on the cancer cells is reduced by 50% or more compared to EGFR expression on control cancer cells not contacted with the binding agent. In some embodiments, EGFR expression on the cancer cells is reduced by 50% or more compared to EGFR expression on control cancer cells contacted with the binding agent. In some embodiments, cell surface removal of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells not contacted with the binding agent. In some embodiments, EGFR expression on the cancer cells is reduced by 20% or more compared to EGFR expression on control cancer cells contacted with the binding agent.

[0021] In some embodiments, the internalization of EGFR in the cancer cells is at least 20% or more compared to the internalization of EGFR in control cancer cells not contacted with the binding agent. In some embodiments, the internalization of EGFR on the cancer cells is at least 20% or more compared to the internalization of EGFR on control cancer cells contacted with the monospecific EGFR-binding agent. In some embodiments, the degradation of EGFR in the cancer cells is at least 20% or more compared to the degradation of EGFR in control cancer cells not contacted with the binding agent. In some embodiments, the cytolysis of EGFR on the cancer cells is at least 20% or more compared to the EGFR on control cancer cells contacted with the monospecific EGFR-binding agent. In some embodiments, the monospecific EGFR-binding agent is cetuximab.

[0022] In some embodiments, the method increases the sensitivity of the cancer cell to a cancer therapeutic agent. In some embodiments, the cancer therapeutic agent is a cytotoxic agent. In some embodiments, the method reduces proliferation of the cancer cell. In some embodiments, the method increases killing of the cancer cell. In some embodiments, the contacting is performed in vivo.

[0023] In another aspect, the disclosure provides a method for treating cancer in a subject, comprising administering to the subject a binding agent, wherein the binding agent comprises: (i) a first binding domain that specifically binds to an endogenous internalizing receptor expressed on a target cell and selected from the group consisting of MUC1, ITGB6, CEACAM5, and CDH17; and (ii) a second binding domain that specifically binds to a target protein comprising EGFR.

[0024] In some embodiments, the endogenous internalizing receptor is MUC1. In some embodiments, the endogenous internalizing receptor is ITGB6. In some embodiments, the endogenous internalizing receptor is CEACAM5. In some embodiments, the endogenous internalizing receptor is CDH17.

[0025] In some embodiments, the cancer is breast cancer, B-cell lymphoma, pancreatic cancer, Hodgkin's lymphoma, ovarian cancer, prostate cancer, mesothelioma, lung cancer, non-Hodgkin's B-cell (B-NHL) lymphoma, melanoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, neuroblastoma, glioma, glioblastoma, bladder cancer, colorectal cancer, or head and neck cancer.

[0026] In some embodiments, the tumor volume of a tumor contacted with the multispecific binding agent is reduced by 20% or more compared to the tumor volume of a tumor not contacted with the bispecific binding agent. In some embodiments, the tumor volume of a tumor contacted with the multispecific binding agent is at least 80% or less in volume compared to the tumor volume of a tumor not contacted with the bispecific binding agent. In some embodiments, the expression of EGFR on cancer cells is reduced by 20% compared to the EGFR expression of cancer cells not contacted with the bispecific binding agent. In some embodiments, the expression of EGFR on cancer cells is reduced by 20% compared to the EGFR expression of cancer cells contacted with the monospecific EGFR binding agent. In some embodiments, the monospecific EGFR binding agent is cetuximab.

[0027] In another aspect, the present disclosure provides a multispecific binding agent comprising: (a) a first binding domain that specifically binds to an endogenous internalizing receptor selected from the group consisting of MUC1, ITGB6, CEACAM5, or CDH17; and (b) a second binding domain that specifically binds to a target protein that is EGFR.

[0028] In some embodiments, the multispecific binding agent is a multispecific antibody, a bispecific antibody, a bispecific diabody, a bispecific Fab2, a bispecific camelid antibody, a bispecific peptibody scFv-Fc, a bispecific IgG, a knob-and-hole bispecific IgG, an Fc-Fab, or a knob-and-hole bispecific Fc-Fab. In some embodiments, the first binding domain comprises a first binding domain variable heavy chain and a first binding domain variable light chain.

[0029] In some embodiments, the endogenous internalizing receptor is MUC1. In some embodiments, the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 71. In some embodiments, the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 71. In some embodiments, the first binding domain variable heavy chain comprises SEQ ID NO: 71. In some embodiments, the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO: 73. In some embodiments, the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO: 73. In some embodiments, the first binding domain variable light chain comprises SEQ ID NO: 73.

[0030] In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 71 and 73. In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 71 and 73. In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that does not comprise any amino acids from the epitope bound by an antibody comprising SEQ ID NOs: 71 and 73.

[0031] In some embodiments, the endogenous internalizing receptor is ITGB6. In some embodiments, the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 287. In some embodiments, the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 287. In some embodiments, the first binding domain variable heavy chain comprises SEQ ID NO: 287. In some embodiments, the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO: 289. In some embodiments, the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO: 289. In some embodiments, the first binding domain variable light chain comprises SEQ ID NO: 289.

[0032] In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 287 and 289. In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 287 and 289. In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that does not include any amino acids from the epitope bound by an antibody comprising SEQ ID NOs: 287 and 289.

[0033] In some embodiments, the endogenous internalizing receptor is CEACAM5. In some embodiments, the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 87. In some embodiments, the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 87. In some embodiments, the first binding domain variable heavy chain comprises SEQ ID NO: 87. In some embodiments, the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO: 89. In some embodiments, the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO: 89. In some embodiments, the first binding domain variable light chain comprises SEQ ID NO: 89.

[0034] In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 87 and 89. In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 87 and 89. In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that does not include any amino acids from the epitope bound by an antibody comprising SEQ ID NOs: 87 and 89.

[0035] In some embodiments, the endogenous internalizing receptor is CDH17. In some embodiments, the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 47. In some embodiments, the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 47. In some embodiments, the first binding domain variable heavy chain comprises SEQ ID NO: 47. In some embodiments, the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO: 49. In some embodiments, the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO: 49. In some embodiments, the first binding domain variable light chain comprises SEQ ID NO: 49.

[0036] In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 47 and 49. In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 47 and 49. In some embodiments, the first binding domain binds to an epitope of an internalizing receptor on a target cell that does not comprise any amino acids from the epitope bound by an antibody comprising SEQ ID NOs: 47 and 49.

[0037] In some embodiments, the second binding domain comprises a second binding domain variable heavy chain. In some embodiments, the second binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 651. In some embodiments, the second binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 651. In some embodiments, the second binding domain variable heavy chain comprises SEQ ID NO: 651.

[0038] In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 80% sequence identity to the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 90% sequence identity to the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that does not contain any amino acids from the epitope to which cetuximab binds.

[0039] In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 80% sequence identity to the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 90% sequence identity to the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that does not contain any amino acids from the epitope to which Mav2 binds.

[0040] In some embodiments, the half-life of the multispecific binding agent is within 20% of the half-life of cetuximab. In some embodiments, the clearance rate of the multispecific binding agent is within 20-95% of the clearance rate of cetuximab. In some embodiments, the Kd of the multispecific binding agent is at least 2-fold but not more than 2-fold lower than the binding affinity of cetuximab to EGFR. In some embodiments, the multispecific binding agent is at least 5-fold but not more than 5-fold lower than the binding affinity of cetuximab to EGFR. In some embodiments, the multispecific binding agent is at least 10-fold but not more than 10-fold lower than the binding affinity of cetuximab to EGFR. In some embodiments, the Kd of the binding affinity of the multispecific binding agent may be within an order of magnitude of the binding affinity of the monovalent binding agent.

[0041] In some embodiments, the Kd of the multispecific binding agent is within ±10% of the binding affinity of cetuximab to EGFR. In some embodiments, the Kd of the multispecific binding agent is within ±20% of the binding affinity of cetuximab to EGFR. In some embodiments, the Kd of the multispecific binding agent is within ±30% of the binding affinity of cetuximab to EGFR. In some embodiments, the Kd of the multispecific binding agent is less than the binding affinity of cetuximab to EGFR. In some embodiments, the Kd of the multispecific binding agent is greater than the binding affinity of cetuximab to EGFR.

[0042] In yet another aspect, the present disclosure provides a method for degrading a target protein on the surface of a target cell, the method comprising contacting an endogenous internalizing receptor and the target protein on the surface of the target cell with a binding agent, wherein the binding agent comprises: (i) a first binding domain that specifically binds to the endogenous internalizing receptor comprising B7-H3; and (ii) a second binding domain that specifically binds to the target protein, wherein the target protein comprises EGFR.

[0043] In some embodiments, the binding agent is a multispecific antibody, a bispecific diabody, a bispecific Fab2, a bispecific camelid antibody, a bispecific peptibody-scFv-Fc, a bispecific IgG, a knob-and-hole bispecific IgG, an Fc-Fab, or a knob-and-hole bispecific Fc-Fab. In some embodiments, the first binding domain binds to an epitope of an endogenous internalizing receptor on a target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 99 and 101. In some embodiments, the first binding domain binds to an epitope of an endogenous internalizing receptor on a target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 99 and 101. In some embodiments, the first binding domain binds to an epitope of an endogenous internalizing receptor on a target cell that does not include any amino acids from the epitope bound by an antibody comprising SEQ ID NOs: 99 and 101.

[0044] In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 80% sequence identity to the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 90% sequence identity to the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that does not contain any amino acids from the epitope to which cetuximab binds.

[0045] In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 80% sequence identity to the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 90% sequence identity to the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that does not contain any amino acids from the epitope to which Mav2 binds.

[0046] In some embodiments, the first binding domain comprises a first binding domain variable heavy chain and a first binding domain variable light chain. In some embodiments, the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 99. In some embodiments, the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 99. In some embodiments, the first binding domain variable heavy chain comprises SEQ ID NO: 99. In some embodiments, the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO: 101. In some embodiments, the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO: 101. In some embodiments, the first binding domain variable light chain comprises SEQ ID NO: 101.

[0047] In some embodiments, the second binding domain comprises a second binding domain variable heavy chain. In some embodiments, the second binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 655. In some embodiments, the second binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 655. In some embodiments, the second binding domain variable heavy chain comprises SEQ ID NO: 655.

[0048] In some embodiments, the endogenous internalizing receptor is recycled to the target cell surface after internalization of the binding agent. In some embodiments, the endogenous internalizing receptor is degraded. In some embodiments, the target cell is a cancer cell. In some embodiments, the cancer cell is selected from the group consisting of breast cancer cells, B-cell lymphoma cells, pancreatic cancer cells, Hodgkin's lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells, non-Hodgkin's B-cell lymphoma (B-NHL) cells, melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, glioma cells, glioblastoma cells, bladder cancer cells, colorectal cancer cells, and head and neck cancer cells.

[0049] In some embodiments, EGFR expression on the cancer cells is reduced after contact with the multispecific binding agent compared to control cancer cells not contacted with the binding agent. In some embodiments, EGFR expression on the cancer cells is reduced by 50% or more compared to EGFR expression on control cancer cells not contacted with the binding agent. In some embodiments, EGFR expression on the cancer cells is reduced by 50% or more compared to EGFR expression on control cancer cells contacted with the monospecific EGFR binding agent. In some embodiments, cell surface ablation of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells not contacted with the binding agent.

[0050] In some embodiments, cell surface removal of EGFR on cancer cells is at least 20% or more compared to EGFR on control cancer cells contacted with the monospecific EGFR-binding agent. In some embodiments, internalization of EGFR in cancer cells is at least 20% or more compared to EGFR on control cancer cells not contacted with the binding agent. In some embodiments, internalization of EGFR on cancer cells is at least 20% or more compared to EGFR on control cancer cells contacted with the monospecific EGFR-binding agent. In some embodiments, degradation of EGFR in cancer cells is at least 20% or more compared to EGFR degradation in control cancer cells not contacted with the binding agent. In some embodiments, cellular degradation of EGFR on cancer cells is at least 20% or more compared to EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

[0051] In some embodiments, the monospecific EGFR-binding agent is cetuximab. In some embodiments, the method increases the sensitivity of the cancer cell to a cancer therapeutic agent. In some embodiments, the cancer therapeutic agent is a cytotoxic agent. In some embodiments, the method reduces proliferation of the cancer cell. In some embodiments, the method increases killing of the cancer cell. In some embodiments, the contacting is performed in vivo.

[0052] In another aspect, the disclosure provides a method for treating cancer in a subject, comprising administering to the subject a binding agent, wherein the binding agent comprises: (i) a first binding domain that specifically binds to an endogenous internalizing receptor expressed on a target cell, the endogenous internalizing receptor being B7-H3; and (ii) a second binding domain that specifically binds to a target protein comprising EGFR.

[0053] In some embodiments, the cancer is breast cancer, B-cell lymphoma, pancreatic cancer, Hodgkin's lymphoma, ovarian cancer, prostate cancer, mesothelioma, lung cancer, non-Hodgkin's B-cell (B-NHL) lymphoma, melanoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, neuroblastoma, glioma, glioblastoma, bladder cancer, colorectal cancer, or head and neck cancer.

[0054] In some embodiments, the tumor volume of a tumor contacted with the multispecific binding agent is reduced by 20% or more compared to the tumor volume of a tumor not contacted with the bispecific binding agent. In some embodiments, the tumor volume of a tumor contacted with the multispecific binding agent is less than 80% or less compared to the tumor volume of a tumor not contacted with the bispecific binding agent. In some embodiments, the expression of EGFR on cancer cells is reduced by 20% compared to the EGFR expression of cancer cells not contacted with the bispecific binding agent. In some embodiments, the expression of EGFR on cancer cells is reduced by 20% compared to the EGFR expression of cancer cells contacted with the monospecific EGFR binding agent. In some embodiments, the monospecific EGFR binding agent is cetuximab.

[0055] In another aspect, the present disclosure provides a multispecific binding agent comprising: (a) a first binding domain that specifically binds to an endogenous internalizing receptor, wherein the first binding domain is B7-H3; and (b) a second binding domain that specifically binds to a target protein, wherein the target protein is EGFR.

[0056] In some embodiments, the multispecific binding agent is a multispecific antibody, a bispecific antibody, a bispecific diabody, a bispecific Fab2, a bispecific camelid antibody, a bispecific peptibody-scFv-Fc, a bispecific IgG, a knob-and-hole bispecific IgG, an Fc-Fab, or a knob-and-hole bispecific Fc-Fab.

[0057] In some embodiments, the first binding domain binds to an epitope of an endogenous internalizing receptor on a target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 99 and 101. In some embodiments, the first binding domain binds to an epitope of an endogenous internalizing receptor on a target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 99 and 101. In some embodiments, the first binding domain binds to an epitope of an endogenous internalizing receptor on a target cell that does not comprise any amino acids from the epitope bound by an antibody comprising SEQ ID NOs: 99 and 101.

[0058] In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 80% sequence identity to the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 90% sequence identity to the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that does not contain any amino acids from the epitope to which cetuximab binds.

[0059] In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 80% sequence identity to the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 90% sequence identity to the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that does not contain any amino acids from the epitope to which Mav2 binds.

[0060] In some embodiments, the first binding domain comprises a first binding domain variable heavy chain and a first binding domain variable light chain. In some embodiments, the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 99. In some embodiments, the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 99. In some embodiments, the first binding domain variable heavy chain comprises SEQ ID NO: 99. In some embodiments, the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO: 101. In some embodiments, the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO: 101. In some embodiments, the first binding domain variable light chain comprises SEQ ID NO: 101.

[0061] In some embodiments, the second binding domain comprises a second binding domain variable heavy chain. In some embodiments, the second binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 655. In the multispecific binding agent of claim 216, the second binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 655. In some embodiments, the second binding domain variable heavy chain comprises SEQ ID NO: 655.

[0062] In some embodiments, the half-life of the multispecific binding agent is within 20% of the half-life of cetuximab. In some embodiments, the clearance rate of the multispecific binding agent is within 20-95% of the clearance rate of cetuximab. In some embodiments, the Kd of the multispecific binding agent is at least 2-fold but not more than 2-fold the binding affinity of cetuximab to EGFR. In some embodiments, the Kd of the multispecific binding agent is at least 5-fold but not more than 5-fold the binding affinity of cetuximab to EGFR. In some embodiments, the Kd of the multispecific binding agent is at least 10-fold but not more than 10-fold the binding affinity of cetuximab to EGFR.

[0063] In some embodiments, the Kd of the binding affinity of the multispecific binding agent may be within an order of magnitude of the binding affinity of the monovalent binding agent. In some embodiments, the Kd of the multispecific binding agent is within ±10% of the binding affinity of cetuximab to EGFR. In some embodiments, the Kd of the multispecific binding agent is within ±20% of the binding affinity of cetuximab to EGFR. In some embodiments, the Kd of the multispecific binding agent is within ±30% of the binding affinity of cetuximab to EGFR. In some embodiments, the Kd of the multispecific binding agent is less than the binding affinity of cetuximab to EGFR. In some embodiments, the Kd of the multispecific binding agent is greater than the binding affinity of cetuximab to EGFR.

[0064] In another aspect, the disclosure provides a method for degrading a target protein on the surface of a target cell, the method comprising contacting an E3 ligase and the target protein on the surface of the target cell with a binding agent, wherein the binding agent comprises: (i) a first binding domain that specifically binds to the E3 ligase, wherein the E3 ligase is RNF43; and (ii) a second binding domain that specifically binds to the target protein, wherein the target protein is EGFR.

[0065] In some embodiments, the binding agent is a multispecific antibody, a bispecific diabody, a bispecific Fab2, a bispecific camelid antibody, a bispecific peptibody scFv-Fc, a bispecific IgG, a knob-and-hole bispecific IgG, an Fc-Fab, or a knob-and-hole bispecific Fc-Fab.

[0066] In some embodiments, the first binding domain binds to an epitope of an endogenous internalizing receptor on a target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 331 and 333. In some embodiments, the first binding domain binds to an epitope of an endogenous internalizing receptor on a target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 331 and 333. In some embodiments, the first binding domain binds to an epitope of an endogenous internalizing receptor on a target cell that does not comprise any amino acids from the epitope bound by an antibody comprising SEQ ID NOs: 331 and 333.

[0067] In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 80% sequence identity to the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 90% sequence identity to the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that does not contain any amino acids from the epitope to which cetuximab binds.

[0068] In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 80% sequence identity to the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 90% sequence identity to the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that does not contain any amino acids from the epitope to which Mav2 binds.

[0069] In some embodiments, the first binding domain comprises a first binding domain variable heavy chain and a first binding domain variable light chain. In some embodiments, the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 331. In some embodiments, the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 331. In some embodiments, the first binding domain variable heavy chain comprises SEQ ID NO: 331. In some embodiments, the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO: 333. In some embodiments, the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO: 333. In some embodiments, the first binding domain variable light chain comprises SEQ ID NO: 333.

[0070] In some embodiments, the second binding domain comprises a second binding domain variable heavy chain. In some embodiments, the second binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 655. In some embodiments, the second binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 655. In some embodiments, the second binding domain variable heavy chain comprises SEQ ID NO: 655.

[0071] In some embodiments, the E3 ligase is degraded. In some embodiments, the target cell is a cancer cell. In some embodiments, the cancer cell is selected from the group consisting of breast cancer cells, B-cell lymphoma cells, pancreatic cancer cells, Hodgkin's lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells, non-Hodgkin's B-cell lymphoma (B-NHL) cells, melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, glioma cells, glioblastoma cells, bladder cancer cells, colorectal cancer cells, and head and neck cancer cells.

[0072] In some embodiments, EGFR expression on the cancer cells is reduced after contact with the bispecific binding agent compared to control cancer cells not contacted with the binding agent. In some embodiments, EGFR expression on the cancer cells is reduced by 50% or more compared to EGFR expression on control cancer cells not contacted with the binding agent. In some embodiments, EGFR expression on the cancer cells is reduced by 50% or more compared to EGFR expression on control cancer cells contacted with the monospecific EGFR-binding agent. In some embodiments, cell surface ablation of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells not contacted with the binding agent. In some embodiments, cell surface ablation of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

[0073] In some embodiments, the internalization of EGFR in the cancer cells is at least 20% or more compared to the internalization of EGFR in control cancer cells not contacted with the binding agent. In some embodiments, the internalization of EGFR on the cancer cells is at least 20% or more compared to the internalization of EGFR on control cancer cells contacted with the monospecific EGFR-binding agent. In some embodiments, the degradation of EGFR in the cancer cells is at least 20% or more compared to the degradation of EGFR in control cancer cells not contacted with the binding agent. In some embodiments, the cytolysis of EGFR on the cancer cells is at least 20% or more compared to the EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

[0074] In some embodiments, the monospecific EGFR-binding agent is cetuximab. In some embodiments, the method increases the sensitivity of the cancer cell to a cancer therapeutic agent. In some embodiments, the cancer therapeutic agent is a cytotoxic agent. In some embodiments, the method reduces proliferation of the cancer cell. In some embodiments, the method increases killing of the cancer cell. In some embodiments, the contacting is performed in vivo.

[0075] In another aspect, the disclosure provides a method for treating cancer in a subject, comprising administering to the subject a binding agent, wherein the binding agent comprises: (i) a first binding domain that specifically binds to an E3 ligase that is RNF43; and (ii) a second binding domain that specifically binds to a target protein including EGFR.

[0076] In some embodiments, the first binding domain comprises a first binding domain variable heavy chain and a first binding domain variable light chain. In some embodiments, the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 331. In some embodiments, the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 331. In some embodiments, the first binding domain variable heavy chain comprises SEQ ID NO: 331. In some embodiments, the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO: 333. In some embodiments, the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO: 333. In some embodiments, the first binding domain variable light chain comprises SEQ ID NO: 333.

[0077] In some embodiments, the second binding domain comprises a second binding domain variable heavy chain. In some embodiments, the second binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 655. In some embodiments, the second binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 655. In some embodiments, the second binding domain variable heavy chain comprises SEQ ID NO: 655.

[0078] In some embodiments, the cancer is breast cancer, B-cell lymphoma, pancreatic cancer, Hodgkin's lymphoma, ovarian cancer, prostate cancer, mesothelioma, lung cancer, non-Hodgkin's B-cell (B-NHL) lymphoma, melanoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, neuroblastoma, glioma, glioblastoma, bladder cancer, colorectal cancer, or head and neck cancer.

[0079] In another aspect, the present disclosure provides a multispecific binding agent comprising: (a) a first binding domain that specifically binds to an E3 ligase, wherein the E3 ligase is RNF43; and (b) a second binding domain that specifically binds to a target protein, wherein the target protein is EGFR.

[0080] In some embodiments, the multispecific binding agent is a multispecific antibody, a bispecific antibody, a bispecific diabody, a bispecific Fab2, a bispecific camelid antibody, a bispecific peptibody-scFv-Fc, a bispecific IgG, a knob-and-hole bispecific IgG, an Fc-Fab, or a knob-and-hole bispecific Fc-Fab.

[0081] In some embodiments, the first binding domain binds to an epitope of RNF43 on a target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 331 and 333. In some embodiments, the first binding domain binds to an epitope of RNF43 on a target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 331 and 333. In some embodiments, the first binding domain binds to an epitope of RNF43 on a target cell that does not include any amino acids from the epitope bound by an antibody comprising SEQ ID NOs: 331 and 333.

[0082] In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 80% sequence identity to the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 90% sequence identity to the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that does not contain any amino acids from the epitope to which cetuximab binds.

[0083] In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 80% sequence identity to the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 90% sequence identity to the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that does not contain any amino acids from the epitope to which Mav2 binds.

[0084] In some embodiments, the first binding domain comprises a first binding domain variable heavy chain and a first binding domain variable light chain. In some embodiments, the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 331. In some embodiments, the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 331. In some embodiments, the first binding domain variable heavy chain comprises SEQ ID NO: 331. In some embodiments, the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO: 333. In some embodiments, the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO: 333. In some embodiments, the first binding domain variable light chain comprises SEQ ID NO: 333.

[0085] In some embodiments, the second binding domain comprises a second binding domain variable heavy chain. In some embodiments, the second binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 655. In some embodiments, the second binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 655. In some embodiments, the second binding domain variable heavy chain comprises SEQ ID NO: 655.

[0086] In another aspect, the disclosure provides a method for degrading a target protein on the surface of a target cell, the method comprising contacting an endogenous internalization receptor and the target protein on the surface of the target cell with a binding agent, wherein the binding agent comprises: (i) a first binding domain that specifically binds to an endogenous internalization receptor selected from the group consisting of LGR5, HER3, LY75, MST1R, MSLN, EpCAM, TNFRSF10B, and CD71; and (ii) a second binding domain that specifically binds to the target protein, wherein the target protein comprises EGFR.

[0087] In some embodiments, the binding agent is a multispecific antibody, a bispecific diabody, a bispecific Fab2, a bispecific camelid antibody, a bispecific peptibody scFv-Fc, a bispecific IgG, a knob-and-hole bispecific IgG, an Fc-Fab, or a knob-and-hole bispecific Fc-Fab.

[0088] In some embodiments, the first binding domain binds to an epitope of an endogenous internalizing receptor on a target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising any one of the variable heavy chain sequences or any one of the variable light chain sequences listed in Table 1 or Table 2. In some embodiments, the first binding domain binds to an epitope of an endogenous internalizing receptor on a target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising any one of the variable heavy chain sequences or any one of the variable light chain sequences listed in Table 1 or Table 2. In some embodiments, the first binding domain binds to an epitope of an endogenous internalizing receptor on a target cell that does not comprise any amino acids from an epitope bound by an antibody comprising any one of the variable heavy chain sequences or any one of the variable light chain sequences listed in Table 1 or Table 2.

[0089] In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 80% sequence identity to the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 90% sequence identity to the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that does not contain any amino acids from the epitope to which cetuximab binds.

[0090] In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 80% sequence identity to the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 90% sequence identity to the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that does not contain any amino acids from the epitope to which Mav2 binds.

[0091] In some embodiments, the first binding domain comprises a first binding domain variable heavy chain and a first binding domain variable light chain. In some embodiments, the first binding domain variable heavy chain comprises at least 80% sequence identity to any one of the variable heavy chain sequences listed in Table 1. In some embodiments, the first binding domain variable heavy chain comprises at least 90% sequence identity to any one of the variable heavy chain sequences listed in Table 1. In some embodiments, the first binding domain variable heavy chain comprises any one of the variable heavy chain sequences listed in Table 1. In some embodiments, the first binding domain variable light chain comprises at least 80% sequence identity to any one of the variable light chain sequences listed in Table 1. In some embodiments, the first binding domain variable light chain comprises at least 90% sequence identity to any one of the variable light chain sequences listed in Table 1. In some embodiments, the first binding domain variable light chain comprises any one of the variable light chain sequences listed in Table 1.

[0092] In some embodiments, the second binding domain comprises a second binding domain variable heavy chain. In some embodiments, the second binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 655. In some embodiments, the second binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 655. In some embodiments, the second binding domain variable heavy chain comprises SEQ ID NO: 655.

[0093] In some embodiments, the endogenous internalizing receptor is recycled to the target cell surface after internalization of the binding agent. In some embodiments, the endogenous internalizing receptor is degraded. In some embodiments, the target cell is a cancer cell. In some embodiments, the cancer cell is selected from the group consisting of breast cancer cells, B-cell lymphoma cells, pancreatic cancer cells, Hodgkin's lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells, non-Hodgkin's B-cell lymphoma (B-NHL) cells, melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, glioma cells, glioblastoma cells, bladder cancer cells, colorectal cancer cells, and head and neck cancer cells.

[0094] In some embodiments, EGFR expression on the cancer cells is reduced after contact with the multispecific binding agent compared to control cancer cells not contacted with the binding agent. In some embodiments, EGFR expression on the cancer cells is reduced by 50% or more compared to EGFR expression on control cancer cells not contacted with the binding agent. In some embodiments, EGFR expression on the cancer cells is reduced by 50% or more compared to EGFR expression on control cancer cells contacted with the monospecific EGFR-binding agent. In some embodiments, cell surface ablation of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells not contacted with the binding agent. In some embodiments, cell surface ablation of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

[0095] In some embodiments, the internalization of EGFR in the cancer cells is at least 20% or more compared to the internalization of EGFR in control cancer cells not contacted with the binding agent. In some embodiments, the internalization of EGFR on the cancer cells is at least 20% or more compared to the internalization of EGFR on control cancer cells contacted with the monospecific EGFR-binding agent. In some embodiments, the degradation of EGFR in the cancer cells is at least 20% or more compared to the degradation of EGFR in control cancer cells not contacted with the binding agent. In some embodiments, the cytolysis of EGFR on the cancer cells is at least 20% or more compared to the EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

[0096] In some embodiments, the monospecific EGFR-binding agent is cetuximab. In some embodiments, the method increases the sensitivity of the cancer cell to a cancer therapeutic agent. In some embodiments, the cancer therapeutic agent is a cytotoxic agent. In some embodiments, the method reduces proliferation of the cancer cell. In some embodiments, the method increases killing of the cancer cell. In some embodiments, the contacting is performed in vivo.

[0097] In another aspect, the disclosure provides a method for treating cancer in a subject, comprising administering to the subject a binding agent, wherein the binding agent comprises: (i) a first binding domain that specifically binds to an endogenous internalizing receptor expressed on a target cell and selected from the group consisting of LGR5, HER3, LY75, MST1R, MSLN, EpCAM, TNFRSF10B, and CD71; and (ii) a second binding domain that specifically binds to a target protein comprising EGFR.

[0098] In some embodiments, the cancer is breast cancer, B-cell lymphoma, pancreatic cancer, Hodgkin's lymphoma, ovarian cancer, prostate cancer, mesothelioma, lung cancer, non-Hodgkin's B-cell (B-NHL) lymphoma, melanoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, neuroblastoma, glioma, glioblastoma, bladder cancer, colorectal cancer, or head and neck cancer. In some embodiments, the tumor volume of a tumor contacted with the multispecific binding agent is reduced by at least 20% or more compared to the tumor volume of a tumor not contacted with the bispecific binding agent. In some embodiments, the tumor volume of a tumor contacted with the multispecific binding agent is reduced by less than 80% or less compared to the tumor volume of a tumor not contacted with the bispecific binding agent. In some embodiments, the expression of EGFR on cancer cells is reduced by at least 20% compared to the EGFR expression of cancer cells not contacted with the bispecific binding agent. In some embodiments, the expression of EGFR on cancer cells is reduced by 20% compared to the EGFR expression of cancer cells contacted with the monospecific EGFR binding agent. In some embodiments, the monospecific EGFR-binding agent is cetuximab.

[0099] In another aspect, the present disclosure provides a multispecific binding agent comprising: (a) a first binding domain that specifically binds to an endogenous internalizing receptor selected from the group consisting of LGR5, HER3, LY75, MST1R, MSLN, EpCAM, TNFRSF10B, and CD71; and (b) a second binding domain that specifically binds to a target protein that is EGFR.

[0100] In some embodiments, the multispecific binding agent is a multispecific antibody, a bispecific antibody, a bispecific diabody, a bispecific Fab2, a bispecific camelid antibody, a bispecific peptibody-scFv-Fc, a bispecific IgG, a knob-and-hole bispecific IgG, an Fc-Fab, or a knob-and-hole bispecific Fc-Fab.

[0101] In some embodiments, the first binding domain binds to an epitope of an endogenous internalizing receptor on a target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising any one of the variable heavy chain sequences or any one of the variable light chain sequences listed in Table 1 or Table 2. In some embodiments, the first binding domain binds to an epitope of an endogenous internalizing receptor on a target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising any one of the variable heavy chain sequences or any one of the variable light chain sequences listed in Table 1 or Table 2. In some embodiments, the first binding domain binds to an epitope of an endogenous internalizing receptor on a target cell that does not comprise any amino acids from an epitope bound by an antibody comprising any one of the variable heavy chain sequences or any one of the variable light chain sequences listed in Table 1 or Table 2.

[0102] In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 80% sequence identity to the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 90% sequence identity to the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that does not contain any amino acids from the epitope to which cetuximab binds.

[0103] In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 80% sequence identity to the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that comprises at least 90% sequence identity to the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of a target protein on a target cell that does not contain any amino acids from the epitope to which Mav2 binds.

[0104] In some embodiments, the first binding domain comprises a first binding domain variable heavy chain and a first binding domain variable light chain. In some embodiments, the first binding domain variable heavy chain comprises at least 80% sequence identity to any one of the variable heavy chain sequences listed in Table 1. In some embodiments, the first binding domain variable heavy chain comprises at least 90% sequence identity to any one of the variable heavy chain sequences listed in Table 1. In some embodiments, the first binding domain variable heavy chain comprises any one of the variable heavy chain sequences listed in Table 1. In some embodiments, the first binding domain variable light chain comprises at least 80% sequence identity to any one of the variable light chain sequences listed in Table 1. In some embodiments, the first binding domain variable light chain comprises at least 90% sequence identity to any one of the variable light chain sequences listed in Table 1. In some embodiments, the first binding domain variable light chain comprises any one of the variable light chain sequences listed in Table 1.

[0105] In some embodiments, the second binding domain comprises a second binding domain variable heavy chain. In some embodiments, the second binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO: 655. In some embodiments, the second binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO: 655. In some embodiments, the second binding domain variable heavy chain comprises SEQ ID NO: 655.

[0106] In some embodiments, the half-life of the multispecific binding agent is within 20% of the half-life of cetuximab. In some embodiments, the clearance rate of the multispecific binding agent is within 20-95% of the clearance rate of cetuximab. In some embodiments, the Kd of the multispecific binding agent is at least 2-fold but not more than 2-fold the binding affinity of cetuximab to EGFR. In some embodiments, the Kd of the multispecific binding agent is at least 5-fold but not more than 5-fold the binding affinity of cetuximab to EGFR. In some embodiments, the Kd of the multispecific binding agent is at least 10-fold but not more than 10-fold the binding affinity of cetuximab to EGFR.

[0107] In some embodiments, the Kd of the binding affinity of the multispecific binding agent may be within an order of magnitude of the binding affinity of the monovalent binding agent. In some embodiments, the Kd of the multispecific binding agent is within ±10% of the binding affinity of cetuximab to EGFR. In some embodiments, the Kd of the multispecific binding agent is within ±20% of the binding affinity of cetuximab to EGFR. In some embodiments, the Kd of the multispecific binding agent is within ±30% of the binding affinity of cetuximab to EGFR. In some embodiments, the Kd of the multispecific binding agent is less than the binding affinity of cetuximab to EGFR. In some embodiments, the Kd of the multispecific binding agent is greater than the binding affinity of cetuximab to EGFR.

[0108] In one aspect, the disclosure provides a method for degrading EGFR protein on a target cell, comprising contacting the EGFR protein and a membrane-bound internalizing protein on the target cell with a bispecific binding agent, wherein contacting the EGFR protein and the membrane-bound internalizing protein with the bispecific binding agent results in internalization and degradation of the EGFR protein, wherein the bispecific binding agent comprises (a) a first binding domain that specifically binds to an extracellular epitope of the membrane-bound internalizing protein; and (b) a second binding domain that specifically binds to an extracellular epitope on the EGFR protein, wherein the membrane-bound internalizing protein is selected from the group consisting of CEACAM5, CEACAM6, HER3, MUC1, CD40, CD50, CD60, CD80, CD90, CD100, CD110, CD120, CD130, CD140, CD150, CD260, CD270, CD280, CD300, CD490, CD510, CD620, CD830, CD90, CD150, CD160, CD170, CD180, CD190, CD290, CD290, CD290, CD290, CD290, CD290, CD290, CD290, CD290, CD290, CD300, CD490, CD190, CD2 ... 205, CD166, PRLR, SLC34A2, ITGB6, LRRC15, MUC16, SLC39A6, AXL, CD40, CD228, MUC5A, ITGB1, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, I-Ag7, ENPP3, CD46, CD56, ROR1, GPR20, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, GPR56, CD71, LGR5, LY75, CD276 / B7-H3, MST1R, MSLN, EpCAM, TNFRSF10B, STEAP1, MELTF, TROP2, CDH17, RNF43 and RNF128.

[0109] In some embodiments, the membrane-bound internalizing protein is selected from CD205, CD166, SLC34A2, ITGB6, LRRC15, and MUC16. In some embodiments, the bispecific binding agent comprises an antibody. In some embodiments, the bispecific binding agent comprises a bispecific antibody.

[0110] In one aspect, the disclosure provides a bispecific binding agent comprising a bispecific antibody or antibody derivative, wherein the bispecific binding agent comprises (a) a first binding domain that specifically binds to an extracellular epitope of an EGFR protein on a target cell; and (b) a second binding domain that specifically binds to an extracellular epitope of a membrane-bound internalized protein on the target cell, wherein the membrane-bound internalized protein is selected from the group consisting of CD205, CD166, SLC34A2, ITGB6, LRRC15, MUC16, SLC39A6, AXL, CD40, CD228, MUC5A, I Bispecific binding agents are provided that are selected from TGB1, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, I-Ag7, ENPP3, CD46, CD56, ROR1, GPR20, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, GPR56, CD71, CECAM5, LGR5, LY75, CD276 / B7-H3, MST1R, MSLN, EpCAM, TNFRSF10B, STEAP1, MELTF, TROP2, CDH17, RNF43, and RNF128.

[0111] In some embodiments, the membrane-bound internalizing protein is CEACAM5. In some embodiments, the membrane-bound internalizing protein is CEACAM6. In some embodiments, the membrane-bound internalizing protein is HER3. In some embodiments, the membrane-bound internalizing protein is MUC1. In some embodiments, the membrane-bound internalizing protein is CD205. In some embodiments, the membrane-bound internalizing protein is CD166. In some embodiments, the membrane-bound internalizing protein is PRLR. In some embodiments, the membrane-bound internalizing protein is SLC34A. In some embodiments, the membrane-bound internalizing protein is ITGB6. In some embodiments, the membrane-bound internalizing protein is LRRC15. In some embodiments, the membrane-bound internalizing protein is MUC16.

[0112] In some embodiments, the membrane-bound internalizing protein is SLC39A6. In some embodiments, the membrane-bound internalizing protein is AXL. In some embodiments, the membrane-bound internalizing protein is MMP14. In some embodiments, the membrane-bound internalizing protein is CMET. In some embodiments, the membrane-bound internalizing protein is CD40. In some embodiments, the membrane-bound internalizing protein is CD228A. In some embodiments, the membrane-bound internalizing protein is CD70. In some embodiments, the membrane-bound internalizing protein is MUC5A. In some embodiments, the membrane-bound internalizing protein is CD44. In some embodiments, the membrane-bound internalizing protein is ITGB1. In some embodiments, the membrane-bound internalizing protein is STn. In some embodiments, the membrane-bound internalizing protein is KAAG1. In some embodiments, the membrane-bound internalizing protein is DLK1. In some embodiments, the membrane-bound internalizing protein is 5T4. In some embodiments, the membrane-bound internalizing protein is SEZ6. In some embodiments, the membrane-bound internalizing protein is CD123. In some embodiments, the membrane-bound internalizing protein is ADAM9. In some embodiments, the membrane-bound internalizing protein is I-Ag7. In some embodiments, the membrane-bound internalizing protein is ENPP3. In some embodiments, the membrane-bound internalizing protein is CD37. In some embodiments, the membrane-bound internalizing protein is CD46. In some embodiments, the membrane-bound internalizing protein is CD56. In some embodiments, the membrane-bound internalizing protein is CD74. In some embodiments, the membrane-bound internalizing protein is IGF1R. In some embodiments, the membrane-bound internalizing protein is ROR1. In some embodiments, the membrane-bound internalizing protein is CDH6. In some embodiments, the membrane-bound internalizing protein is ROR2. In some embodiments, the membrane-bound internalizing protein is GPR20. In some embodiments, the membrane-bound internalizing protein is TM4SF1. In some embodiments, the membrane-bound internalizing protein is B7-H4.In some embodiments, the membrane-bound internalization protein is ALPP. In some embodiments, the membrane-bound internalization protein is LY6E. In some embodiments, the membrane-bound internalization protein is CLDN18. In some embodiments, the membrane-bound internalization protein is LY6G6D. In some embodiments, the membrane-bound internalization protein is GPR56. In some embodiments, the membrane-bound internalization protein is CD71.

[0113] In some embodiments, the membrane-bound internalizing protein is LGR5. In some embodiments, the membrane-bound internalizing protein is LY75. In some embodiments, the membrane-bound internalizing protein is CD276 / B7-H3. In some embodiments, the membrane-bound internalizing protein is MST1R. In some embodiments, the membrane-bound internalizing protein is MSLN. In some embodiments, the membrane-bound internalizing protein is EpCAM. In some embodiments, the membrane-bound internalizing protein is TNFRSF10B. In some embodiments, the membrane-bound internalizing protein is STEAP1. In some embodiments, the membrane-bound internalizing protein is MELTF. In some embodiments, the membrane-bound internalizing protein is TROP2. In some embodiments, the membrane-bound internalizing protein is CDH17. In some embodiments, the membrane-bound internalizing protein is RNF43. In some embodiments, the membrane-bound internalizing protein is RNF43

[0114] In some embodiments, the bispecific binding agent comprises a knob-and-hole bispecific IgG. In some embodiments, the bispecific binding agent does not comprise an antibody-drug conjugate.

[0115] In another aspect, the present disclosure provides a pharmaceutical composition comprising a bispecific binding agent of the present disclosure and a pharmaceutically acceptable excipient.

[0116] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a bispecific binding agent of the present disclosure or a pharmaceutical composition comprising a bispecific binding agent of the present disclosure and a pharmaceutically acceptable excipient.

[0117] In another aspect, the disclosure provides a method of arresting the growth of a target cell, the method comprising contacting the cell with a bispecific binding agent of the disclosure. In some embodiments, the cell is a cancer cell. Incorporation by Reference

[0118] All publications, patents, and patent applications mentioned herein are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or supersede any such conflicting material.

[0119] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also referred to herein as "drawings" and "figures"). [Brief explanation of the drawings]

[0120] [Figure 1] FIG. 1 depicts a method of the present disclosure in which degradation of a target protein 112 (ie, EGFR) is mediated by binding of a bifunctional binding agent 101.

[0121] [Figure 2-1]Figures 2A-2D depict the percentage of cell surface ablation of EGFR in multiple cell types upon treatment with different concentrations of various bispecific antibodies, with the affinity of the resolving monoclonal antibody (Ab) varying from 1 to 1800 nM. Figure 2A depicts the percentage of EGFR cell surface ablation in NCI-H1975 cells treated with 50 nM of bispecific antibody. Figure 2B depicts the percentage of EGFR cell surface ablation in NCI-H1975 cells treated with 500 nM of bispecific antibody. Figure 2C depicts the percentage of EGFR cell surface ablation in HT29 cells treated with 50 nM of bispecific antibody. Figure 2D depicts the percentage of EGFR cell surface ablation in HT29 cells treated with 500 nM of bispecific antibody. [Figure 2-2] Same as above.

[0122] [Figure 3-1] Figures 3A-3B depict the percentage of EGFR cell surface ablation on target cells treated with various bispecific antibodies. Figure 3A depicts the percentage of EGFR cell surface ablation on NCIH1975 target cells treated with various bispecific antibodies at 500 nM concentrations. Figure 3B depicts the percentage of EGFR cell surface ablation on HT29 target cells treated with various bispecific antibodies at 500 nM concentrations. [Figure 3-2] Same as above.

[0123] [Figure 4] Figures 4A-4B depict cell surface removal of EGFR. Figure 4A depicts cell surface removal of EGFR on target cells when treated with various bispecific antibodies in which antibodies against the EGFR target bind to different epitopes. Figure 4B depicts cell surface removal of EGFR on target cells when treated with various bispecific antibodies in which antibodies against a degrading agent bind to different epitopes.

[0124] [Figure 5]FIG. 5 depicts the internalization of EGFR on target cells when treated with various bispecific antibodies, where the bispecific induces internalization on either single-armed mAbs targeting either the target or degraders.

[0125] [Figure 6-1] Figures 6A-6B depict the internalization and degradation of EGFR on target cells upon treatment with various bispecific antibodies. Figure 6A depicts the internalization of EGFR on target cells upon treatment with various bispecific antibodies. Figure 6B depicts the whole-cell degradation of EGFR on target cells upon treatment with various bispecific antibodies. [Figure 6-2] Same as above.

[0126] [Figure 7] Figures 7A-7B depict the amount of EGFR in target cells treated with various bispecific antibodies. Figure 7A is an image of a Western blot depicting the amount of EGFR protein on target cells when treated with various bispecific antibodies. Figure 7B depicts the whole-cell degradation of EGFR on target cells when treated with various bispecific antibodies.

[0127] [Figure 8] Figures 8A-8B depict the amount of EGFR degraded in target cells treated with various bispecific antibodies. Figure 8A is an image of a Western blot depicting the amount of total EGFR protein on target cells when treated with different concentrations of various bispecific antibodies. Figure 8B depicts the percentage of EGFR degradation on target cells when treated with different concentrations of various bispecific antibodies.

[0128] [Figure 9-1]Figures 9A-9C depict EGFR degradation in target cells treated with various bispecific antibodies. Figure 9A depicts flow cytometry binding analysis by fluorescence in target cells treated with various bispecific antibodies. Figure 9B is a schematic depicting an exemplary mechanism of EGFR degradation. Figure 9C is an image of immunofluorescence staining in cancer spheroids. [Figure 9-2] Same as above.

[0129] [Figure 10-1] Figures 10A-10G depict EGFR expression in target cells treated with various bispecific antibodies. Figure 10A is an image of a Western blot depicting the amount of EGFR protein and phosphorylated EGFR protein on target cells when treated with various bispecific antibodies. Figure 10B is an image of a Western blot depicting the amount of EGFR protein and phosphorylated EGFR protein on target cells when treated with various bispecific antibodies. Figure 10C is an image of a Western blot depicting the amount of EGFR protein and phosphorylated EGFR protein on target cells when treated with various concentrations of various bispecific antibodies. Figure 10D is an image of tumor spheroids. Figure 10E depicts quantification of tumor spheroids in samples treated with various bispecific antibodies. Figure 10F depicts quantification of tumor spheroids in samples treated with various bispecific antibodies. Figure 10G depicts quantification of tumor spheroids in samples treated with various bispecific antibodies. [Figure 10-2] Same as above. [Figure 10-3] Same as above.

[0130] [Figure 11-1]Figures 11A-11H depict cancer endpoints in animals treated with bispecific antibodies. Figure 11A is a schematic depicting an exemplary workflow and treatment regimen. Figure 11B depicts tumor volume over time in animals treated with bispecific antibodies. Figure 11C depicts tumor volume in animals treated with different concentrations of bispecific antibodies. Figure 11D depicts tumor volume over time in animals treated with bispecific antibodies. Figure 11E is an image depicting EGFR expression in cells treated with various bispecific antibodies. Figure 11F depicts quantification of EGFR in cells treated with various bispecific antibodies. Figure 11G depicts p-EGFR expression in cells treated with various bispecific antibodies. Figure 11H depicts quantification of p-EGFR in cells treated with various bispecific antibodies. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above.

[0131] [Figure 12-1] Figures 12A-12C depict pharmacokinetic endpoints in animals treated with bispecific antibodies. Figure 12A is a schematic depicting an exemplary workflow and treatment regimen. Figure 12B depicts serum concentrations of various bispecific antibodies over time in mice. Figure 12C depicts serum concentrations of various bispecific antibodies over time in mice. [Figure 12-2] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0132] Detailed Description The present disclosure generally relates to multispecific binding agents that bind to both a target protein and a membrane-bound internalization protein or a membrane-bound degradation protein present on the surface of a target cell. In some embodiments, the present disclosure provides a method for degrading a target protein, comprising contacting the target protein with a binding agent that simultaneously binds to the membrane-bound internalization protein, resulting in the target protein's internalization within the cell and subsequent degradation. In other embodiments, the present disclosure provides a method for degrading a target protein, comprising contacting the target protein with a binding agent that simultaneously binds to the membrane-bound degradation protein, resulting in the target protein's degradation. definition

[0133] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications mentioned herein are incorporated by reference.

[0134] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0135] The terms "administer," "administered," "administers," and "administering" are defined as providing a composition to a subject by a route known in the art, including, but not limited to, intravenous, intraarterial, intrathecal, oral, parenteral, perineural, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, intraperitoneal, or nerve root sheath routes of administration. In certain embodiments, the oral route of administering the composition can be used. The terms "administer," "administered," "administers," and "administering" a Therapeutic Protein should be understood to mean providing a Therapeutic Protein of the present disclosure or a prodrug of a Therapeutic Protein of the present disclosure to an individual in need thereof.

[0136] The term "humanizing" refers to the replacement or substitution of certain amino acids in antibodies or nanobodies derived from a non-human species, particularly in the framework regions and constant domains of the heavy and / or light chains, to avoid or minimize an immune response in humans.

[0137] As used herein, the term "complementarity-determining region" or "CDR" in the context of antibodies or nanobodies refers to the variable region of either the heavy (H) or light (L) chain (also abbreviated as VH and VL, respectively) and contains amino acid sequences capable of specifically binding to antigenic targets. These CDR regions are responsible for the antibody's basic specificity for a particular antigenic determinant. Such regions are also referred to as "hypervariable regions." Although CDRs represent noncontiguous stretches of amino acids within the variable region, the positional arrangement of these key amino acid sequences within the variable heavy and light chain regions has been found to have similar arrangements within the amino acid sequences of the variable chains, regardless of species. The variable heavy and light chains of all canonical antibodies each have three CDR regions (termed L1, L2, L3, H1, H2, and H3) that are noncontiguous with each other for each light (L) and heavy (H) chain. Nanobodies, in particular, comprise a single amino acid chain that can generally be considered to comprise four "framework sequences or regions" or FRs and three "complementarity-determining regions" or CDRs. Nanobodies have three CDR regions (termed CDR1, CDR2, and CDR3), each of which is non-contiguous with the others. The delineation of FR and CDR sequences is based on the IMGT proprietary numbering system for V and V-like domains.

[0138] As used herein, the terms "nucleic acid molecule," "polynucleotide," "polynucleic acid," and "nucleic acid" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules may be linear or circular.

[0139] As used herein, "nanobody" (Nb) refers to the smallest antigen-binding fragment or single variable domain ("VHH") derived from a naturally occurring heavy-chain antibody, as known to those skilled in the art. They are derived from heavy-chain-only antibodies found, for example, in camelid antibodies. In this regard, nanobodies generally comprise a single amino acid chain that can be considered to include four "framework sequences" constituting a "scaffold" and three "complementarity-determining regions" or CDRs (as defined herein above). It should be noted that, as used herein, the term "nanobody," in its broadest sense, is not limited to a particular biological source or a particular method of preparation.

[0140] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0141] As used herein, the phrase "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient.

[0142] As used herein, the terms "polypeptide," "protein," and "peptide" are used interchangeably herein and refer to polymeric forms of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones.

[0143] The terms "subject," "individual," and "patient" are used interchangeably and may refer to humans as well as non-human mammals (e.g., non-human primates, dogs, horses, cats, pigs, cows, ungulates, rabbits, rodents, etc.). In various embodiments, a subject may be a human (e.g., an adult male, adult female, adolescent male, adolescent female, boy, girl) under the supervision of a physician or other medical professional in a hospital, as an outpatient, or in other clinical settings. In certain embodiments, a subject may not be under the supervision or prescription of a physician or other medical professional.

[0144] As used herein, the phrase "subject in need thereof" refers to a subject suffering from or at risk of a condition, as described below, that is to be treated prophylactically or therapeutically with a therapeutic protein described herein.

[0145] As used herein, the term "specificity" refers to the ability of a protein binding domain, in particular an immunoglobulin or immunoglobulin fragment, such as a nanobody, to preferentially bind to one antigen over a different antigen, and does not necessarily imply high affinity.

[0146] As used herein, "treatment" or "treating" refers to an approach for obtaining a beneficial or desired result with respect to a disease, disorder, or medical condition, including, but not limited to, a therapeutic benefit and / or a preventative benefit. In certain embodiments, treating or treating involves administering a therapeutic protein or composition disclosed herein to a subject. Therapeutic benefit may include eradication or amelioration of the underlying disorder being treated. Therapeutic benefit may also be achieved along with eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such as observing an improvement in a subject, even though the subject may still be afflicted with the underlying disorder. In certain embodiments, for preventative benefit, a composition is administered to a subject at risk of developing a particular disease or reporting one or more physiological symptoms of a disease, even if a diagnosis of the disease has not been made. Treating may include, for example, reducing, delaying, or alleviating the severity of one or more symptoms of a disease or condition, or it may include reducing the frequency with which a symptom of a disease, defect, disorder, or adverse condition, etc., is experienced by the patient. Treating can be used herein to refer to a method that results in some level of treatment or improvement of a disease or condition, and may contemplate a range of outcomes directed toward that end, including, but not limited to, prevention of the condition altogether.

[0147] In certain embodiments, the term "prevent" or "preventing" in relation to a disease or disorder may refer to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in a treated sample compared to an untreated control sample, or delays the onset of or reduces the severity of one or more symptoms of the disorder or condition compared to an untreated control sample.

[0148] As used herein, the term "therapeutic effect" encompasses therapeutic and / or prophylactic benefits as described above. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying the onset of or eliminating symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[0149] As used herein, the term "degradation protein" or "degrader protein" may encompass a variety of moieties, including, but not limited to, a membrane-bound internalizing protein, an internalizing receptor, a membrane-bound degradation receptor, a degradation receptor, a surface moiety configured to internalize a binding agent, a surface moiety configured to degrade a binding agent, combinations thereof, or variants thereof.

[0150] As used herein, the term "internalizing protein" may encompass a variety of moieties, including, but not limited to, membrane-bound internalizing proteins, internalizing receptors, surface moieties configured to internalize binding agents, combinations thereof, or variants thereof. How to degrade the EGFR protein

[0151] The epidermal growth factor receptor (EGFR) is a transmembrane protein that is a receptor for extracellular protein ligands of the epidermal growth factor family (EGF family). EGFR is activated by the binding of these specific ligands, including epidermal growth factor (EGF) and transforming growth factor alpha (TGFα). Abnormal EGFR function and / or expression is involved in cancer, where it causes enhanced cell growth and division, leading to tumor growth and invasion.

[0152] Mutations resulting in EGFR overexpression (also known as upregulation or amplification) are associated with several cancers, including lung adenocarcinoma, anal cancer, glioblastoma, and epithelial tumors of the head and neck. Mutations, amplification, or misregulation of EGFR or family members are involved in approximately 30% of all epithelial cancers. Many of these somatic mutations, including EGFR, result in its constant activation, leading to uncontrolled cell division. Therefore, degradation of EGFR in cancer is a promising treatment modality for cancer.

[0153] The present disclosure provides a method for degrading EGFR protein on a target cell, as shown in Figure 1. The method utilizes a multispecific binding agent 101 that specifically binds to both (1) an extracellular epitope on the EGFR protein 112 and (2) an extracellular epitope on a membrane-bound internalizing protein 113 on the target cell 111. The bispecific binding agent 101 comprises a first binding domain 102 that selectively binds to the EGFR protein 112 and a second binding domain 103 that selectively binds to the membrane-bound internalizing protein 113. The simultaneous binding of the multispecific binding agent 101 to the EGFR protein 112 and the membrane-bound internalizing protein 113 results in the internalization of both the EGFR protein 112 and the membrane-bound internalizing protein 113 in the target cell 111. After internalization, the EGFR protein 112 is degraded by the target cell 111 (e.g., by transport to lysosomes).

[0154] In some embodiments, the membrane-bound internalizing protein is a cell surface protein that is internalized upon binding of a binding agent (e.g., an antibody) to the protein. In some embodiments, the membrane-bound internalizing protein is CEACAM5, CEACAM6, HER3, MUC1, CD205, CD166, PRLR, SLC34A2, ITGB6, LRRC15, MUC16, SLC39A6, AXL, CD40, CD228, MUC5A, ITGB1, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, I-Ag7, ENP P3, CD46, CD56, ROR1, GPR20, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, GPR56, CD71, LGR5, LY75, CD276 / B7-H3, MST1R, MSLN, EpCAM, TNFRSF10B, STEAP1, MELTF, TROP2, CDH17, RNF43 and RNF128.

[0155] The present disclosure also provides a method for degrading EGFR protein on a target cell, as shown in Figure 9B. The method utilizes a multispecific binding agent that specifically binds to both (1) an extracellular epitope on the EGFR protein and (2) an extracellular epitope on a membrane-bound degradation protein on the target cell. The multispecific binding agent comprises a first binding domain that selectively binds to the EGFR protein and a second binding domain that selectively binds to the membrane-bound degradation protein. Simultaneous binding of the multispecific binding agent to the EGFR protein and the membrane-bound degradation protein results in degradation of both the EGFR protein and the membrane-bound degradation protein.

[0156] In some embodiments, the membrane-bound degradation protein is a cell surface protein that degrades upon binding of a binding agent (e.g., an antibody) to the protein, hi some embodiments, the membrane-bound degradation protein is RNF43.

[0157] In one aspect, the present disclosure provides a method for degrading EGFR protein on a target cell, comprising: contacting an EGFR protein and a membrane-bound internalizing protein on the target cell with the bispecific binding agent, wherein contacting the EGFR protein and the membrane-bound internalizing protein with the bispecific binding agent results in internalization and degradation of the EGFR protein; the bispecific binding agent comprises: (a) a first binding domain that specifically binds to an extracellular epitope of a membrane-bound internalized protein; and (b) a second binding domain that specifically binds to an extracellular epitope on an EGFR protein; Membrane-bound internalized proteins include CEACAM5, CEACAM6, HER3, MUC1, CD205, CD166, PRLR, SLC34A2, ITGB6, LRRC15, MUC16, SLC39A6, AXL, CD40, CD228, MUC5A, ITGB1, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, I-Ag7, ENPP3, CD46, C D56, ROR1, GPR20, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, GPR56, CD71, LGR5, LY75, CD276 / B7-H3, MST1R, MSLN, EpCAM, TNFRSF10B, STEAP1, MELTF, TROP2, CDH17, RNF43 and RNF128.

[0158] In some embodiments, the multispecific binding agent comprises an antibody. In some embodiments, the multispecific binding agent comprises a multispecific antibody. In some embodiments, the multispecific binding agent comprises a bispecific antibody. In some embodiments, the multispecific binding agent comprises an IgG antibody. In some embodiments, the multispecific binding agent comprises a multispecific IgG antibody. In some embodiments, the multispecific binding agent comprises a knob-and-hole bispecific IgG. In some embodiments, the multispecific binding agent is not an antibody-drug conjugate ("ADC"). In some embodiments, the multispecific binding agent comprises a bispecific binding agent. In some embodiments, the multispecific binding agent comprises a bispecific antibody. In some embodiments, the multispecific binding agent comprises a bispecific diabody. In some embodiments, the multispecific binding agent comprises a bispecific Fab2. In some embodiments, the multispecific binding agent comprises a bispecific camelid antibody. In some embodiments, the multispecific binding agent comprises a bispecific peptibody scFv-Fc. In some embodiments, the multispecific binding agent comprises an Fc-Fab. In some embodiments, the multispecific binding agent comprises a knob-and-hole bispecific Fc-Fab. Multispecific Binding Agents

[0159] The multispecific binding agents of the present disclosure comprise at least two binding domains, one specific for a membrane-bound internalization protein or a membrane-bound degradation protein, and the other specific for an EGFR protein. Multispecific binding agents of the present disclosure include, but are not limited to, agents in which the membrane-bound internalization or degradation protein binding domain and the EGFR-binding domain are independently selected from an antibody (or antibody half), a nanobody, a minibody, a Fab fragment, a single-chain variable fragment (scFv), and a single-domain antibody (sdAb), or functional fragments thereof. These two binding domains may be the same type of molecule or may be different. For example, the multispecific binding agents of the present disclosure include, but are not limited to, a multispecific binding agent having an IgG that binds to a membrane-bound internalization or degradation protein and an scFv domain that binds to EGFR. The binding domains of a multispecific binding agent can be connected by a covalent bond, a non-covalent interaction, or a combination thereof.

[0160] Multispecific binding agents generally may take the form of proteins, glycoproteins, lipoproteins, phosphoproteins, etc. Some multispecific binding agents of the present disclosure take the form of multispecific antibodies, bispecific antibodies, or antibody derivatives. In some embodiments, the target protein-binding domain is selected from the group consisting of a half antibody, nanobody, or minibody, F(ab')2 fragment, Fab fragment, single-chain variable fragment (scFv), and single-domain antibody (sdAb), or functional fragments thereof. The binding domains may together take the form of bispecific antibodies, bispecific diabodies, bispecific camelid antibodies, or bispecific peptibodies, etc. Antibody derivatives need not be derived from a specific wild-type antibody. For example, one skilled in the art can generate and select small proteins having binding domains similar to the complementarity-determining regions (CDRs) of an antibody using known techniques such as phage display. In some embodiments, the antigen-binding portion comprises an scFv. The binding domain may also be derived from a soluble or membrane-bound, natural or synthetic ligand or receptor that specifically binds to the EGFR protein.

[0161] Multispecific antibodies can be prepared by known methods. Embodiments of the present disclosure include "knobs-into-hole" bispecific antibodies, in which the otherwise symmetric dimerization region of a bispecific binding agent is modified to become asymmetric. For example, a knobs-into-hole bispecific IgG specific for antigens A and B can be modified so that the Fc portion of the A binding chain has one or more protrusions ("knobs") and the Fc portion of the B binding chain has one or more cavities ("holes"), where the knobs and holes are positioned to interact. This reduces homodimerization (AA and BB antibodies) and promotes heterodimerization, which is desirable for bispecific binding agents. See, for example, Y. Xu et al., mAbs (2015) 7(1):231-42. In some embodiments, the bispecific binding agent has a knobs-into-hole design. In some embodiments, the "knobs" include a T336W modification in the CH3 domain, i.e., threonine at position 336 is replaced by tryptophan. In some embodiments, the "hole" comprises one or a combination of T366S, L368A, and Y407V. In some embodiments, the "hole" comprises T366S, L368A, and Y407V.

[0162] In some embodiments, the multispecific binding agent comprises an FcRn receptor recognition domain to facilitate the return of the bispecific binding agent to the extracellular space when the bispecific binding agent is internalized.

[0163] In another aspect, the disclosure provides a multispecific binding agent comprising a bispecific antibody or antibody derivative, wherein the bispecific binding agent: a) a first binding domain that specifically binds to an extracellular epitope of the EGFR protein of a target cell; and b) a second binding domain that specifically binds to an extracellular epitope of a membrane-bound, internalized protein on a target cell; Includes; and RNF128. Membrane-bound internalized proteins

[0164] The disclosed methods and multispecific binding agents utilize membrane-bound internalizing proteins that cause the internalization and / or degradation of EGFR proteins. The present disclosure utilizes the unique function of membrane-bound internalizing proteins, which internalize upon binding of a binding agent to a protein. By simultaneously binding to EGFR using a first binding domain and to the membrane-bound internalizing protein using a second binding domain, the multifunctional binding agent internalizes the EGFR protein together with the membrane-bound internalizing protein into target cells. Once internalized, the EGFR protein is sequestered and / or degraded (e.g., by lysosomal degradation) within the target cells.

[0165] Membrane-bound internalizing proteins for use in the methods and bifunctional binding agents of the present disclosure include cell surface proteins that are internalized upon binding of a binding agent (e.g., an antibody) to the protein. Such membrane-bound internalizing proteins generally include cell surface proteins currently targeted by antibody-drug conjugates, which rely on internalization of the antibody-protein complex to ensure release of the conjugated drug. Examples of such membrane-bound internalizing proteins useful for the methods of the present disclosure include, for example, CEACAM5 (i.e., CEA cell adhesion molecule 5), CEACAM6 (i.e., CEA cell adhesion molecule 6), HER3 (i.e., receptor tyrosine protein kinase erbB-3), MUC1 (i.e., mucin 1), CD205 (i.e., lymphocyte antigen 75), CD166 (i.e., activated lymphocyte cell adhesion molecule, also known as ALCAM), PRLR (i.e., prolactin receptor), SLC34A2 (i.e., solute carrier family 34 member 2), ITGB6 (i.e., integrin subunit beta 6), LRRC15 (i.e., leucine-rich repeat-containing protein 15), and MUC16 (i.e., mucin 16). These proteins have been shown to be internalized into cells upon binding of a binding agent (e.g., an antibody) to an extracellular epitope of the protein.

[0166] In some embodiments, the membrane-bound internalization protein is selected from the group consisting of SLC39A6 (i.e., solute carrier family 39 member 6), AXL (i.e., AXL receptor tyrosine kinase, also known as tyrosine protein kinase receptor UFO), CD40 (i.e., CD40 molecule, also known as tumor necrosis factor receptor superfamily member 5), CD228 (i.e., melanotransferrin), MUC5A (i.e., mucin 5AC, oligomeric mucus / gel forming), ITGB1 (i.e., integrin subunit beta 1), STn, KAAG1 (i.e., kidney-associated DCDC2 antisense RNA 1), DLK1 (i.e., delta-like noncanonical Notch ligand 1), 5T4 (i.e., trophoblast glycoprotein), SEZ6 (i.e., seizure-associated 6 homolog), ADAM9 (i.e., ADAM metallopeptidase domain 9), I-Ag7 (i.e., MHC class II molecule Ag7), ENPP3 (i.e., extracellular nucleotide pyrophosphatase / phosphodiesterase 3), CD46 (i.e., CD46 molecule), CD56 (i.e., neuronal junction receptor tyrosine kinase-like orphan receptor 1), GPR20 (i.e., G protein-coupled receptor 20), TM4SF1 (i.e., transmembrane 4L-size family member 1), B7-H4 (i.e., V-Set domain-containing T cell activation inhibitory factor 1), ALPP (i.e., alkaline phosphatase, placenta), LY6E (i.e., lymphocyte antigen 6 family member E), CLDN18 (i.e., claudin 18), LY6G6D (i.e., lymphocyte antigen 6 family member G6D), GPR5 6 (i.e., adhesion G protein-coupled receptor G1), CD71 (transferrin receptor-1), B7-H3 (i.e., B7 homolog 3 protein or cluster of differentiation antigen 276 or CD276), CDH17 (i.e., cadherin 17), LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5), LY75 (lymphocyte antigen 75 or CD205), MST1R (macrophage-stimulating 1 receptor), MSLN (mesothelin), EpCAM (epithelial cell adhesion molecule), TNFRSF10B (TNF receptor superfamily member 10b),The protein is selected from STEAP1 (STEAP family member 1), MELTF (melanotransferrin), TROP2 (tumor-associated calcium signaling factor 2), RNF43 (RING finger protein 43), and RNF128 (RING finger protein 128). These proteins have been shown to be internalized into cells upon binding of a binding agent (e.g., an antibody) to an extracellular epitope of the protein.

[0167] In some embodiments, the membrane-bound internalizing protein is selected from CEACAM5, CEACAM6, HER3, MUC1, CD205, CD166, PRLR, SLC34A2, ITGB6, LRRC15, MUC16, SLC39A6, AXL, CD40, CD228, MUC5A, ITGB1, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, I-AG7, ENPP3, CD46, CD56, ROR1, GPR20, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, GPR56, and CD71. In some embodiments, the membrane-bound internalizing protein is selected from CD205, CD166, SLC34A2, ITGB6, LRRC15, MUC16, SLC39A6, AXL, CD40, CD228, MUC5A, ITGB1, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, I-AG7, ENPP3, CD46, CD56, ROR1, GPR20, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, GPR56, and CD71.

[0168] In some embodiments, the membrane-bound internalizing protein is selected from SLC34A2, ITGB6, LRRC15, MUC16, SLC39A6, AXL, CD40, CD228, MUC5A, ITGB1, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, I-AG7, ENPP3, CD46, CD56, ROR1, GPR20, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, GPR56, and CD71. In some embodiments, the membrane-bound internalizing protein is selected from CD205, CD166, LRRC15, MUC16, SLC39A6, AXL, CD40, CD228, MUC5A, ITGB1, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, I-AG7, ENPP3, CD46, CD56, ROR1, GPR20, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, GPR56, and CD71. In some embodiments, the membrane-bound internalizing protein is selected from CD205, CD166, SLC34A2, ITGB6, SLC39A6, AXL, CD40, CD228, MUC5A, ITGB1, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, I-AG7, ENPP3, CD46, CD56, ROR1, GPR20, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, GPR56, and CD71.

[0169] In some embodiments, the membrane-bound internalizing protein is selected from SLC39A6, AXL, CD40, CD228, MUC5A, ITGB1, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, I-AG7, ENPP3, CD46, CD56, ROR1, GPR20, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, GPR56, and CD71. In some embodiments, the membrane-bound internalizing protein is selected from CD228, MUC5A, ITGB1, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, I-AG7, ENPP3, CD46, CD56, ROR1, GPR20, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, GPR56, and CD71.

[0170] In some embodiments, the membrane-bound internalizing protein is selected from SLC39A6, AXL, CD40, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, I-AG7, ENPP3, CD46, CD56, ROR1, GPR20, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, GPR56, and CD71. In some embodiments, the membrane-bound internalizing protein is selected from SLC39A6, AXL, CD40, CD228, MUC5A, ITGB1, 5T4, SEZ6, ADAM9, I-AG7, ENPP3, CD46, CD56, ROR1, GPR20, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, GPR56, and CD71. In some embodiments, the membrane-bound internalizing protein is selected from SLC39A6, AXL, CD40, CD228, MUC5A, ITGB1, STn, KAAG1, DLK1, I-AG7, ENPP3, CD46, CD56, ROR1, GPR20, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, GPR56, and CD71.

[0171] In some embodiments, the membrane-bound internalizing protein is selected from SLC39A6, AXL, CD40, CD228, MUC5A, ITGB1, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, CD56, ROR1, GPR20, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, GPR56, and CD71. In some embodiments, the membrane-bound internalizing protein is selected from SLC39A6, AXL, CD40, CD228, MUC5A, ITGB1, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, I-AG7, ENPP3, CD46, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, GPR56, and CD71. In some embodiments, the membrane-bound internalizing protein is selected from SLC39A6, AXL, CD40, CD228, MUC5A, ITGB1, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, I-AG7, ENPP3, CD46, CD56, ROR1, GPR20, LY6E, CLDN18, LY6G6D, GPR56, and CD71. In some embodiments, the membrane-bound internalizing protein is selected from SLC39A6, AXL, CD40, CD228, MUC5A, ITGB1, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, I-AG7, ENPP3, CD46, CD56, ROR1, GPR20, TM4SF1, B7-H4, and ALPP.

[0172] In some embodiments, the membrane-bound internalizing protein is selected from CD205, CD166, CD40, CD228, CD46, CD56, and CD71. In some embodiments, the membrane-bound internalizing protein is selected from SLC34A2, ITGB6, LRRC15, MUC16, SLC39A6, AXL, MUC5A, ITGB1, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, I-AG7, ENPP3, ROR1, GPR20, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, and GPR56.

[0173] In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, SLC34A2, ITGB6, LRRC15 and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD166, SLC34A2, ITGB6, LRRC15 and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, SLC34A2, ITGB6, LRRC15 and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, ITGB6, LRRC15 and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, SLC34A2, LRRC15 and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, SLC34A2, LRRC15 and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, SLC34A2, ITGB6 and MUC16. In some embodiments, the membrane-bound internalizing protein is selected from CD205, CD166, SLC34A2, ITGB6, and LRRC15.

[0174] In some embodiments, the membrane-bound internalizing protein is selected from B7-H3, CDH17, MUC1, ITGB6, RNF43, and CECAM5. In some embodiments, the membrane-bound internalizing protein is selected from B7-H3, CDH17, MUC1, ITGB6, and CECAM5.

[0175] In some embodiments, the membrane-bound internalization protein is selected from CDH17, MUC1, ITGB6, and CECAM5. In some embodiments, the membrane-bound internalization protein is selected from B7-H3, MUC1, ITGB6, and CECAM5. In some embodiments, the membrane-bound internalization protein is selected from B7-H3, CDH17, ITGB6, and CECAM5. In some embodiments, the membrane-bound internalization protein is selected from B7-H3, CDH17, MUC1, and CECAM5. In some embodiments, the membrane-bound internalization protein is selected from MUC1, ITGB6, and CECAM5. In some embodiments, the membrane-bound internalization protein is selected from CD166, SLC34A2, ITGB6, LRRC15, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from SLC34A2, ITGB6, LRRC15, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD166, ITGB6, LRRC15 and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD166, SLC34A2, LRRC15 and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD166, SLC34A2, ITGB6 and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD166, SLC34A2, ITGB6 and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD166, SLC34A2, ITGB6 and LRRC15.

[0176] In some embodiments, the membrane-bound internalization protein is selected from SLC34A2, ITGB6, LRRC15 and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, ITGB6, LRRC15 and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, SLC34A2, LRRC15 and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, SLC34A2, ITGB6 and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, SLC34A2, ITGB6 and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, SLC34A2, ITGB6 and LRRC15.

[0177] In some embodiments, the membrane-bound internalization protein is selected from CD166, ITGB6, LRRC15, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, ITGB6, LRRC15, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, ITGB6, LRRC15, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, LRRC15, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, LRRC15, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, ITGB6, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, ITGB6, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, ITGB6, and LRRC15.

[0178] In some embodiments, the membrane-bound internalization protein is selected from CD166, SLC34A2, LRRC15, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, SLC34A2, LRRC15, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, LRRC15, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, SLC34A2, LRRC15, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, SLC34A2, LRRC15, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, SLC34A2, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, SLC34A2, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, SLC34A2, and LRRC15.

[0179] In some embodiments, the membrane-bound internalization protein is selected from CD166, SLC34A2, ITGB6, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, SLC34A2, ITGB6, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, ITGB6, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, SLC34A2, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, SLC34A2, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, SLC34A2, ITGB6, and MUC16. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, SLC34A2, and ITGB6.

[0180] In some embodiments, the membrane-bound internalization protein is selected from CD166, SLC34A2, ITGB6 and LRRC15. In some embodiments, the membrane-bound internalization protein is selected from CD205, SLC34A2, ITGB6 and LRRC15. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, ITGB6 and LRRC15. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, SLC34A2 and LRRC15. In some embodiments, the membrane-bound internalization protein is selected from CD205, CD166, SLC34A2 and ITGB6.

[0181] In some embodiments, the membrane-bound internalization protein is CD205 or CD166. In some embodiments, the membrane-bound internalization protein is CD205 or SLC34A2. In some embodiments, the membrane-bound internalization protein is CD205 or ITGB6. In some embodiments, the membrane-bound internalization protein is CD205 or LRRC15. In some embodiments, the membrane-bound internalization protein is CD205 or MUC16.

[0182] In some embodiments, the membrane-bound internalization protein is CD166 or CD205. In some embodiments, the membrane-bound internalization protein is CD166 or SLC34A2. In some embodiments, the membrane-bound internalization protein is CD166 or ITGB6. In some embodiments, the membrane-bound internalization protein is CD166 or LRRC15. In some embodiments, the membrane-bound internalization protein is CD166 or MUC16.

[0183] In some embodiments, the membrane-bound internalization protein is SLC34A2 or CD205. In some embodiments, the membrane-bound internalization protein is SLC34A2 or CD166. In some embodiments, the membrane-bound internalization protein is SLC34A2 or ITGB6. In some embodiments, the membrane-bound internalization protein is SLC34A2 or LRRC15. In some embodiments, the membrane-bound internalization protein is SLC34A2 or MUC16.

[0184] In some embodiments, the membrane-bound internalization protein is ITGB6 or CD205. In some embodiments, the membrane-bound internalization protein is ITGB6 or CD166. In some embodiments, the membrane-bound internalization protein is ITGB6 or SLC34A2. In some embodiments, the membrane-bound internalization protein is ITGB6 or LRRC15. In some embodiments, the membrane-bound internalization protein is ITGB6 or MUC16.

[0185] In some embodiments, the membrane-bound internalization protein is LRRC15 or CD205. In some embodiments, the membrane-bound internalization protein is LRRC15 or CD166. In some embodiments, the membrane-bound internalization protein is LRRC15 or SLC34A2. In some embodiments, the membrane-bound internalization protein is LRRC15 or ITGB6. In some embodiments, the membrane-bound internalization protein is LRRC15 or MUC16.

[0186] In some embodiments, the membrane-bound internalization protein is MUC16 or CD205. In some embodiments, the membrane-bound internalization protein is MUC16 or CD166. In some embodiments, the membrane-bound internalization protein is MUC16 or SLC34A2. In some embodiments, the membrane-bound internalization protein is MUC16 or ITGB6. In some embodiments, the membrane-bound internalization protein is MUC16 or LRRC15.

[0187] In some embodiments, the membrane-bound internalizing protein is CD205. In some embodiments, the membrane-bound internalizing protein is CD166. In some embodiments, the membrane-bound internalizing protein is SLC34A2. In some embodiments, the membrane-bound internalizing protein is ITGB6. In some embodiments, the membrane-bound internalizing protein is LRRC15. In some embodiments, the membrane-bound internalizing protein is MUC16.

[0188] In some embodiments, the membrane-bound internalizing protein is CEACAM5. In some embodiments, the membrane-bound internalizing protein is CEACAM6. In some embodiments, the membrane-bound internalizing protein is HER3. In some embodiments, the membrane-bound internalizing protein is MUC1. In some embodiments, the membrane-bound internalizing protein is CD205. In some embodiments, the membrane-bound internalizing protein is CD166. In some embodiments, the membrane-bound internalizing protein is PRLR. In some embodiments, the membrane-bound internalizing protein is SLC34A2. In some embodiments, the membrane-bound internalizing protein is ITGB6. In some embodiments, the membrane-bound internalizing protein is LRRC15. In some embodiments, the membrane-bound internalizing protein is MUC16.

[0189] In some embodiments, the membrane-bound internalizing protein is selected from SLC39A6. In some embodiments, the membrane-bound internalizing protein is selected from AXL. In some embodiments, the membrane-bound internalizing protein is selected from CD40. In some embodiments, the membrane-bound internalizing protein is selected from CD228. In some embodiments, the membrane-bound internalizing protein is selected from MUC5A. In some embodiments, the membrane-bound internalizing protein is selected from ITGB1. In some embodiments, the membrane-bound internalizing protein is selected from STn. In some embodiments, the membrane-bound internalizing protein is selected from KAAG1.

[0190] In some embodiments, the membrane-bound internalizing protein is selected from DLK1. In some embodiments, the membrane-bound internalizing protein is selected from 5T4. In some embodiments, the membrane-bound internalizing protein is selected from SEZ6. In some embodiments, the membrane-bound internalizing protein is selected from ADAM9. In some embodiments, the membrane-bound internalizing protein is selected from I-AG7. In some embodiments, the membrane-bound internalizing protein is selected from ENPP3. In some embodiments, the membrane-bound internalizing protein is selected from CD46. In some embodiments, the membrane-bound internalizing protein is selected from CD56. In some embodiments, the membrane-bound internalizing protein is selected from ROR1.

[0191] In some embodiments, the membrane-bound internalizing protein is selected from GPR20. In some embodiments, the membrane-bound internalizing protein is selected from TM4SF1. In some embodiments, the membrane-bound internalizing protein is selected from B7-H4. In some embodiments, the membrane-bound internalizing protein is selected from ALPP. In some embodiments, the membrane-bound internalizing protein is selected from LY6E. In some embodiments, the membrane-bound internalizing protein is selected from CLDN18. In some embodiments, the membrane-bound internalizing protein is selected from LY6G6D. In some embodiments, the membrane-bound internalizing protein is selected from GPR56. In some embodiments, the membrane-bound internalizing protein is selected from CD71.

[0192] In some embodiments, the membrane-bound internalizing protein is LGR5. In some embodiments, the membrane-bound internalizing protein is LY75. In some embodiments, the membrane-bound internalizing protein is CD276 / B7-H3. In some embodiments, the membrane-bound internalizing protein is MST1R. In some embodiments, the membrane-bound internalizing protein is MSLN. In some embodiments, the membrane-bound internalizing protein is EpCAM. In some embodiments, the membrane-bound internalizing protein is TNFRSF10B. In some embodiments, the membrane-bound internalizing protein is TEAP1. In some embodiments, the membrane-bound internalizing protein is MELTF. In some embodiments, the membrane-bound internalizing protein is TROP2. In some embodiments, the membrane-bound internalizing protein is CDH17. In some embodiments, the membrane-bound internalizing protein is RNF43. In some embodiments, the membrane-bound internalizing protein is RNF43.

[0193] In some embodiments, the membrane-bound internalizing protein is recycled to the target cell surface after internalization of the binding agent, hi some embodiments, the membrane-bound internalizing protein is degraded. Membrane-bound degradation proteins

[0194] The methods and multispecific binding agents of the present disclosure can utilize membrane-bound degradation proteins to cause degradation of the EGFR protein. The present disclosure can use membrane-bound degradation proteins to cause ubiquitination upon binding of the binding agent to the membrane-bound degradation protein. Furthermore, by binding to EGFR with a first binding domain and binding to the membrane-bound degradation protein using a second binding domain, the multifunctional binding agent can degrade the EGFR protein together with the membrane-bound degradation protein.

[0195] Membrane-bound degradation proteins for use in the methods and bifunctional binding agents of the present disclosure may include cell surface proteins that are degraded upon binding and / or internalization of a binding agent (e.g., an antibody) to the protein. Such membrane-bound degradation proteins may include cell surface proteins targeted by antibody-drug conjugates, which may rely on the degradation of the antibody-protein complex to ensure the release of the conjugated drug. Examples of such membrane-bound degradation proteins useful for the methods of the present disclosure include, for example, TROP2. In some embodiments, the membrane-bound degradation protein is an E3 ligase. In some embodiments, the membrane-bound degradation protein is RNF43 (i.e., ring finger protein 43). First Binding Region

[0196] In some embodiments, the first binding domain is derived from an antibody against a membrane-bound internalization protein or a degradation protein. Such antibodies are known to those skilled in the art and can be incorporated into the methods and bispecific binding agents of the present disclosure. For example, in some embodiments, the complementarity determining region ("CDR") of a known antibody against a membrane-bound internalization protein of interest or a membrane-bound degradation protein of interest can be incorporated into the multispecific binding agent and method of the present disclosure using known techniques. Exemplary antibodies suitable for incorporation into the methods and multispecific binding agents of the present disclosure include those described below.

[0197] Antibodies targeting CEACAM5 are known in the art, including, for example, the CC4 antibody disclosed in Zheng, Chaogu, et al., "A novel anti-CEACAM5 monoclonal antibody, CC4, suppresses colorectal tumor growth and enhances NK cell-mediated tumor immunity." PloS one 6.6 (2011): e21146. Additional antibodies targeting CEACAM5 suitable for use in the present disclosure include, for example, the anti-CEACAM5 antibodies MN-14, MN-15, and MN-3 described in Blumenthal, Rosalyn D., Hans J. Hansen, and David M. Goldenberg. "Inhibition of adhesion, invasion, and metastasis by antibodies targeting CEACAM6 (NCA-90) and CEACAM5 (Carcinoembryonic Antigen)." Cancer research 65.19 (2005): 8809-8817.

[0198] For example, antibodies targeting CEACAM6, including anti-CEACAM6 antibodies, sdAb, 2Ab, and 4Ab, as described in Wu, Shang-Jung, et al. "Migration and invasion of NSCLC suppressed by the downregulation of Src / focal adhesion kinase using single, double, and tetra domain anti-CEACAM6 antibodies." Translational oncology 14.7 (2021): 101057, are known in the art. Additional antibodies targeting CEACAM6 that are suitable for use in the present disclosure include, for example, the anti-CEACAM6 antibodies MN-3 and MN-15, described in Blumenthal, Rosalyn D., Hans J. Hansen, and David M. Goldenberg. "Inhibition of adhesion, invasion, and metastasis by antibodies targeting CEACAM6 (NCA-90) and CEACAM5 (Carcinoembryonic Antigen)." Cancer research 65.19 (2005): 8809-8817.

[0199] Antibodies targeting HER3 (also known as ErbB-3) are known in the art, including, for example, the anti-HER3 antibody GSK2849330, described in Gan, Hui K., et al. "A phase I, first-in-human study of GSK2849330, an anti-HER3 monoclonal antibody, in HER3-expressing solid tumors." The oncologist 26.10 (2021): e1844-e1853. Further examples of anti-HER3 antibodies include, for example, patritumab (U3-1287), which is described in Hashimoto, Yuuri, et al. "A Novel HER3-Targeting Antibody-Drug Conjugate, U3-1402, Exhibits Potent Therapeutic Efficacy through the Delivery of Cytotoxic Payload by Efficient Internalization Preclinical Evaluation of U3-1402, a HER3-Targeting ADC." Clinical Cancer Research 25.23 (2019): 7151-7161.

[0200] Antibodies that target MUC1 are known in the art, including, for example, the anti-MUC1 antibodies MY.1E12, KL6, 5E5, and TAB004 described in Bose, Mukulika, and Pinku Mukherjee. "Potential of anti-MUC1 antibodies as a targeted therapy for gastrointestinal cancers." Vaccines 8.4 (2020): 659.

[0201] Antibodies that target CD205 are known in the art, including, for example, the anti-CD205 antibody MEN1309 / OBT076 described in, for example, Rieke, Damian T., and Ulrich Keller. "A CD205-directed antibody drug conjugate - lymphoma precision oncology or sophisticated chemotherapy?" Haematologica 105.11 (2020): 2504.

[0202] Antibodies that target CD166 are known in the art, such as the anti-CD166 antibody CX-2009 described in Boni, Valentina, et al. "Praluzatamab ravtansine, a CD166-targeting antibody-drug conjugate, in patients with advanced solid tumors: an open-label phase 1 / 2 trial of praluzatamab ravtansine in patients with advanced tumors." Clinical Cancer Research (2022).

[0203] Antibodies targeting PRLR are known in the art, for example, the anti-PRLR antibody ABBV-176 described in Anderson, Mark G., et al. "ABBV-176, a PRLR antibody drug conjugate with a potent DNA-damaging PBD cytotoxin and enhanced activity with PARP inhibition." BMC cancer 21.1 (2021): 1-11. Additional antibodies targeting CEACAM6 suitable for use in the present disclosure include, for example, the anti-CEACAM6 antibody LFA102 described in Damiano, Jason S., et al. "Neutralization of Prolactin Receptor Function by Monoclonal Antibody LFA102, a Novel Potential Therapeutic for the Treatment of Breast Cancer Preclinical Development of Anti-PRLR Antibody LFA102." Molecular cancer therapeutics 12.3 (2013): 295-305.

[0204] Antibodies that target SCL34A2 are known in the art, for example, the anti-NaPi2b antibody described in Lin, Kedan, et al. "Preclinical Development of an Anti-NaPi2b (SLC34A2) Antibody-Drug Conjugate as a Therapeutic for Non-Small Cell Lung and Ovarian Cancers Preclinical Development of NaPi2b Antibody-Drug Conjugate," Clinical Cancer Research 21.22 (2015): 5139-5150. Another antibody suitable for incorporation into the bispecific binding agent of the present disclosure is the anti-SCL34A2 antibody MX35 described in Yin, Beatrice WT, et al. "Monoclonal antibody MX35 detects the membrane transporter NaPi2b (SLC34A2) in human carcinomas," Cancer immunity 8.1 (2008).

[0205] Antibodies targeting ITGB6 are known in the art, including, for example, the antibody SGN-B6A described in Patnaik, Amita, et al. "A phase 1 study of SGN-B6A, an antibody-drug conjugate targeting integrin beta-6, in patients with advanced solid tumors (SGN-B6A-001, Trial in Progress)." (2021). Another antibody suitable for incorporation into the present disclosure is the anti-ITGB6 antibody TPS3144-TPS3144 described in Zheng, Xiaoxia, et al. "Silencing of ITGB6 inhibits the progression of cervical carcinoma via regulating JAK / STAT3 signaling pathway." Annals of Translational Medicine 9.9 (2021).

[0206] Antibodies that target LRRC15 are known in the art, including, for example, the anti-LRRC15 antibody, ABBV-085, described in, for example, Demetri, George D., et al. "First-in-Human Phase I Study of ABBV-085, an Antibody-Drug Conjugate Targeting LRRC15, in Sarcoma and Other Advanced Solid Tumors Phase I Study of ABBV-085, an LRRC15-Targeting ADC." Clinical Cancer Research 27.13 (2021): 3556-3566; and Slemmons, Katherine K., et al. "LRRC15 antibody-drug conjugates show promise as osteosarcoma therapeutics in preclinical studies." Pediatric blood & cancer 68.2 (2021): e28771.

[0207] Antibodies targeting MUC16 are known in the art, including, for example, the anti-MUC16 antibody OC125 described in, for example, Rao, Thapi Dharma, et al. "Novel monoclonal antibodies against the proximal (carboxy-terminal) portions of MUC16." Applied immunohistochemistry & molecular morphology: AIMM / official publication of the Society for Applied Immunohistochemistry 18.5 (2010): 462. Additional anti-MUC16 antibodies include those described, for example, in Aithal, Abhijit, et al. "MUC16 as a novel target for cancer therapy." Expert opinion on therapeutic targets 22.8 (2018): 675-686; and Rao, Thapi Dharma, et al. "Antibodies against specific MUC16 glycosylation sites inhibit ovarian cancer growth." ACS chemical biology 12.8 (2017): 2085-2096.

[0208] Antibodies targeting SLC39A6 are known in the art, including the anti-SLC39A6 antibody described in Cui, Shen, et al., "SLC39A6: a potential target for diagnosis and therapy of esophageal carcinoma," Journal of Translational Medicine 13 (2015): 321. Additional anti-SLC39A6 antibodies include those described in Sussman, Smith, et al., "SGN-LIV1A: A novel antibody-drug conjugate targeting LIV-1 for the treatment of metastatic breast cancer," Mol Chancer Ther (2014) 13 (12): 2991-3000; and Wan and Wang, "Role of SLC39A in the development and progression of liver cancer," Oncology Letters 23.3. (2022): 77.

[0209] Antibodies targeting AXL are known in the art, including the AXL-specific antibody described in, for example, Vajkoczy, Knyazev, et al. "Dominant-negative inhibition of the Axl receptor tyrosine kinase suppresses brain tumor cell growth, invasion, and prolongs survival." Proceedings of the National Academy of Sciences 103.15 (2006): 5799-5804. Additional anti-AXL antibodies include, for example, the anti-AXL antibody 20G7-D9 described in, for example, Leconet, Chentouf, et al. "Therapeutic activity of anti-AXL antibody against triple-negative breast cancer patient-derived xenografts and metastasis." Clin Cancer Research 23.11 (2017):2806-2816.

[0210] For example, antibodies that target CD40 are known in the art, including the anti-CD40 antibody described in Xu, Gao, et al. "Repulsive guidance molecule a blockade exerts the immunoregulatory function in DCs stimulated with ABP and LPS." Human vaccines & immunotherapeutics 12.8 (2016): 2169-2180. Additional anti-CD40 antibodies include, for example, those described in Silvin, Chapuis, et al. "Elevated calprotectin and abnormal myeloid cell subsets discriminate severe from mild COVID-19." Cell 182.6 (2020): 1401-1418; and Ceglia, Zurawski, et al. "Anti-CD40 Antibody Fused to CD40 Ligand Is a Superagonist Platform for Adjuvant Intrinsic DC-Targeting Vaccines." Frontiers in immunology 12:786144 (2021).

[0211] Antibodies targeting CD228 are known in the art, including the anti-MELTF antibody described in Sawaki, Kanda, et al. "Level of melanotransferrin in tissue and sera serves as a prognostic marker of gastric cancer." Anticancer Research 39.11 (2019): 6125-6133. Additional anti-CD228 antibodies include those described in Singh, Eyford, et al. "Discovery of a Highly Conserved Peptide in the Iron Transporter Melanotransferrin that Traverses an Intact Blood-Brain Barrier and Localizes in Neural Cells." Frontiers in neuroscience 15: 596976. (2021): 473.

[0212] For example, antibodies targeting MUC5A are known in the art, including the anti-MUC5A antibody MUC5:TR-3A described in Zuhdi Alimam, Piazza, et al. "Muc-5 / 5ac mucin messenger RNA and protein expression is a marker of goblet cell metaplasia in murine airways." American Journal of Respiratory Cell and Molecular Biology 22.3 (2000): 253-260. Additional anti-MUC5 antibodies include, for example, those described in Wang, Jin, et al. "Expression of survivin, MUC2, and MUC5 in colorectal cancer and their association with clinicopathological characteristics," Oncology Letters 14.1 (2017): 1011-1016; and Reis, David, et al. "Immunohistochemical study of MUC5AC expression in human gastric carcinomas using a novel monoclonal antibody," International journal of cancer 74.1 (1997): 112-121.

[0213] Antibodies targeting ITGB1 are known in the art, including the anti-ITGB1 antibody described in Du, Yang, et al. "The circular RNA circSKA3 binds integrin β1 to induce invadopodium formation enhancing breast cancer invasion." Molecular Therapy 28.5 (2020): 1287-1298. Additional anti-ITGB1 antibodies include those described in Kawahara, Niwa, et al. "Integrin β1 is an essential factor in vasculogenic mimicry of human cancer cells." Cancer science 109.8 (2018): 2490-2496; and Wang and Li. "Ropivacaine inhibits the proliferation and migration of colorectal cancer cells through ITGB1." Bioengineered 12.1 (2021): 44-53.

[0214] Antibodies targeting STn are known in the art, including the anti-STn antibody described in Prendergast, da Silva, et al. "Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates demonstrate tumor specificity and anti-tumor activity." mAbs 9,4 (2017): 615-627. Additional anti-STn antibodies include those described in Eavarone, David A. et al. "Humanized anti-Sialyl-Tn antibodies for the treatment of ovarian carcinoma." PloS one 13,7 (2018) e0201314.27.

[0215] Antibodies that target KAAG1 are known in the art, including, for example, the anti-KAAG1 antibody, anti-KAAG1 AB-3A, described in US Pat. No. 9,393,302 B2.

[0216] For example, antibodies targeting DLK1 are known in the art, including the anti-DLK1 antibody and anti-DLK1 SIP(EB3) described in Bujak, Ritz, et al. "A monoclonal antibody to human Dlk1 reveals differential expression in cancer and absence in healthy tissues." Antibodies 4.2 (2015): 71-87. Further anti-DLKL antibodies include, for example, those described in Takagi, Zhao, et al. "Delta-like 1 homolog (DLK1) as a possible therapeutic target and its application to radioimmunotherapy using 125I-labeled anti-DLK1 antibody in lung cancer models (HOT1801 and FIGHT004)." Lung Cancer 153 (2021): 134-142; and Huang, Zhang, et al. "Up-regulation of DLK1 as an imprinted gene could contribute to human hepatocellular carcinoma." Carcinogenesis 28.5 (2007): 1094-1103.

[0217] Antibodies targeting 5T4, including the anti-5T4 antibody and anti-5T4 IgG1 described in Shapiro, Vaishampayan, et al. "First-in-human trial of an anti-5T4 antibody-monomethylauristatin conjugate, PF-06263507, in patients with advanced solid tumors," Investigational New Drugs 35.3 (2017): 315-323, are known in the art. Additional anti-5T4 antibodies include those described in Owens, Sheard, et al. "Preclinical assessment of CAR T-cell therapy targeting the tumor antigen 5T4 in ovarian cancer," Journal of Immunotherapy 41.3 (2018): 130-140.

[0218] Antibodies targeting SEZ6 are known in the art, including the anti-SEZ6 antibody described in, for example, Jiang, Chen, et al. "Correlation between human seizure-related gene 6 variants and idiopathic generalized epilepsy in a Southern Chinese Han population." Neural Regeneration Research 7.2 (2012): 96-100. Additional anti-SEZ6 antibodies include, for example, those described in Kuhn, Koroniak, et al. "Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons." The EMBO journal 31.14 (2012): 3157-3168.

[0219] Antibodies targeting ADAM9 are known in the art, including the anti-ADAM9 antibody described in, for example, Mazzocca, Coppari, et al. "A secreted form of ADAM9 promotes carcinoma invasion through tumor-stromal interactions." Cancer research 65.11 (2005): 4728-4738. Additional anti-ADAM9 antibodies include, for example, those described in Zigrino, Mauch, et al. "Adam-9 expression and regulation in human skin melanoma and melanoma cell lines." International journal of cancer 116.6 (2005): 853-859; and Kim, Jeung, et al. "The effect of disintegrin-metalloproteinase ADAM9 in gastric cancer progression." Molecular cancer therapeutics 13.12 (2014): 3074-3085.

[0220] For example, antibodies targeting I-Ag7 are known in the art, including the anti-I-Ag7 antibody described in Zhang, Crawford, et al. "Monoclonal antibody blocking the recognition of an insulin peptide-MHC complex modulates type 1 diabetes." Proceedings of the National Academy of Sciences 111.7 (2014): 2656-2661. Additional antibodies targeting I-Ag7 include, for example, those described in Noorchashm, Hooman, et al. "I-Ag7-mediated antigen presentation by B lymphocytes is critical in overcoming a checkpoint in T cell tolerance to islet β cells of nonobese diabetic mice." The Journal of Immunology 163.2 (1999): 743-750; and Gardiner, Richards, et al. "Conformation of MHC class II I-Ag7 is sensitive to the P9 anchor amino acid in bound peptide." International Immunology 19.9 (2007): 1103-1113.

[0221] Antibodies targeting ENPP3 are known in the art, including the anti-ENPP3 antibody described in Boggavarapu, Lalitkumar, et al. "Compartmentalized gene expression profiling of receptive endometrium reveals progesterone-regulated ENPP3 is differentially expressed and secreted in glycosylated form," Scientific Reports 6.1 (2016): 1-13. Additional anti-ENPP3 antibodies include those described in Schiechl, Hermann, et al. "Basophils trigger fibroblast activation in cardiac allograft fibrosis development," American Journal of Transplantation 16.9 (2016): 2574-2588.

[0222] Antibodies targeting CD46 are known in the art, including the anti-CD46 antibody YS5 described in Su, Liu, et al. "Targeting CD46 for both adenocarcinoma and neuroendocrine prostate cancer," JCI insight 3.17 (2018) e121497. Additional anti-CD46 antibodies include those described in Carver-Ward, Hollanders, et al. "Progesterone does not potentiate the acrosome reaction in human spermatozoa: flow cytometric analysis using CD46 antibody," Human reproduction 11.1 (1996): 121-126; and Krey, Himmelreich, et al. "Function of bovine CD46 as a cellular receptor for bovine viral diarrhea virus is determined by complement control protein 1," Journal of virology 80.8 (2006): 3912-3922.

[0223] Antibodies targeting CD56 are known in the art, including the anti-CD56 antibody described in Silvin, Chapuis, et al. "Elevated calprotectin and abnormal myeloid cell subsets discriminate severe from mild COVID-19." Cell 182.6 (2020): 1401-1418. Additional anti-CD46 antibodies include those described in Zhan, Guo, et al. "Glioma stem-like cells evade interferon suppression through MBD3 / NuRD complex-mediated STAT1 downregulation." The Journal of experimental medicine 217,5 (2020): e20191340; and Feng, Wang et al. "Differential killing of CD56-expressing cells by drug-conjugated human antibodies targeting membrane-distal and membrane-proximal non-overlapping epitopes." mAbs 8.4 (2016): 799-810.

[0224] Antibodies targeting ROR1 are known in the art, including the anti-ROR1 antibody anti-ROR1 4A5 described in Balakrishnan, Goodpaster, et al. "Analysis of ROR1 Protein Expression in Human Cancer and Normal Tissues," Clinical Cancer Research 23.12 (2017): 3061-3071. Additional anti-ROR1 antibodies include those described in, for example, Baskar, Wiestner et al. "Targeting malignant B cells with an immunotoxin against ROR1," mAbs. 4.3 (2012) 349-361; and Zhang, Chen et al. "ROR1 is expressed in human breast cancer and associated with enhanced tumor-cell growth," PloS one 7,3 (2012): e31127.

[0225] Antibodies targeting GPR20 are known in the art, including the anti-GPR20 antibody described in Wheway, Schmidts, et al. "An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes." Nature cell biology 17,8 (2015): 1074-1087. Additional anti-GPR20 antibodies include those described in Iida, Ahmed, et al. "Identification and Therapeutic Targeting of GPR20, Selectively Expressed in Gastrointestinal Stromal Tumors, with DS-6157a, a First-in-Class Antibody-Drug Conjugate." Cancer Discovery 11.6 (2021): 1508-1523.

[0226] For example, antibodies targeting TM4SF1 are known in the art, including the anti-TM4SF1 antibody described in Zacharias, Frank, et al. "Regeneration of the lung alveolus by an evolutionarily conserved epithelial progenitor." Nature 555,7695 (2018): 251-255. Additional antibodies targeting TM4SF1 include, for example, the anti-TM4SF1 antibody 8G4 described in Lin, Merley, et al. "TM4SF1: a new vascular therapeutic target in cancer." Angiogenesis 17,4 (2014): 897-907, and the anti-TM4SF1 antibody described in Wang, Sun, et al. "B7-H3 suppresses doxorubicin-induced senescence-like growth arrest in colorectal cancer through the AKT / TM4SF1 / SIRT1 pathway" Cell death & disease 12,5 (2021): 453.

[0227] For example, antibodies targeting B7-H4 are known in the art, including the anti-B7-H4 antibody described in Podojil, Glaser, et al. "Antibody targeting of B7-H4 enhances the immune response in urothelial carcinoma." Oncoimmunology 9,1 (2020): 1744897. Additional antibodies targeting B7-H4 include, for example, those described in Miao and Sun, "Development of a novel anti-B7-H4 antibody enhances anti-tumor immune response of human T cells." Biomedicine & pharmacotherapy 141 (2021): 111913; and Dangaj, Lanitis, et al., "Novel Recombinant Human B7-H4 Antibodies Overcome Tumor Immune Escape to Potentiate T-Cell Antitumor Responses Overcoming B7-H4-Mediated T-Cell Inhibition." Cancer research 73.15 (2013): 4820-4829.

[0228] Antibodies targeting ALPP are known in the art, including the anti-ALPP antibody, anti-ALPP SP15, described in Zwolanek, Satue, et al., "Tracking mesenchymal stem cell contributions to regeneration in an immunocompetent cartilage regeneration model," JCI Insight 2.20 (2017) e87322. Additional antibodies targeting ALPP include those described in Chen, Chen, et al., "Placental alkaline phosphatase promotes Zika virus replication by stabilizing viral proteins through BIP," MBio 11.5 (2020): e01716-20; and Odorfer, Egerbacher, et al., "Hematopoietic bone marrow cells participate in endothelial, but not epithelial or mesenchymal cell renewal in adult rats," Journal of Cellular and Molecular Medicine 15.10 (2011): 2232-2244.

[0229] For example, antibodies targeting LY6E are known in the art, including the anti-LY6E antibody described in Mar, Rinkenberger, et al. "LY6E mediates an evolutionarily conserved enhancement of viral infection by targeting a late entry step." Nature communications 9.1 (2018): 1-14. Additional antibodies targeting LY6E include, for example, anti-LY6E MTS35, an anti-LY6E antibody described in Langford, Outhwaite, et al. "Deletion of the Syncytin A receptor Ly6e impairs syncytiotrophoblast fusion and placental morphogenesis causing embryonic lethality in mice." Scientific reports 8,1 (2018): 3961; and anti-LY6E 9B12, an anti-LY6E antibody described in Dela Cruz Chuh, Josefa, et al. "Preclinical optimization of Ly6E-targeted ADCs for increased durability and efficacy of anti-tumor response." MAbs 13.1 (2021).

[0230] Antibodies targeting CLDN18 are known in the art, including the anti-CLDN18 antibody described in Tureci, Mitnacht-Kraus, et al. "Characterization of zolbetuximab in pancreatic cancer models," Oncoimmunology 8.1 (2019): e1523096. Additional anti-CLDN18 antibodies include those described in Matsusaka, Ushiku, et al. "Coupling CDH17 and CLDN18 markers for comprehensive membrane-targeted detection of human gastric cancer," Oncotarget 7,39 (2016): 64168-64181.

[0231] For example, antibodies targeting LY6G6D are known in the art, including the anti-LY6G6D antibody described in Sewda, Coppola, et al. "Cell-surface markers for colon adenoma and adenocarcinoma." Oncotarget 7,14 (2016): 17773-89. Further examples of anti-LY6G6D antibodies include the anti-LY6G6D antibody, anti-LY6G6D clone 10C1, described in Corrales, Hipp, et al. "LY6G6D is a selectively expressed colorectal cancer antigen that can be used for targeting a therapeutic T-cell response by a T-cell engager." Frontiers in immunology 13 (2022): 1008764; and the anti-LY6G6D antibody described in Wang, Sun, et al. "Novel Anti-LY6G6D / CD3 T Cell-Dependent Bispecific Antibody for the Treatment of Colorectal Cancer." Molecular Cancer Therapeutics 21:6 (2022): 974-985.

[0232] For example, antibodies targeting GPR56 are known in the art, including the anti-GPR56 antibody anti-GPR56 10C7 described in Chatterjee, Zhang, et al. "Anti-GPR56 monoclonal antibody potentiates GPR56-mediated Src-Fak signaling to modulate cell adhesion." Journal of Biological Chemistry 296 (2021) 100261. Further anti-GPR56 antibodies include, for example, those described in Iguchi, Sakata, et al. "Orphan G protein-coupled receptor GPR56 regulates neural progenitor cell migration via a Gα12 / 13 and Rho pathway." Journal of Biological Chemistry 283.21 (2008): 14469-14478; and Chen, Yang, et al. "GPR56 is essential for testis development and male fertility in mice." Developmental Dynamics 239.12 (2010): 3358-3367.

[0233] Antibodies targeting CD71 are known in the art, including, for example, the anti-CD71 antibody anti-Tfr1 H68.4 described in Byrne, et al. "Ferristatin II promotes degradation of transferrin receptor-1 in vitro and in vivo." PLoS One 8.7 (2013): e70199. Additional anti-CD71 antibodies include those described, for example, in Hamamichi, et al. "Novel method for screening functional antibodies with comprehensive analysis of their immunoliposomes." Scientific reports 11.1 (2021): 1-13; and Kono, et al. "Morphological definition of CD71-positive reticulocytes by various staining techniques and electron microscopy compared to reticulocytes detected by an automated hematology analyzer." Clinica Chimica Acta 404.2 (2009): 105-110.

[0234] The antibodies described above are merely examples and are not meant to limit the scope of the present disclosure in any way. Additional binding agents, including antibodies, suitable for incorporation into the methods and bispecific binding agents of the present disclosure will be apparent to those of skill in the art.

[0235] While aspects of the present disclosure have been described with reference to disclosed embodiments, those skilled in the art will readily appreciate that the specific examples disclosed are merely illustrative of these aspects and are not intended to limit the disclosure in any way. Various modifications can be made without departing from the spirit of the disclosure.

[0236] In some embodiments, the first binding domain comprises a heavy chain (HC) sequence, a variable heavy chain (VH) sequence, a light chain (LC) sequence, and a variable light chain (VL) sequence. In some embodiments, the first binding domain comprises a HC sequence and a VH sequence. A first binding domain comprising a HC sequence, a VH sequence, a LC sequence, and a VL sequence may comprise one or more sequences listed in Table 1 or Table 2. A first binding domain comprising a HC sequence, a VH sequence, a LC sequence, and a VL sequence may comprise at least 70% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, a first binding domain comprising a HC sequence, a VH sequence, a LC sequence, and a VL sequence comprises at least 75% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, the first binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 80% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, the first binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 85% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, the first binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 90% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, the first binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 91% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, the first binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 92% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, the first binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 93% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, the first binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 94% sequence identity to one or more sequences listed in Table 1 or Table 2.In some cases, the first binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 95% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, the first binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 96% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, the first binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 97% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, the first binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 98% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, the first binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 99% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, the first binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 99.5% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, the first binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 99.9% sequence identity to one or more sequences listed in Table 1 or Table 2.

[0237] In some embodiments, the first binding domain comprises an antibody comprising a heavy chain (HC) sequence, a variable heavy chain (VH) sequence, a light chain (LC) sequence, and a variable light chain (VL) sequence. In some embodiments, the first binding domain comprises an antibody comprising a HC sequence and a VH sequence. A first binding domain comprising an antibody comprising a HC sequence, a VH sequence, a LC sequence, and a VL sequence may comprise one or more sequences listed in Table 1 or Table 2. A first binding domain comprising an antibody comprising a HC sequence, a VH sequence, a LC sequence, and a VL sequence may comprise at least 70% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, a first binding domain comprising an antibody comprising a HC sequence, a VH sequence, a LC sequence, and a VL sequence comprises at least 75% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, a first binding domain comprising an antibody comprising an HC sequence, a VH sequence, an LC sequence, and a VL sequence comprises at least 80% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, a first binding domain comprising an antibody comprising an HC sequence, a VH sequence, a LC sequence, and a VL sequence comprises at least 85% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, a first binding domain comprising an antibody comprising an HC sequence, a VH sequence, a LC sequence, and a VL sequence comprises at least 90% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, a first binding domain comprising an antibody comprising an HC sequence, a VH sequence, a LC sequence, and a VL sequence comprises at least 91% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, a first binding domain comprising an antibody comprising an HC sequence, a VH sequence, an LC sequence, and a VL sequence comprises at least 92% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, a first binding domain comprising an antibody comprising an HC sequence, a VH sequence, an LC sequence, and a VL sequence comprises at least 93% sequence identity to one or more sequences listed in Table 1 or Table 2.In some cases, a first binding domain comprising an antibody comprising an HC sequence, a VH sequence, an LC sequence, and a VL sequence comprises at least 94% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, a first binding domain comprising an antibody comprising an HC sequence, a VH sequence, a LC sequence, and a VL sequence comprises at least 95% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, a first binding domain comprising an antibody comprising an HC sequence, a VH sequence, a LC sequence, and a VL sequence comprises at least 96% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, a first binding domain comprising an antibody comprising an HC sequence, a VH sequence, a LC sequence, and a VL sequence comprises at least 97% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, a first binding domain comprising an antibody comprising an HC sequence, a VH sequence, an LC sequence, and a VL sequence comprises at least 98% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, a first binding domain comprising an antibody comprising an HC sequence, a VH sequence, a LC sequence, and a VL sequence comprises at least 99% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, a first binding domain comprising an antibody comprising an HC sequence, a VH sequence, a LC sequence, and a VL sequence comprises at least 99.5% sequence identity to one or more sequences listed in Table 1 or Table 2. In some cases, a first binding domain comprising an antibody comprising an HC sequence, a VH sequence, a LC sequence, and a VL sequence comprises at least 99.9% sequence identity to one or more sequences listed in Table 1 or Table 2.

[0238] In some embodiments, the antibody that targets an internalizing receptor protein comprises a sequence listed in Table 1. In some embodiments, the antibody that targets an internalizing receptor protein comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or at least 99.9% sequence identity to a sequence listed in Table 1.

[0239] In some cases, an antibody targeting an internalizing receptor protein may bind to the same epitope as any one of the antibodies listed in Table 1. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 70% sequence identity to an epitope bound by any one of the antibodies listed in Table 1. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 75% sequence identity to an epitope bound by any one of the antibodies listed in Table 1. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 80% sequence identity to an epitope bound by any one of the antibodies listed in Table 1. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 85% sequence identity to an epitope bound by any one of the antibodies listed in Table 1. An antibody that targets an internalizing receptor protein may bind to an epitope that comprises about 90% sequence identity to the epitope bound by any one of the antibodies listed in Table 1. An antibody that targets an internalizing receptor protein may bind to an epitope that comprises about 95% sequence identity to the epitope bound by any one of the antibodies listed in Table 1. An antibody that targets an internalizing receptor protein may bind to an epitope that comprises about 99% sequence identity to the epitope bound by any one of the antibodies listed in Table 1.

[0240] In some embodiments, an antibody targeting an internalized receptor protein may bind with an affinity similar to any one of the antibodies listed in Table 1 (Table 5 lists the affinities of certain monovalent binders). Table 5 lists the monovalent Kd for certain cell surface monovalent proteins. In certain embodiments, the multispecific binding agent has a Kd that is less than, greater than, within 10%, 20%, 30%, 40%, 50%, 75%, or 100% of the binding affinity of the monovalent binding agent. For example, Table 5 lists the monovalent binding affinities for certain CD71 monovalent binding agents. When these CD71-binding arms are incorporated into monovalent binding agents of the present disclosure, the binding affinity of the multispecific binding agent may be within an order of magnitude, or more than twofold to twofold less than the binding affinity of the monovalent binding agent. For example, the binding affinity of the monovalent binding agent has a Kd of 0.1 nM to 100 nM. When incorporated into a multispecific binding agent, the Kd may be within the same range. Alternatively, the binding affinity may be slightly higher than the monovalent binding affinity, but not less than two times higher. The binding affinity may be not less than one-third higher than the monovalent binding affinity, but not more than three times higher.

[0241] The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 70% sequence identity to an epitope to which any one of the antibodies listed in Table 1 binds with similar affinity as any one of the antibodies listed in Table 1. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 75% sequence identity to an epitope to which any one of the antibodies listed in Table 1 binds with similar affinity as any one of the antibodies listed in Table 1. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 80% sequence identity to an epitope to which any one of the antibodies listed in Table 1 binds with similar affinity as any one of the antibodies listed in Table 1. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 85% sequence identity to an epitope to which any one of the antibodies listed in Table 1 binds with similar affinity as any one of the antibodies listed in Table 1. The antibody that targets an internalizing receptor protein may bind to an epitope that comprises about 90% sequence identity to an epitope to which any one of the antibodies listed in Table 1 binds with similar affinity as any one of the antibodies listed in Table 1. The antibody that targets an internalizing receptor protein may bind to an epitope that comprises about 95% sequence identity to an epitope to which any one of the antibodies listed in Table 1 binds with similar affinity as any one of the antibodies listed in Table 1. The antibody that targets an internalizing receptor protein may bind to an epitope that comprises about 99% sequence identity to an epitope to which any one of the antibodies listed in Table 1 binds with similar affinity as any one of the antibodies listed in Table 1.

[0242] In some embodiments, an antibody targeting an internalizing receptor protein may bind to the same epitope as any one of the antibodies listed in Table 1 but with a different affinity compared to any one of the antibodies listed in Table 1. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 70% sequence identity to an epitope to which any one of the antibodies listed in Table 1 binds with a different affinity compared to any one of the antibodies listed in Table 1. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 75% sequence identity to an epitope to which any one of the antibodies listed in Table 1 binds with a different affinity compared to any one of the antibodies listed in Table 1. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 80% sequence identity to an epitope to which any one of the antibodies listed in Table 1 binds with a different affinity compared to any one of the antibodies listed in Table 1. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 85% sequence identity to an epitope to which any one of the antibodies listed in Table 1 binds with a different affinity compared to any one of the antibodies listed in Table 1. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 90% sequence identity to an epitope to which any one of the antibodies listed in Table 1 binds with a different affinity compared to any one of the antibodies listed in Table 1. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 95% sequence identity to an epitope to which any one of the antibodies listed in Table 1 binds with a different affinity compared to any one of the antibodies listed in Table 1. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 99% sequence identity to an epitope to which any one of the antibodies listed in Table 1 binds with a different affinity compared to any one of the antibodies listed in Table 1.

[0243] An antibody targeting an internalizing receptor protein may bind to an epitope that includes an epitope different from the epitope bound by any one of the antibodies listed in Table 1. An antibody targeting an internalizing receptor protein may bind to an epitope that includes an epitope different from the epitope bound by any one of the antibodies listed in Table 1, and that does not bind to any of the same amino acids on the internalizing receptor protein. An antibody targeting an internalizing receptor protein may bind to an epitope that includes an epitope different from the epitope bound by any one of the antibodies listed in Table 1, and that binds to any one or more of the same amino acids on the internalizing receptor protein. An antibody targeting an internalizing receptor protein may bind to an epitope that includes an epitope different from the epitope bound by any one of the antibodies listed in Table 1, and that binds to any two or more of the same amino acids on the internalizing receptor protein. An antibody that targets an internalizing receptor protein may bind to an epitope that binds to any three or more of the same amino acids on the internalizing receptor protein, including an epitope that is different from the epitope bound by any one of the antibodies listed in Table 1. An antibody that targets an internalizing receptor protein may bind to an epitope that binds to any four or more of the same amino acids on the internalizing receptor protein, including an epitope that is different from the epitope bound by any one of the antibodies listed in Table 1. An antibody that targets an internalizing receptor protein may bind to an epitope that binds to any five or more of the same amino acids on the internalizing receptor protein, including an epitope that is different from the epitope bound by any one of the antibodies listed in Table 1. An antibody that targets an internalizing receptor protein may bind to an epitope that includes an epitope that is different from the epitope bound by any one of the antibodies listed in Table 1, and that binds to any six or more of the same amino acids on the internalizing receptor protein.An antibody that targets an internalizing receptor protein may bind to an epitope that includes an epitope that is different from the epitope bound by any one of the antibodies listed in Table 1 and that binds to any seven or more of the same amino acids on the internalizing receptor protein. An antibody that targets an internalizing receptor protein may bind to an epitope that includes an epitope that is different from the epitope bound by any one of the antibodies listed in Table 1 and that binds to any eight or more of the same amino acids on the internalizing receptor protein. An antibody that targets an internalizing receptor protein may bind to an epitope that includes an epitope that is different from the epitope bound by any one of the antibodies listed in Table 1 and that binds to any nine or more of the same amino acids on the internalizing receptor protein. An antibody that targets an internalizing receptor protein may bind to an epitope that includes an epitope that is different from the epitope bound by any one of the antibodies listed in Table 1, and that binds to any 10 or more of the same amino acids on the internalizing receptor protein.

[0244] In some embodiments, the antibody targeting the degrader protein comprises a sequence listed in Table 1. In some embodiments, the antibody targeting the degrader protein comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or at least 99.9% sequence identity to a sequence listed in Table 1.

[0245] In some cases, an antibody targeting a degrader protein may bind to the same epitope as any one of the antibodies listed in Table 1. An antibody targeting a degrader protein may bind to an epitope that comprises about 70% sequence identity to the epitope bound by any one of the antibodies listed in Table 1. An antibody targeting a degrader protein may bind to an epitope that comprises about 75% sequence identity to the epitope bound by any one of the antibodies listed in Table 1. An antibody targeting a degrader protein may bind to an epitope that comprises about 80% sequence identity to the epitope bound by any one of the antibodies listed in Table 1. An antibody targeting a degrader protein may bind to an epitope that comprises about 85% sequence identity to the epitope bound by any one of the antibodies listed in Table 1. An antibody targeting a degrader protein may bind to an epitope that comprises about 90% sequence identity to the epitope bound by any one of the antibodies listed in Table 1. The antibody targeting the degrader protein may bind to an epitope that comprises about 95% sequence identity to the epitope bound by any one of the antibodies listed in Table 1. The antibody targeting the degrader protein may bind to an epitope that comprises about 99% sequence identity to the epitope bound by any one of the antibodies listed in Table 1.

[0246] An antibody targeting a degrader protein may bind to an epitope that includes an epitope different from the epitope bound by any one of the antibodies listed in Table 1. An antibody targeting a degrader protein may bind to an epitope that includes an epitope different from the epitope bound by any one of the antibodies listed in Table 1, and that does not bind to any of the same amino acids on the degrader protein. An antibody targeting a degrader protein may bind to an epitope that includes an epitope different from the epitope bound by any one of the antibodies listed in Table 1, and that binds to any one or more of the same amino acids on the degrader protein. An antibody targeting a degrader protein may bind to an epitope that includes an epitope different from the epitope bound by any one of the antibodies listed in Table 1, and that binds to any two or more of the same amino acids on the degrader protein. An antibody targeting a degrader protein may bind to an epitope that binds to any three or more of the same amino acids on a degrader protein, including an epitope that is different from the epitope bound by any one of the antibodies listed in Table 1. An antibody targeting a degrader protein may bind to an epitope that binds to any four or more of the same amino acids on a degrader protein, including an epitope that is different from the epitope bound by any one of the antibodies listed in Table 1. An antibody targeting a degrader protein may bind to an epitope that binds to any five or more of the same amino acids on a degrader protein, including an epitope that is different from the epitope bound by any one of the antibodies listed in Table 1. An antibody targeting a degrader protein may bind to an epitope that binds to any six or more of the same amino acids on a degrader protein, including an epitope that is different from the epitope bound by any one of the antibodies listed in Table 1.An antibody targeting a degrader protein may bind to an epitope that binds to any seven or more of the same amino acids on a degrader protein, including an epitope that is different from the epitope bound by any one of the antibodies listed in Table 1. An antibody targeting a degrader protein may bind to an epitope that binds to any eight or more of the same amino acids on a degrader protein, including an epitope that is different from the epitope bound by any one of the antibodies listed in Table 1. An antibody targeting a degrader protein may bind to an epitope that binds to any nine or more of the same amino acids on a degrader protein, including an epitope that is different from the epitope bound by any one of the antibodies listed in Table 1. An antibody targeting a degrader protein may bind to an epitope that binds to any ten or more of the same amino acids on a degrader protein, including an epitope that is different from the epitope bound by any one of the antibodies listed in Table 1. Table 1. Exemplary antibody sequences targeting internalizing receptor or degrader proteins. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

[0247] The sequences listed in Table 1 (SEQ ID NOs: 1 to 353) are amino acid molecules. The sequences listed in Table 1 (SEQ ID NOs: 1 to 353) are amino acid molecules that are synthetic constructs. The sequences listed in Table 1 (SEQ ID NOs: 1 to 353) for HC sequences (heavy chain), VH sequences (variable heavy chain sequences), LC sequences (light chain), and VL sequences (variable light chain sequences) are amino acid molecules that are synthetic constructs.

[0248] In some embodiments, the antibody that targets an internalizing receptor protein comprises a sequence listed in Table 2. In some embodiments, the antibody that targets an internalizing receptor protein comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or at least 99.9% sequence identity to a sequence listed in Table 2.

[0249] In some cases, an antibody targeting an internalizing receptor protein may bind to the same epitope as any one of the antibodies listed in Table 2. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 70% sequence identity to an epitope bound by any one of the antibodies listed in Table 2. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 75% sequence identity to an epitope bound by any one of the antibodies listed in Table 2. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 80% sequence identity to an epitope bound by any one of the antibodies listed in Table 2. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 85% sequence identity to an epitope bound by any one of the antibodies listed in Table 2. The antibody that targets the internalizing receptor protein may bind to an epitope that comprises about 90% sequence identity to the epitope bound by any one of the antibodies listed in Table 2. The antibody that targets the internalizing receptor protein may bind to an epitope that comprises about 95% sequence identity to the epitope bound by any one of the antibodies listed in Table 2. The antibody that targets the internalizing receptor protein may bind to an epitope that comprises about 99% sequence identity to the epitope bound by any one of the antibodies listed in Table 2.

[0250] In some embodiments, an antibody targeting an internalizing receptor protein may bind with an affinity similar to any one of the antibodies listed in Table 2 (Table 5 lists the affinities of certain monovalent binders). Table 5 lists the monovalent Kd for certain internalizing receptor monovalent proteins. In certain embodiments, the multispecific binding agent has a Kd that is less than, greater than, within 10%, 20%, 30%, 40%, 50%, 75%, or 100% of the binding affinity of the monovalent binding agent. For example, Table 5 lists the monovalent binding affinities for certain CD71 monovalent binding agents. When these CD71-binding arms are incorporated into the monovalent binding agents of the present disclosure, the binding affinity of the multispecific binding agent may be within an order of magnitude, or more than twofold to twofold less than the binding affinity of the monovalent binding agent. For example, the binding affinity of the monovalent binding agent has a Kd of 0.1 nM to 100 nM. When incorporated into a multispecific binding agent, the Kd may be within the same range. Alternatively, the binding affinity may be slightly higher than the monovalent binding affinity, but not less than two-fold higher. The binding affinity may be not less than three-fold higher than the monovalent binding affinity.

[0251] In some embodiments, an antibody targeting an internalizing receptor protein may bind to the same epitope as any one of the antibodies listed in Table 2 that binds with similar affinity as any one of the antibodies listed in Table 2. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 70% sequence identity to an epitope to which any one of the antibodies listed in Table 2 binds with similar affinity as any one of the antibodies listed in Table 2. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 75% sequence identity to an epitope to which any one of the antibodies listed in Table 2 binds with similar affinity as any one of the antibodies listed in Table 2. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 80% sequence identity to an epitope to which any one of the antibodies listed in Table 2 binds with similar affinity as any one of the antibodies listed in Table 2. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 85% sequence identity to an epitope to which any one of the antibodies listed in Table 2 binds with similar affinity as any one of the antibodies listed in Table 2. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 90% sequence identity to an epitope to which any one of the antibodies listed in Table 2 binds with similar affinity as any one of the antibodies listed in Table 2. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 95% sequence identity to an epitope to which any one of the antibodies listed in Table 2 binds with similar affinity as any one of the antibodies listed in Table 2. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 99% sequence identity to an epitope to which any one of the antibodies listed in Table 2 binds with similar affinity as any one of the antibodies listed in Table 2.

[0252] In some embodiments, an antibody targeting an internalizing receptor protein may bind to the same epitope as any one of the antibodies listed in Table 2, but with a different affinity compared to any one of the antibodies listed in Table 2. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 70% sequence identity to an epitope to which any one of the antibodies listed in Table 2 binds with a different affinity compared to any one of the antibodies listed in Table 2. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 75% sequence identity to an epitope to which any one of the antibodies listed in Table 2 binds with a different affinity compared to any one of the antibodies listed in Table 2. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 80% sequence identity to an epitope to which any one of the antibodies listed in Table 2 binds with a different affinity compared to any one of the antibodies listed in Table 2. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 85% sequence identity to an epitope to which any one of the antibodies listed in Table 2 binds with a different affinity compared to any one of the antibodies listed in Table 2. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 90% sequence identity to an epitope to which any one of the antibodies listed in Table 2 binds with a different affinity compared to any one of the antibodies listed in Table 2. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 95% sequence identity to an epitope to which any one of the antibodies listed in Table 2 binds with a different affinity compared to any one of the antibodies listed in Table 2. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 99% sequence identity to an epitope to which any one of the antibodies listed in Table 2 binds with a different affinity compared to any one of the antibodies listed in Table 2.

[0253] An antibody targeting an internalizing receptor protein may bind to an epitope that includes an epitope different from the epitope bound by any one of the antibodies listed in Table 2. An antibody targeting an internalizing receptor protein may bind to an epitope that includes an epitope different from the epitope bound by any one of the antibodies listed in Table 2, and that does not bind to any of the same amino acids on the internalizing receptor protein. An antibody targeting an internalizing receptor protein may bind to an epitope that includes an epitope different from the epitope bound by any one of the antibodies listed in Table 2, and that binds to any one or more of the same amino acids on the internalizing receptor protein. An antibody targeting an internalizing receptor protein may bind to an epitope that includes an epitope different from the epitope bound by any one of the antibodies listed in Table 2, and that binds to any two or more of the same amino acids on the internalizing receptor protein. An antibody that targets an internalizing receptor protein may bind to an epitope that binds to any three or more of the same amino acids on the internalizing receptor protein, including an epitope that is different from the epitope bound by any one of the antibodies listed in Table 2. An antibody that targets an internalizing receptor protein may bind to an epitope that binds to any four or more of the same amino acids on the internalizing receptor protein, including an epitope that is different from the epitope bound by any one of the antibodies listed in Table 2. An antibody that targets an internalizing receptor protein may bind to an epitope that binds to any five or more of the same amino acids on the internalizing receptor protein, including an epitope that is different from the epitope bound by any one of the antibodies listed in Table 2. An antibody that targets an internalizing receptor protein may bind to an epitope that includes an epitope that is different from the epitope bound by any one of the antibodies listed in Table 2, and that binds to any six or more of the same amino acids on the internalizing receptor protein.An antibody that targets an internalizing receptor protein may bind to an epitope that includes an epitope that is different from the epitope bound by any one of the antibodies listed in Table 2 and that binds to any seven or more of the same amino acids on the internalizing receptor protein. An antibody that targets an internalizing receptor protein may bind to an epitope that includes an epitope that is different from the epitope bound by any one of the antibodies listed in Table 2 and that binds to any eight or more of the same amino acids on the internalizing receptor protein. An antibody that targets an internalizing receptor protein may bind to an epitope that includes an epitope that is different from the epitope bound by any one of the antibodies listed in Table 2 and that binds to any nine or more of the same amino acids on the internalizing receptor protein. Antibodies that target internalizing receptor proteins may bind to epitopes that include epitopes that are different from the epitopes bound by any one of the antibodies listed in Table 2, and that bind to any 10 or more of the same amino acids on the internalizing receptor protein.

[0254] In some embodiments, the antibody targeting the degrader protein comprises a sequence listed in Table 2. In some embodiments, the antibody targeting the degrader protein comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or at least 99.9% sequence identity to a sequence listed in Table 2.

[0255] In some cases, an antibody targeting a degrader protein may bind to the same epitope as any one of the antibodies listed in Table 2. An antibody targeting a degrader protein may bind to an epitope that comprises about 70% sequence identity to the epitope bound by any one of the antibodies listed in Table 2. An antibody targeting a degrader protein may bind to an epitope that comprises about 75% sequence identity to the epitope bound by any one of the antibodies listed in Table 2. An antibody targeting a degrader protein may bind to an epitope that comprises about 80% sequence identity to the epitope bound by any one of the antibodies listed in Table 2. An antibody targeting a degrader protein may bind to an epitope that comprises about 85% sequence identity to the epitope bound by any one of the antibodies listed in Table 2. An antibody targeting a degrader protein may bind to an epitope that comprises about 90% sequence identity to the epitope bound by any one of the antibodies listed in Table 2. The antibody targeting the degrader protein may bind to an epitope that comprises about 95% sequence identity to the epitope bound by any one of the antibodies listed in Table 2. The antibody targeting the degrader protein may bind to an epitope that comprises about 99% sequence identity to the epitope bound by any one of the antibodies listed in Table 2.

[0256] An antibody targeting a degrader protein may bind to an epitope that includes an epitope different from the epitope bound by any one of the antibodies listed in Table 2. An antibody targeting a degrader protein may bind to an epitope that includes an epitope different from the epitope bound by any one of the antibodies listed in Table 2, and that does not bind to any of the same amino acids on the degrader protein. An antibody targeting a degrader protein may bind to an epitope that includes an epitope different from the epitope bound by any one of the antibodies listed in Table 2, and that binds to any one or more of the same amino acids on the degrader protein. An antibody targeting a degrader protein may bind to an epitope that includes an epitope different from the epitope bound by any one of the antibodies listed in Table 2, and that binds to any two or more of the same amino acids on the degrader protein. An antibody targeting a degrader protein may bind to an epitope that binds to any three or more of the same amino acids on a degrader protein, the epitope comprising an epitope different from that bound by any one of the antibodies listed in Table 2. An antibody targeting a degrader protein may bind to an epitope that binds to any four or more of the same amino acids on a degrader protein, the epitope comprising an epitope different from that bound by any one of the antibodies listed in Table 2. An antibody targeting a degrader protein may bind to an epitope that binds to any five or more of the same amino acids on a degrader protein, the epitope comprising an epitope different from that bound by any one of the antibodies listed in Table 2. An antibody targeting a degrader protein may bind to an epitope that binds to any six or more of the same amino acids on a degrader protein, the epitope comprising an epitope different from that bound by any one of the antibodies listed in Table 2.An antibody targeting a degrader protein may bind to an epitope that binds to any seven or more of the same amino acids on a degrader protein, including an epitope that is different from the epitope bound by any one of the antibodies listed in Table 2. An antibody targeting a degrader protein may bind to an epitope that binds to any eight or more of the same amino acids on a degrader protein, including an epitope that is different from the epitope bound by any one of the antibodies listed in Table 2. An antibody targeting a degrader protein may bind to an epitope that binds to any nine or more of the same amino acids on a degrader protein, including an epitope that is different from the epitope bound by any one of the antibodies listed in Table 2. An antibody targeting a degrader protein may bind to an epitope that binds to any ten or more of the same amino acids on a degrader protein, including an epitope that is different from the epitope bound by any one of the antibodies listed in Table 2. Table 2. Additional exemplary antibody sequences targeting internalizing receptor or degrader proteins. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8]

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

Table 2-24

Table 2-25

[0257] The sequences listed in Table 2 (SEQ ID NOs: 354-633; 684-723) are amino acid molecules. The sequences listed in Table 2 (SEQ ID NOs: 354-633; 684-723) are amino acid molecules that are synthetic constructs. The sequences listed in Table 2 for HC sequences (heavy chains), VH sequences (variable heavy chain sequences), LC sequences (light chains), and VL sequences (variable light chain sequences) (SEQ ID NOs: 354-633; 684-723) are amino acid molecules that are synthetic constructs. Second binding region

[0258] In some embodiments, the second binding domain (i.e., the EGFR-binding domain) comprises an EGFR-binding domain (e.g., a CDR that specifically binds to EGFR) derived from an anti-EGFR antibody. Such antibodies are known to those of skill in the art and can be incorporated into the methods and bispecific binding agents of the present disclosure. Antibodies targeting EGFR are known in the art, and include, for example, the following anti-EGFR antibodies: (i) cetuximab, described, for example, in P. Kirkpatrick, et al., "Cetuximab," Nature Reviews Drug Discovery, 3(7) (2004): 549; (ii) panitumumab, described, for example, in L. Saltz, et al., "Panitumamab," Nature Reviews Drug Discovery, 5(12) (2006): 987; (iii) nimotuzumab, described, for example, in MS Ramakrishnan, "Nimotuzumab, a promising therapeutic monoclonal for treatment of tumors of epithelial origin." mAbs 1(1) (2009):41; and (iv) necitumumab, described, for example, in DR Tabernero, "Necitumumab, a fully human IgG1 mAb directed against the EGFR for the potential treatment of cancer." Current Opinions in Investigational Drugs, 11(12) (2000): 1434.

[0259] The antibodies described above are merely examples and are not meant to limit the scope of the present disclosure in any way. Additional binding agents, including antibodies, suitable for incorporation into the methods and bispecific binding agents of the present disclosure will be apparent to those of skill in the art.

[0260] In some embodiments, the second binding domain binds to a mutant EGFR protein. In some embodiments, the second binding domain selectively binds to a mutant EGFR protein.

[0261] In some embodiments, the second binding domain comprises a heavy chain (HC) sequence, a variable heavy chain (VH) sequence, a light chain (LC) sequence, and a variable light chain (VL) sequence. In some embodiments, the second binding domain comprises a HC sequence and a VH sequence. A second binding domain comprising a HC sequence, a VH sequence, a LC sequence, and a VL sequence may comprise one or more sequences listed in Table 3. A second binding domain comprising a HC sequence, a VH sequence, a LC sequence, and a VL sequence may comprise at least 70% sequence identity to one or more sequences listed in Table 3. In some cases, a second binding domain comprising a HC sequence, a VH sequence, a LC sequence, and a VL sequence comprises at least 75% sequence identity to one or more sequences listed in Table 3. In some cases, a second binding domain comprising a HC sequence, a VH sequence, a LC sequence, and a VL sequence comprises at least 80% sequence identity to one or more sequences listed in Table 3. In some cases, the second binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 85% sequence identity to one or more sequences listed in Table 3. In some cases, the second binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 90% sequence identity to one or more sequences listed in Table 3. In some cases, the second binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 91% sequence identity to one or more sequences listed in Table 3. In some cases, the second binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 92% sequence identity to one or more sequences listed in Table 3. In some cases, the second binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 93% sequence identity to one or more sequences listed in Table 3. In some cases, the second binding domain comprising the HC, VH, LC, and VL sequences comprises at least 94% sequence identity to one or more sequences listed in Table 3.In some cases, the second binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 95% sequence identity to one or more sequences listed in Table 3. In some cases, the second binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 96% sequence identity to one or more sequences listed in Table 3. In some cases, the second binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 97% sequence identity to one or more sequences listed in Table 3. In some cases, the second binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 98% sequence identity to one or more sequences listed in Table 3. In some cases, the second binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 99% sequence identity to one or more sequences listed in Table 3. In some cases, the second binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 99.5% sequence identity to one or more sequences listed in Table 3. In some cases, the second binding domain comprising the HC sequence, the VH sequence, the LC sequence, and the VL sequence comprises at least 99.9% sequence identity to one or more sequences listed in Table 3.

[0262] In some embodiments, the EGFR-targeting antibody comprises a sequence listed in Table 3. In some embodiments, the EGFR-targeting antibody comprises a sequence listed in Table 3. In some embodiments, the EGFR-targeting antibody comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or at least 99.9% sequence identity to a sequence listed in Table 3.

[0263] In some embodiments, the second binding domain comprises at least 70% sequence identity to cetuximab. In some embodiments, the second binding domain comprises at least 75% sequence identity to cetuximab. In some embodiments, the second binding domain comprises at least 80% sequence identity to cetuximab. In some embodiments, the second binding domain comprises at least 85% sequence identity to cetuximab. In some embodiments, the second binding domain comprises at least 90% sequence identity to cetuximab. In some embodiments, the second binding domain comprises at least 91% sequence identity to cetuximab. In some embodiments, the second binding domain comprises at least 92% sequence identity to cetuximab. In some embodiments, the second binding domain comprises at least 93% sequence identity to cetuximab. In some embodiments, the second binding domain comprises at least 94% sequence identity to cetuximab. In some embodiments, the second binding domain comprises at least 95% sequence identity to cetuximab. In some embodiments, the second binding domain comprises at least 96% sequence identity to cetuximab. In some embodiments, the second binding domain comprises at least 97% sequence identity to cetuximab. In some embodiments, the second binding domain comprises at least 98% sequence identity to cetuximab. In some embodiments, the second binding domain comprises at least 99% sequence identity to cetuximab. In some embodiments, the second binding domain comprises at least 99.5% sequence identity to cetuximab. In some embodiments, the second binding domain comprises at least 99.9% sequence identity to cetuximab.

[0264] In some embodiments, the second binding domain comprises at least 70% sequence identity to murine adenovirus 2 (Mav2). In some embodiments, the second binding domain comprises at least 75% sequence identity to Mav2. In some embodiments, the second binding domain comprises at least 80% sequence identity to Mav2. In some embodiments, the second binding domain comprises at least 85% sequence identity to Mav2. In some embodiments, the second binding domain comprises at least 90% sequence identity to Mav2. In some embodiments, the second binding domain comprises at least 91% sequence identity to Mav2. In some embodiments, the second binding domain comprises at least 92% sequence identity to Mav2. In some embodiments, the second binding domain comprises at least 93% sequence identity to Mav2. In some embodiments, the second binding domain comprises at least 94% sequence identity to Mav2. In some embodiments, the second binding domain comprises at least 95% sequence identity to Mav2. In some embodiments, the second binding domain comprises at least 96% sequence identity to Mav2. In some embodiments, the second binding domain comprises at least 97% sequence identity to Mav2. In some embodiments, the second binding domain comprises at least 98% sequence identity to Mav2. In some embodiments, the second binding domain comprises at least 99% sequence identity to Mav2. In some embodiments, the second binding domain comprises at least 99.5% sequence identity to Mav2. In some embodiments, the second binding domain comprises at least 99.9% sequence identity to Mav2. As described herein, h7D12 hIgG1 is Mav2.

[0265] In some embodiments, the second binding domain binds to an epitope of EGFR on a target cell that comprises at least 70% sequence identity to the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of EGFR on a target cell that comprises at least 80% sequence identity to the epitope to which cetuximab binds. In some cases, the second binding domain binds to an epitope of EGFR on a target cell that comprises at least 90% sequence identity to the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of EGFR on a target cell that comprises at least 95% sequence identity to the epitope to which cetuximab binds.

[0266] In some embodiments, the second binding domain binds to an epitope of EGFR on a target cell that does not contain any amino acids from the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of EGFR on a target cell that contains 1, 2, 3, 4, 5, or 6 amino acids from the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of EGFR on a target cell that contains one or more amino acids from the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of EGFR on a target cell that contains two or more amino acids from the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of EGFR on a target cell that contains three or more amino acids from the epitope to which cetuximab binds. In some embodiments, the second binding domain binds to an epitope of EGFR on a target cell that comprises four or more amino acids from the epitope to which cetuximab binds.

[0267] In some embodiments, the second binding domain binds to an epitope of EGFR on a target cell that comprises at least 70% sequence identity to the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of EGFR on a target cell that comprises at least 80% sequence identity to the epitope to which Mav2 binds. In some cases, the second binding domain binds to an epitope of EGFR on a target cell that comprises at least 90% sequence identity to the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of EGFR on a target cell that comprises at least 95% sequence identity to the epitope to which Mav2 binds.

[0268] In some embodiments, the second binding domain binds to an epitope of EGFR on a target cell that does not contain any amino acids from the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of EGFR on a target cell that contains 1, 2, 3, 4, 5, or 6 amino acids from the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of EGFR on a target cell that contains one or more amino acids from the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of EGFR on a target cell that contains two or more amino acids from the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of EGFR on a target cell that contains three or more amino acids from the epitope to which Mav2 binds. In some embodiments, the second binding domain binds to an epitope of EGFR on a target cell that comprises four or more amino acids from the epitope to which Mav2 binds.

[0269] In some embodiments, the epitope of EGFR comprises the following amino acids of human EGFR (UniProt ID: P00533): P373, R377, L406, Q407, Q432, H433, Q435, F436, V441, S442, I462, S464, G465, K467, K489, I490, I491, S492, N493, G495, and N497. Antibodies targeting EGFR may target an epitope including amino acids P373, R377, L406, Q407, Q432, H433, Q435, F436, V441, S442, I462, S464, G465, K467, K489, I490, I491, S492, N493, G495, and N497 of human EGFR. In some embodiments, the antibody targeting amino acids P373, R377, L406, Q407, Q432, H433, Q435, F436, V441, S442, I462, S464, G465, K467, K489, I490, I491, S492, N493, G495, and N497 of human EGFR comprises cetuximab. In some embodiments, the epitope of EGFR comprises the following amino acids of human EGFR: L349, H370, L372, P373, V374, R377, D379, F381, T382, Q408, H433, S442. The antibody targeting EGFR may target an epitope including amino acids L349, H370, L372, P373, V374, R377, D379, F381, T382, Q408, H433, and S442 of human EGFR. In some embodiments, the antibody targeting amino acids L349, H370, L372, P373, V374, R377, D379, F381, T382, Q408, H433, and S442 of human EGFR comprises Mav2 (h7D12 hIgG1).

[0270] In some embodiments, an antibody targeting an internalized receptor protein may bind to the same epitope as cetuximab with similar affinity. An antibody targeting an internalized receptor protein may bind to an epitope that contains about 70% sequence identity to an epitope that cetuximab binds to with similar affinity. An antibody targeting an internalized receptor protein may bind to an epitope that contains about 75% sequence identity to an epitope that cetuximab binds to with similar affinity. An antibody targeting an internalized receptor protein may bind to an epitope that contains about 80% sequence identity to an epitope that cetuximab binds to with similar affinity. An antibody targeting an internalized receptor protein may bind to an epitope that contains about 85% sequence identity to an epitope that cetuximab binds to with similar affinity. The antibody targeting the internalizing receptor protein may bind to an epitope that contains about 90% sequence identity to the epitope to which cetuximab binds with similar affinity to cetuximab. The antibody targeting the internalizing receptor protein may bind to an epitope that contains about 95% sequence identity to the epitope to which cetuximab binds with similar affinity to cetuximab. The antibody targeting the internalizing receptor protein may bind to an epitope that contains about 99% sequence identity to the epitope to which cetuximab binds with similar affinity to cetuximab.

[0271] In some embodiments, an antibody targeting an internalized receptor protein may bind to the same epitope as cetuximab but with a different affinity compared to cetuximab. An antibody targeting an internalized receptor protein may bind to an epitope that contains about 70% sequence identity to an epitope to which cetuximab binds with a different affinity compared to cetuximab. An antibody targeting an internalized receptor protein may bind to an epitope that contains about 75% sequence identity to an epitope to which cetuximab binds with a different affinity compared to cetuximab. An antibody targeting an internalized receptor protein may bind to an epitope that contains about 80% sequence identity to an epitope to which cetuximab binds with a different affinity compared to cetuximab. An antibody targeting an internalized receptor protein may bind to an epitope that contains about 85% sequence identity to an epitope to which cetuximab binds with a different affinity compared to cetuximab. The antibody targeting the internalized receptor protein may bind to an epitope that contains about 90% sequence identity to an epitope to which cetuximab binds with a different affinity compared to cetuximab. The antibody targeting the internalized receptor protein may bind to an epitope that contains about 95% sequence identity to an epitope to which cetuximab binds with a different affinity compared to cetuximab. The antibody targeting the internalized receptor protein may bind to an epitope that contains about 99% sequence identity to an epitope to which cetuximab binds with a different affinity compared to cetuximab.

[0272] In some embodiments, an antibody targeting an internalizing receptor protein may bind to the same epitope as Mav2, with an affinity similar to that of Mav2. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 70% sequence identity to an epitope to which Mav2 binds with an affinity similar to that of Mav2. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 75% sequence identity to an epitope to which Mav2 binds with an affinity similar to that of Mav2. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 80% sequence identity to an epitope to which Mav2 binds with an affinity similar to that of Mav2. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 85% sequence identity to an epitope to which Mav2 binds with an affinity similar to that of Mav2. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 90% sequence identity to an epitope to which Mav2 binds with similar affinity to Mav2. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 95% sequence identity to an epitope to which Mav2 binds with similar affinity to Mav2. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 99% sequence identity to an epitope to which Mav2 binds with similar affinity to Mav2.

[0273] In some embodiments, an antibody targeting an internalized receptor protein may bind to the same epitope as Mav2, but with a different affinity compared to Mav2. An antibody targeting an internalized receptor protein may bind to an epitope that contains about 70% sequence identity to an epitope to which Mav2 binds with a different affinity compared to Mav2. An antibody targeting an internalized receptor protein may bind to an epitope that contains about 75% sequence identity to an epitope to which Mav2 binds with a different affinity compared to Mav2. An antibody targeting an internalized receptor protein may bind to an epitope that contains about 80% sequence identity to an epitope to which Mav2 binds with a different affinity compared to Mav2. An antibody targeting an internalized receptor protein may bind to an epitope that contains about 85% sequence identity to an epitope to which Mav2 binds with a different affinity compared to Mav2. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 90% sequence identity to an epitope to which Mav2 binds with a different affinity compared to Mav2. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 95% sequence identity to an epitope to which Mav2 binds with a different affinity compared to Mav2. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 99% sequence identity to an epitope to which Mav2 binds with a different affinity compared to Mav2.

[0274] In some cases, the antibody targeting EGFR may bind to the same epitope as cetuximab or Mav2 (h7D12 hIgG1). The antibody targeting EGFR may bind to an epitope that shares about 70% sequence identity with the epitope bound by cetuximab or Mav2 (h7D12 hIgG1). The antibody targeting EGFR may bind to an epitope that shares about 75% sequence identity with the epitope bound by cetuximab or Mav2 (h7D12 hIgG1). The antibody targeting EGFR may bind to an epitope that shares about 80% sequence identity with the epitope bound by cetuximab or Mav2 (h7D12 hIgG1). The antibody targeting EGFR may bind to an epitope that shares about 85% sequence identity with the epitope bound by cetuximab or Mav2 (h7D12 hIgG1). The antibody targeting EGFR may bind to an epitope that comprises about 90% sequence identity to the epitope bound by cetuximab or Mav2 (h7D12 hIgG1). The antibody targeting EGFR may bind to an epitope that comprises about 95% sequence identity to the epitope bound by cetuximab or Mav2 (h7D12 hIgG1). The antibody targeting EGFR may bind to an epitope that comprises about 99% sequence identity to the epitope bound by cetuximab or Mav2 (h7D12 hIgG1).

[0275] The antibody targeting EGFR may bind to an epitope comprising an epitope different from the epitope bound by cetuximab or Mav2 (h7D12 hIgG1). The antibody targeting EGFR may bind to an epitope comprising an epitope different from the epitope bound by cetuximab or Mav2 (h7D12 hIgG1), which does not bind to any of the same amino acids on EGFR. The antibody targeting EGFR may bind to an epitope comprising an epitope different from the epitope bound by cetuximab or Mav2 (h7D12 hIgG1), which binds to any one or more of the same amino acids on EGFR. The antibody targeting EGFR may bind to an epitope comprising an epitope different from the epitope bound by cetuximab or Mav2 (h7D12 hIgG1), which binds to any two or more of the same amino acids on EGFR. The antibody targeting EGFR may bind to an epitope that includes an epitope that is different from the epitope bound by cetuximab or Mav2 (h7D12 hIgG1) and that binds to any three or more of the same amino acids on EGFR. The antibody targeting EGFR may bind to an epitope that includes an epitope that is different from the epitope bound by cetuximab or Mav2 (h7D12 hIgG1) and that binds to any four or more of the same amino acids on EGFR. The antibody targeting EGFR may bind to an epitope that includes an epitope that is different from the epitope bound by cetuximab or Mav2 (h7D12 hIgG1) and that binds to any five or more of the same amino acids on EGFR. Antibodies that target EGFR may bind to epitopes that include epitopes that are different from those bound by cetuximab or Mav2 (h7D12 hIgG1) and that bind to any six or more of the same amino acids on EGFR.The antibody targeting EGFR may bind to an epitope that includes an epitope that is different from the epitope bound by cetuximab or Mav2 (h7D12 hIgG1) and that binds to any seven or more of the same amino acids on EGFR. The antibody targeting EGFR may bind to an epitope that includes an epitope that is different from the epitope bound by cetuximab or Mav2 (h7D12 hIgG1) and that binds to any eight or more of the same amino acids on EGFR. The antibody targeting EGFR may bind to an epitope that includes an epitope that is different from the epitope bound by cetuximab or Mav2 (h7D12 hIgG1) and that binds to any nine or more of the same amino acids on EGFR. Antibodies that target EGFR may bind to epitopes that include epitopes that are different from those bound by cetuximab or Mav2 (h7D12 hIgG1) and that bind to any 10 or more of the same amino acids on EGFR.

[0276] In some cases, the antibody targeting EGFR may bind to the same epitope as any one of the antibodies listed in Table 3. The antibody targeting EGFR may bind to an epitope that comprises about 70% sequence identity to the epitope bound by any one of the antibodies listed in Table 3. The antibody targeting EGFR may bind to an epitope that comprises about 75% sequence identity to the epitope bound by any one of the antibodies listed in Table 3. The antibody targeting EGFR may bind to an epitope that comprises about 80% sequence identity to the epitope bound by any one of the antibodies listed in Table 3. The antibody targeting EGFR may bind to an epitope that comprises about 85% sequence identity to the epitope bound by any one of the antibodies listed in Table 3. The antibody targeting EGFR may bind to an epitope that comprises about 90% sequence identity to the epitope bound by any one of the antibodies listed in Table 3. The antibody that targets EGFR may bind to an epitope that comprises about 95% sequence identity to the epitope bound by any one of the antibodies listed in Table 3. The antibody that targets EGFR may bind to an epitope that comprises about 99% sequence identity to the epitope bound by any one of the antibodies listed in Table 3.

[0277] In some embodiments, an antibody targeting an internalizing receptor protein may bind to the same epitope as any one of the antibodies listed in Table 3 that binds with similar affinity as any one of the antibodies listed in Table 3. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 70% sequence identity to an epitope to which any one of the antibodies listed in Table 3 binds with similar affinity as any one of the antibodies listed in Table 3. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 75% sequence identity to an epitope to which any one of the antibodies listed in Table 3 binds with similar affinity as any one of the antibodies listed in Table 3. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 80% sequence identity to an epitope to which any one of the antibodies listed in Table 3 binds with similar affinity as any one of the antibodies listed in Table 3. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 85% sequence identity to an epitope to which any one of the antibodies listed in Table 3 binds with similar affinity as any one of the antibodies listed in Table 3. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 90% sequence identity to an epitope to which any one of the antibodies listed in Table 3 binds with similar affinity as any one of the antibodies listed in Table 3. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 95% sequence identity to an epitope to which any one of the antibodies listed in Table 3 binds with similar affinity as any one of the antibodies listed in Table 3. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 99% sequence identity to an epitope to which any one of the antibodies listed in Table 3 binds with similar affinity as any one of the antibodies listed in Table 3.

[0278] In some embodiments, an antibody targeting an internalizing receptor protein may bind to the same epitope as any one of the antibodies listed in Table 3, but with a different affinity compared to any one of the antibodies listed in Table 3. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 70% sequence identity to an epitope to which any one of the antibodies listed in Table 3 binds with a different affinity compared to any one of the antibodies listed in Table 3. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 75% sequence identity to an epitope to which any one of the antibodies listed in Table 3 binds with a different affinity compared to any one of the antibodies listed in Table 3. An antibody targeting an internalizing receptor protein may bind to an epitope that comprises about 80% sequence identity to an epitope to which any one of the antibodies listed in Table 3 binds with a different affinity compared to any one of the antibodies listed in Table 3. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 85% sequence identity to an epitope to which any one of the antibodies listed in Table 3 binds with a different affinity compared to any one of the antibodies listed in Table 3. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 90% sequence identity to an epitope to which any one of the antibodies listed in Table 3 binds with a different affinity compared to any one of the antibodies listed in Table 3. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 95% sequence identity to an epitope to which any one of the antibodies listed in Table 3 binds with a different affinity compared to any one of the antibodies listed in Table 3. The antibody targeting the internalizing receptor protein may bind to an epitope that comprises about 99% sequence identity to an epitope to which any one of the antibodies listed in Table 3 binds with a different affinity compared to any one of the antibodies listed in Table 3.

[0279] In some embodiments, an antibody targeting an internalizing receptor protein may bind with an affinity similar to any one of the antibodies listed in Table 3 (Table 5 lists the affinities of certain monovalent binders). Table 5 lists the monovalent Kd for certain internalizing receptor monovalent proteins. In certain embodiments, the multispecific binding agent has a Kd that is less than, greater than, within 10%, 20%, 30%, 40%, 50%, 75%, or 100% of the binding affinity of the monovalent binding agent. For example, Table 5 lists the monovalent binding affinities for certain CD71 monovalent binding agents. When these CD71-binding arms are incorporated into the monovalent binding agents of the present disclosure, the binding affinity of the multispecific binding agent may be within an order of magnitude, or more than twofold to twofold less than the binding affinity of the monovalent binding agent. For example, the binding affinity of the monovalent binding agent has a Kd of 0.1 nM to 100 nM. When incorporated into a multispecific binding agent, the Kd may be within the same range. Alternatively, the binding affinity may be slightly higher than the monovalent binding affinity, but not less than two-fold higher. The binding affinity may be not less than three-fold higher than the monovalent binding affinity.

[0280] An antibody that targets EGFR may bind to an epitope that includes an epitope different from the epitope bound by any one of the antibodies listed in Table 3. An antibody that targets EGFR may bind to an epitope that includes an epitope different from the epitope bound by any one of the antibodies listed in Table 3, and that does not bind to any of the same amino acids on EGFR. An antibody that targets EGFR may bind to an epitope that includes an epitope different from the epitope bound by any one of the antibodies listed in Table 3, and that binds to any one or more of the same amino acids on EGFR. An antibody that targets EGFR may bind to an epitope that includes an epitope different from the epitope bound by any one of the antibodies listed in Table 3, and that binds to any two or more of the same amino acids on EGFR. An antibody targeting EGFR may bind to an epitope that includes an epitope that is different from the epitope bound by any one of the antibodies listed in Table 3, and that binds to any three or more of the same amino acids on EGFR. An antibody targeting EGFR may bind to an epitope that includes an epitope that is different from the epitope bound by any one of the antibodies listed in Table 3, and that binds to any four or more of the same amino acids on EGFR. An antibody targeting EGFR may bind to an epitope that includes an epitope that is different from the epitope bound by any one of the antibodies listed in Table 3, and that binds to any five or more of the same amino acids on EGFR. An antibody targeting EGFR may bind to an epitope that includes an epitope that is different from the epitope bound by any one of the antibodies listed in Table 3, and that binds to any six or more of the same amino acids on EGFR. An antibody that targets EGFR may bind to an epitope that includes an epitope that is different from the epitope bound by any one of the antibodies listed in Table 3, and that binds to any seven or more of the same amino acids on EGFR.An antibody that targets EGFR may bind to an epitope that includes an epitope that is different from the epitope bound by any one of the antibodies listed in Table 3, and that binds to any eight or more of the same amino acids on EGFR. An antibody that targets EGFR may bind to an epitope that includes an epitope that is different from the epitope bound by any one of the antibodies listed in Table 3, and that binds to any nine or more of the same amino acids on EGFR. An antibody that targets EGFR may bind to an epitope that includes an epitope that is different from the epitope bound by any one of the antibodies listed in Table 3, and that binds to any ten or more of the same amino acids on EGFR. Table 3. Exemplary antibody sequences targeting EGFR. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0281] The sequences listed in Table 3 (SEQ ID NOs: 634-655; 724-725) are amino acid molecules. The sequences listed in Table 3 (SEQ ID NOs: 634-655; 724-725) are amino acid molecules that are synthetic constructs. The sequences listed in Table 3 for HC sequences (heavy chain), VH sequences (variable heavy chain sequences), LC sequences (light chain), and VL sequences (variable light chain sequences) (SEQ ID NOs: 634-655; 724-725) are amino acid molecules that are synthetic constructs. synthesis

[0282] Multispecific binding agents are synthesized using recombinant DNA and protein expression techniques. For example, suitable DNA sequences encoding heavy and light chain constant domains are widely available for synthesizing DNA encoding the dual IgG of the present disclosure. Sequences encoding selected variable domains are inserted by standard methods, and the resulting nucleic acids encoding full-length heavy and light chains are transformed into suitable host cells and expressed. Alternatively, nucleic acids can be expressed in cell-free expression systems, which can provide more control over oxidation and reduction conditions, pH, folding, glycosylation, etc.

[0283] The binding activity of engineered antibodies of the present disclosure can be assayed by any suitable method known in the art. For example, the binding activity of engineered antibodies of the present disclosure can be determined, for example, by Scatchard analysis (Munsen et al., Analyt Biochem (1980) 107:220-39). Specific binding can be assessed using techniques known in the art, including, but not limited to, competitive ELISA, BIACORE® assay, and / or KINEXA® assay. Antibodies that preferentially or specifically bind (used interchangeably herein) to a target antigen or target epitope are terms well understood in the art, and methods for determining such specific or preferential binding are also known in the art. An antibody is said to exhibit specific or preferential binding if it reacts or associates with a particular antigen or epitope more frequently, rapidly, with a longer duration, and / or with greater affinity than it reacts or associates with alternative antigens or epitopes. An antibody specifically or preferentially binds to a target if it binds with greater affinity, avidity, ease, and / or duration than it binds to other substances. Also, an antibody specifically or preferentially binds to a target in a sample if it binds to that target with greater affinity, avidity, ease, and / or duration than it binds to other substances present in the sample. For example, an antibody that specifically or preferentially binds to a HER2 epitope is an antibody that binds to this epitope with greater affinity, avidity, ease, and / or duration than it binds to other HER2 epitopes or non-HER2 epitopes. It is also understood by reading this definition that, for example, an antibody that specifically or preferentially binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. Thus, specific binding and preferential binding do not necessarily require (but may include) exclusive binding. nucleic acid molecule

[0284] In one aspect, some embodiments disclosed herein relate to nucleic acid molecules comprising nucleotide sequences encoding the multispecific binding agents of the present disclosure, including expression cassettes and expression vectors containing these nucleic acid molecules operably linked to heterologous nucleic acid sequences, such as, for example, regulatory sequences that direct the in vivo expression of the protein in a host cell.

[0285] Also provided herein are vectors, plasmids, or viruses that contain one or more nucleic acid molecules that encode any of the binding agents disclosed herein.Nucleic acid molecules can be contained in vectors, for example, that can direct their expression in cells that the vector transforms / transduces.Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art, commercially available, or can be easily prepared by those skilled in the art. See, for example, Sambrook, J., & Russell, DW (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russell, DW (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (collectively referred to herein as "Sambrook"); Ausubel, FM (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (with addendums up to 2014); Bollag, DM et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, MG et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997).The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, KB, Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, EA (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, SL et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (with addenda through 2014); and Makrides, SC (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences BV, the disclosures of which are incorporated herein by reference. Method for binding to target cancer cells

[0286] In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds to a membrane-bound internalization or degradation protein and a second binding domain that specifically binds to EGFR contacts target cancer cells. In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds to a membrane-bound internalization or degradation protein and a second binding domain that specifically binds to EGFR contacts bladder cancer cells. In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds to a membrane-bound internalization or degradation protein and a second binding domain that specifically binds to EGFR contacts colon cancer cells. In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds to a membrane-bound internalization or degradation protein and a second binding domain that specifically binds to EGFR contacts rectal cancer cells. In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds to a membrane-bound internalization or degradation protein and a second binding domain that specifically binds to EGFR contacts lymphoma cancer cells. In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds to a membrane-bound internalization or degradation protein and a second binding domain that specifically binds to EGFR is contacted with lung cancer cells. In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds to a membrane-bound internalization or degradation protein and a second binding domain that specifically binds to EGFR is contacted with non-small cell lung cancer cells. In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds to a membrane-bound internalization or degradation protein and a second binding domain that specifically binds to EGFR is contacted with head and neck cancer cells.

[0287] In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds a membrane-bound internalization or degradation protein and a second binding domain that specifically binds EGFR contacts a target cancer cell and reduces EGFR expression on the cancer cell by at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%. In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds a membrane-bound internalization or degradation protein and a second binding domain that specifically binds EGFR contacts a target cancer cell and reduces EGFR expression on the cancer cell by about 40% to 80%, about 50% to 80%, about 60% to 80%, about 70% to 80%, about 40% to 70%, about 50% to 70%, about 60% to 70%, about 40% to 60%, or about 50% to 60%. In some embodiments, the expression of EGFR on the target cells is determined relative to the expression of EGFR on control cancer cells that have not been contacted with the binding agent.

[0288] In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds a membrane-bound internalization or degradation protein and a second binding domain that specifically binds EGFR contacts a target cancer cell and increases surface elimination of EGFR on the target cancer cell by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%. In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds a membrane-bound internalization or degradation protein and a second binding domain that specifically binds EGFR on a target cancer cell increases cell surface elimination of EGFR by about 20-90%, about 30-90%, about 40-90%, about 50-90%, about 60-90%, about 70-90%, about 80-90%, about 20-80%, about 30-80%, about 40-80%, about 50-80%, about 60-80%, about 70-80%, about 20-70%, about 30-70%, about 40-70%, about 50-70%, about 60-70%, about 20-60%, about 30-60%, about 40-60%, about 50-60%, about 20-50%, about 30-50%, about 40-50%, about 20-40%, about 30-40%, or about 20-30% increase. In some embodiments, cell surface ablation of EGFR on target cells is determined compared to cell surface ablation of EGFR on control cancer cells not contacted with the binding agent. In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds to a membrane-bound internalization or degradation protein and a second binding domain that specifically binds to EGFR contacts a target cancer cell and increases the internalization of EGFR on the target cancer cell by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds to a membrane-bound internalization or degradation protein on a target cancer cell and a second binding domain that specifically binds to EGFR inhibits EGFR internalization by about 20-90%, about 30-90%, about 40-90%, about 50-90%, about 60-90%, about 70-90%, about 80-90%, about 20-80%, about In some embodiments, the internalization of EGFR on target cells is determined relative to the internalization of EGFR on control cancer cells not contacted with the binding agent.

[0289] In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds a membrane-bound internalization or degradation protein and a second binding domain that specifically binds EGFR contacts a target cancer cell and increases EGFR degradation on the target cancer cell by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%. In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds a membrane-bound internalization or degradation protein and a second binding domain that specifically binds EGFR on a target cancer cell increases EGFR degradation by about 20-90%, about 30-90%, about 40-90%, about 50-90%, about 60-90%, about 70-90%, about 80-90%, about 20-8 ... In some embodiments, the degradation of EGFR on target cells is determined relative to the degradation of EGFR on control cancer cells not contacted with the binding agent.

[0290] In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds a membrane-bound internalization or degradation protein and a second binding domain that specifically binds EGFR contacts a target cancer cell and increases the sensitivity of the cancer cell to a cancer therapeutic agent. In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds a membrane-bound internalization or degradation protein and a second binding domain that specifically binds EGFR contacts a target cancer cell and increases the sensitivity of the cancer cell to a cytotoxic agent. In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds a membrane-bound internalization or degradation protein and a second binding domain that specifically binds EGFR contacts a target cancer cell and reduces the proliferation of the target cancer cell. In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds a membrane-bound internalization or degradation protein and a second binding domain that specifically binds EGFR contacts a target cancer cell and increases the killing of the cancer cell.

[0291] In some embodiments, a multispecific binding agent comprising a first binding domain that specifically binds to a membrane-bound internalizing or degradation protein and a second binding domain that specifically binds to EGFR is contacted with a target cancer cell in vivo. Pharmaceutical Composition

[0292] In some embodiments, the multispecific binding agents, nucleic acids, and recombinant cells of the present disclosure can be incorporated into compositions, including pharmaceutical compositions. Such compositions typically include the multispecific binding agent and a pharmaceutically acceptable excipient, such as a carrier. The multispecific binding agents of the present disclosure can be administered using formulations used to administer antibodies and antibody-based therapeutics, or formulations based thereon.

[0293] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Administration of multispecific binding agents

[0294] Administration of any one or more of the therapeutic compositions, eg, multispecific binding agents and pharmaceutical compositions, described herein can be used to treat an individual with a neoplastic disease, such as cancer.

[0295] Thus, in one aspect, provided herein is a method for inhibiting the activity of target cells in an individual, comprising administering to the individual a first therapy comprising one or more of the multispecific binding agents and pharmaceutical compositions provided herein, wherein the first therapy inhibits the activity of the target cells by degrading the target surface protein. For example, the activity of the target cells can be inhibited when their proliferation is reduced, when their pathological or pathogenic behavior is reduced, when they are destroyed or killed, etc. Generally, the target cells of the disclosed methods can be any cancer cells.

[0296] In some embodiments, a method for treating cancer in a subject includes administering to the subject a binding agent, the binding agent comprising a first binding domain that specifically binds to a membrane-bound internalization or degradation protein expressed on a target cell, and a second binding domain that specifically binds to a target protein comprising EGFR.

[0297] In some embodiments, a method for treating cancer in a subject comprises administering to the subject a binding agent, wherein the binding agent comprises a first binding domain that specifically binds to a membrane-bound internalization or degradation protein expressed on a target cell and a second binding domain that specifically binds to a target protein comprising EGFR, wherein the method results in a reduction of EGFR expression on the target cell by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more.

[0298] In some embodiments, the method of treating cancer comprises reducing the expression of EGFR on target cells. In some embodiments, the method of treating cancer comprises reducing the expression of EGFR on target cells. In some embodiments, the method of treating cancer comprises administering a multispecific binding agent as an individual treatment. In some embodiments, the method of treating cancer comprises administering a multispecific binding agent as a combination treatment. In some embodiments, the combination treatment comprises administering the multispecific binding agent before, after, or simultaneously with an additional treatment. In some embodiments, the additional treatment comprises a standard of care treatment. In some embodiments, non-limiting examples of standard of care treatment include cytotoxic agents, immunotherapy, radiation, chemotherapy, surgery, hormonal therapy, or a combination thereof.

[0299] In some embodiments, the methods of treating cancer include treatment of breast cancer, B-cell lymphoma, pancreatic cancer, Hodgkin's lymphoma, ovarian cancer, prostate cancer, mesothelioma, lung cancer, non-Hodgkin's B-cell (B-NHL) lymphoma, melanoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, neuroblastoma, glioma, glioblastoma, bladder cancer, colorectal cancer, or head and neck cancer.

[0300] In some embodiments, a method for treating cancer in a subject comprises administering to the subject a binding agent, wherein the binding agent comprises a first binding domain that specifically binds to a membrane-bound internalization or degradation protein expressed on a target cell and a second binding domain that specifically binds to a target protein comprising EGFR, wherein the method results in a reduction in tumor volume of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, at least 100%, at least 125%, at least 150%, or more. In some embodiments, the tumor volume of a tumor contacted with the multispecific binding agent is determined compared to the tumor volume of a tumor not contacted with the multispecific binding agent. In some embodiments, the tumor volume of a tumor contacted with the multispecific binding agent is determined compared to the tumor volume of a tumor contacted with cetuximab.

[0301] In some embodiments, a method for treating cancer in a subject comprises administering to the subject a binding agent, wherein the binding agent comprises a first binding domain that specifically binds to a membrane-bound internalization or degradation protein expressed on a target cell and a second binding domain that specifically binds to a target protein comprising EGFR, wherein the method results in a tumor volume of a tumor contacted with the multispecific binding agent that is smaller than the tumor volume of a tumor not contacted with the multispecific binding agent. In some embodiments, a method for treating cancer in a subject comprises administering to the subject a binding agent, wherein the binding agent comprises a first binding domain that specifically binds to a membrane-bound internalization or degradation protein expressed on a target cell and a second binding domain that specifically binds to a target protein comprising EGFR, wherein the method results in a tumor volume of a tumor contacted with the multispecific binding agent that is smaller than the tumor volume of a tumor contacted with cetuximab.

[0302] In some embodiments, the half-life of the multispecific binding agent is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110% or longer than the half-life of cetuximab. In some embodiments, the clearance rate of the multispecific binding agent is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110% or higher than the clearance rate of cetuximab.

[0303] In some embodiments, the bispecific binding agents disclosed herein can be compared with other bispecific binding agents. In some cases, the other bispecific binding agents may target membrane-bound proteins other than EGFR. In some cases, the other bispecific binding agents may target proteins other than EGFR. In some cases, the other bispecific binding agents may bind to the RSV F protein. In some embodiments, the binding domain configured to bind to the RSV F protein comprises the sequence listed in Table 4. Table 4. Exemplary binding agents targeting the RSV F protein [Table 4]

[0304] The sequences listed in Table 4 (SEQ ID NOs: 656-659) are amino acid molecules. The sequences listed in Table 4 (SEQ ID NOs: 656-659) are amino acid molecules that are synthetic constructs. The sequences listed in Table 4 (SEQ ID NOs: 656-659) for the HC sequence (heavy chain), VH sequence (variable heavy chain sequence), LC sequence (light chain), and VL sequence (variable light chain sequence) are amino acid molecules that are synthetic constructs. [Example]

[0305] The following examples are illustrative, but not limiting, of the scope of the compositions, devices, and methods disclosed herein. Cell line:

[0306] Cells are grown in complete growth medium and maintained at 37° C. and 5% CO 2 . Example 1 Expression of bispecific antibodies.

[0307] Bispecific binders are expressed and purified from mammalian cells (e.g., Expi293F, ExpiCHO-S) using transient transfection according to the manufacturer's protocol. At designated time points (e.g., 4–14 days), the medium is collected by centrifugation at 4,000 × g for 20 minutes. Tagged bispecific binders and knob half IgG are purified by Ni-NTA or Protein A affinity chromatography, buffer-exchanged into PBS containing 20% glycerol, concentrated, and snap-frozen for storage at -80°C. IgG and hole half IgG are purified by Protein A affinity chromatography and buffer-exchanged into PBS containing 20% glycerol. Knob and hole half IgGs are recombined under reducing conditions (e.g., 10 mM Tris pH 7.5, 100 mM NaCl, 20% 800 mM L-Arg pH 10 + 200-fold excess reduced glutathione) and then purified by Ni-NTA affinity chromatography, buffer exchanged into PBS containing 20% glycerol, concentrated, and snap frozen for storage at -80° C. Purity and integrity of all proteins are assessed by SDS-PAGE and SEC. Example 2 Generation of stable cell lines.

[0308] An epitope-tagged receptor (e.g., EGFR) at the N-terminus (e.g., alpha, HA, Myc, etc.) is cloned into the pLVX lentiviral vector. Lentivirus is produced by transfecting HEK293T cells with a standard packaging vector. Stable cell lines expressing the epitope-tagged receptor are selected with puromycin and expression is verified by flow cytometry using an anti-epitope tag primary antibody. Example 3 Decomposition experiment.

[0309] Cells (e.g., human cancer cell lines, primary human immune cells, or stable cell lines generated herein) are seeded (e.g., in 6-, 12-, 24-, 48-, 96-, or 348-well plates) and grown to approximately 70% confluence before treatment. The medium is aspirated, and the cells are treated with a bispecific binding agent (e.g., including any of the antibodies disclosed herein) or a control antibody in complete growth medium (concentration range: 0.001-1000 nM; time range: 0-7 days). After incubation at 37°C, the cells are washed with phosphate-buffered saline (PBS). Samples are then tested following Western blotting, intracellular Western blotting, or flow cytometry protocols to quantify target protein levels. Example 4 Quantification of EGFR levels by Western blotting.

[0310] Cells are lifted with Versene and collected by centrifugation at 300 × g for 5 min at 4 °C. Cell pellets are lysed in 1 × RIPA buffer containing cOmplete mini protease inhibitor cocktail (Sigma-Aldrich) for 30 min at 4 °C. Lysates are centrifuged at 2,000 (for 96-well plates) or 16,000 × g for 10 min at 4 °C. 4 × NuPAGE LDS sample buffer (Invitrogen) and 2-mercaptoethanol (BME) are added to the lysates and boiled for 10 min. Equal amounts of lysates are loaded onto 4-12% Bis-Tris gels and run at 200 V for 37 min. Gels are incubated in 20% ethanol for 10 min and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes are blocked in PBS containing 0.1% Tween-20 + 5% bovine serum albumin (BSA) for 30 minutes at room temperature with gentle shaking. The membranes are incubated with the respective dilutions of primary antibodies in PBS + 0.2% Tween-20 + 5% BSA for 1 hour at room temperature with gentle shaking. The membranes are washed four times with Tris-buffered saline (TBS) + 0.1% Tween-20, then co-incubated with secondary antibodies in PBS + 0.2% Tween-20 + 5% BSA for 1 hour at room temperature. The membranes are washed four times with TBS + 0.1% Tween-20, then washed with PBS. The membranes are imaged using an Odyssey CLx Imager (LI-COR). Band intensity is quantified using Image Studio software (LI-COR). Example 5 Quantification of EGFR levels by intracellular Western blotting.

[0311] A fixative solution (e.g., 4% paraformaldehyde in PBS) is added to the cells and incubated for 20 minutes at room temperature without agitation. The fixative solution is then removed, and the cells are washed with PBS. A permeabilization solution (e.g., 0.1% Triton®-X100 in PBS) is added to the cells and incubated for 20 minutes with shaking. The permeabilization solution is removed, and the cells are incubated in blocking buffer for 1 hour at room temperature with shaking. The blocking buffer is removed, and the cells are incubated with primary antibody for 2 hours with shaking. The cells are washed four times with TBS + 0.1% Tween®-20. The cells are then incubated with secondary antibody for 1 hour with shaking. The cells are then washed four times with TBS + 0.1% Tween®-20. The wash solution is removed, and the plate is imaged using an Odyssey CLx Imager (LI-COR). Well intensities are quantified using Empiria Studio software (LI-COR). Example 6 Quantification of EGFR levels by flow cytometry.

[0312] Cells are lifted with Versene and collected by centrifugation at 300 x g for 5 minutes at 4°C. The cell pellet is washed with cold PBS and centrifuged at 300 x g for 5 minutes. Cells are blocked with cold PBS + 3% BSA and centrifuged (300 x g for 5 minutes). Cells are incubated with primary antibody diluted in PBS + 3% BSA for 30 minutes at 4°C. Cells are washed three times with cold PBS + 3% BSA and secondary antibody (if applicable) diluted in PBS + 3% BSA is added and incubated for 30 minutes at 4°C. Cells are washed three times with cold PBS + 3% BSA and resuspended in cold PBS. Flow cytometry is performed on a CytoFLEX cytometer (Beckman Coulter) and gating is performed on single and live cells before acquisition of 10,000 cells. Analysis is performed using the FlowJo software package. Example 7 Cell surface removal of EGFR using a bispecific binding agent that binds to EGFR and a degrader protein.

[0313] To determine EGFR cell surface depletion by bispecific binders that bind to EGFR and degrader proteins, we performed cell surface depletion assays using an EGFR x CD71 bispecific antibody (an antibody that binds to EGFR and CD71; Figures 2A-2D). All EGFR-targeting bispecific binders contained Mav2 as the EGFR-binding domain. The CD71-binding domain was varied to determine its distinctive effect on EGFR degradation, such as the CD71 epitope or the antibody's binding affinity for CD71. The CD71 binding domains tested include EPI511-1 (ABBV2029(EPKSC)), EPI1015-1 (ABBV2029(GGGGS)), EPI867-1 (AF-20D), EPI873-1 (h15G11v5), EPI874-1 (h15G11v5-52A), EPI875-1 (h15G11v5-53A), EPI876-1 (h15G11v5-53A / 92A), EPI1094-1 (h15G11v5-92A), and EPI1095-1 (h15G11v5-52A / 92A) (Table 5). As described in Singh et al., Mol Cancer Ther (2022) 21 (8): 1326-1336 (EPI511 and EPI1015) and US20210087288A1 (EPI873, EPI874, and EPI1094), the binding affinities of the monovalent binding domains for CD71 ranged from about 1 nM to about 1800 nM. Table 5. Information on tested EGFRxCD71 bispecific constructs [Table 5]

[0314] Additionally, cetuximab (RG001-3) was tested as an EGFR-only control. An IgG1 isotype control against EGFR (RG196-1) was used as a non-targeting control. Palivizumab / Mav2 (RSV x EGFR bispecific) (EPI733-1) was tested as a single-arm Mav2 control, with a second arm that did not bind to target cells. Palivizumab / Mav2 served as a baseline for comparison when determining the efficacy of other tested constructs. Table 6. EGFR molecular information [Table 6-1] [Table 6-2]

[0315] Various constructs were tested at concentrations of 50 nM and 500 nM in the non-small cell lung cancer cell line NCIH1975 (Figures 2A and 2B) or the colorectal cancer cell line HT29 (Figures 2C and 2D). For these assays, NCIH1975 or HT29 cells were seeded into 96-well plates and incubated overnight at 37°C and 5% CO2. The following morning, cells were treated with 50 or 500 nM of antibody. Twenty-four hours after treatment, cells were harvested using a dissociation reagent, stained with a fluorescently labeled anti-EGFR antibody, and acquired on a Cytek Northern Lights flow cytometer. The percentage of EGFR cell surface removal was calculated using untreated control samples after background was established using an isotype control.

[0316] The EGFRxCD71 bispecific antibody induced cell surface removal of EGFR at 50 nM (Figure 2A) and 500 nM (Figure 2B) in NCIH1975 cells and at 50 nM (Figure 2C) and 500 nM (Figure 2D) in HT29 cells. The EGFRxCD71 bispecific binder induced higher levels of EGFR cell surface removal than the standard therapeutic molecules cetuximab and palivizumab across multiple cell lines (NCIH1975 and HT29) with a single-arm Mav2 antibody. This effect persisted across molecular attributes, including binding affinities ranging from approximately 1 to approximately 1800 nM and multiple epitopes. For example, the transferrin receptor non-blocking EPI876-1 (Mav2 x h15G11v5-53A / 92A) bispecific binder, with a Kd of approximately 600 nM, showed similar cell surface clearance as the EPI511-1 (Mav2 x ABBV2029) bispecific binder, with a Kd of approximately 1 nM, indicating that EpiTACs bearing degrader arms with different affinities and epitope binding can induce similar target degradation.

[0317] This demonstrates the ability to tailor the activity of bispecific antibodies by altering the epitope and / or affinity of the binding agent. Furthermore, this data demonstrates the effectiveness of bispecific binding agents that bind to both EGFR and degrader proteins to remove EGFR from the surface of target cells in multiple contexts. Example 8 Screening of EGFR cell surface ablation to identify effective degrader protein binding domains for EGFR-targeting bispecific antibodies.

[0318] To identify degrader protein binding domains on EGFR-targeting bispecific binders that result in enhanced EGFR cell surface clearance, we performed a screen using 72 bispecific binders (Figures 3A-3B). The 72 bispecific binders bound to 20 unique degrader proteins. For many degrader proteins, multiple binding domains binding to different epitopes were tested. Additionally, cetuximab (RG001-3), an IgG1 isotype control (RG196-1), and EGFRxRSV (EPI733-1) were tested as EGFR-only, negative, and single-arm Mav2 controls, respectively, with EGFRxRSV serving as a baseline for comparison. In this example, R001-3 corresponds to EPI431 (commercially available cetuximab (MedChemExpress)), and RG196 corresponds to EPI1102 (commercially available human IgG1 isotype control (Bio X Cell)). Positive controls for the assay included EGFRxcMet (EPI818, amivantimab) and EGFRxLGR5 (EPI1097), with an EGFRxCD71 molecule (EPI511-1) used as a control to track inter-assay variability. The bispecific binding agents used in this example contain binding arms comprising the sequences listed in Table 7. Table 7. Binding Arm 1 Targets and Sequences [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7]

Table 7-8

Table 7-9

Table 7-10

Table 7-11

Table 7-12

Table 7-13

Table 7-14

Table 7-15

Table 7-16

Table 7-17

Table 7-18

Table 7-19

Table 7-20

Table 7-21

Table 7-22

Table 7-23

Table 7-24

[0319] The bispecific binding agents used in this example comprise a second binding arm comprising a sequence listed in Table 8. The sequences listed in Table 8 were paired with each of the antibodies listed in Table 7. Table 8. Binding arm 2 targets and sequences [Table 8]

[0320] Screening was performed on both NCIH1975 (non-small cell lung cancer) and HT29 (colorectal cancer) cell lines using the method described in Example 8. Briefly, cells were seeded into 96-well plates and incubated overnight at 37°C and 5% CO2. The following morning, cells were treated with 500 nM of the bispecific binder or control antibody. 24 hours after treatment, cells were harvested using a dissociation reagent, stained with a fluorescently labeled anti-EGFR antibody, and acquired on a Cytek Northern Lights flow cytometer. The percentage of EGFR cell surface removal was calculated using untreated control samples after background was established using an isotype control.

[0321] Screening results identified a group of degrader proteins and specific molecular epitopes that, when paired with EGFR in a bispecific antibody format, showed improved ability to induce EGFR cell surface removal compared to palivizumab x Mav2, a bispecific binder pairing EGFR with a non-targeting control arm (Figures 3A and 3B). Several effective bispecific antibodies were generated that were unique to the cancer type of the cell line used. In NCIH1975 cells (non-small cell lung cancer), MUC1, ITGB6, and TROP2 were effective in removing EGFR from the cell surface (Figure 3A). In HT29 (colorectal cancer), CD276, RNF43, and MST1R were effective in removing EGFR from the cell surface (Figure 3B). Furthermore, EpCAM, CD71, LGR5, and HER3 were effective in removing EGFR from the cell surface across cell lines. These experiments demonstrate the cell specificity of EGFR degradation using various bispecific antibody pairs. Example 9 EGFR cell surface depletion of bispecific antibodies targeting non-Mav2 EGFR.

[0322] While most molecules included in the screening used Mav2 as the EGFR-binding arm of the bispecific antibody, binders using EgB4 as the EGFR-binding arm were also tested in the cell surface depletion assay (Figure 4). EgB4 is an EGFR-binding arm with the same binding affinity for EGFR as Mav2 (9.8 nM), but does not have EGF-blocking properties, indicating a difference in epitope compared to Mav2. Mav2 and EgB4 binders were delivered in a VHH (sdAb) format. Durigotuzumab (HER3xEGFR) or hu15G11v5 (CD71) binding domains were used for the degrader protein-binding arm. Additionally, RG001-3 (cetuximab), RG196-3 (IgG1 isotype control), EPI733-2 (Mav2 x RSV), and EPI484-1 (EgB4 x RSV) were tested for comparison (Table 9; Table 10). Table 9. EGFR molecular information [Table 9] Table 10. Sequence information for exemplary EgB4 bispecific binding agents [Table 10-1] [Table 10-2] [Table 10-3]

[0323] The sequences listed in Table 10 (SEQ ID NOs: 660 to 683) are amino acid molecules. The sequences listed in Table 10 (SEQ ID NOs: 660 to 683) are amino acid molecules that are synthetic constructs. The sequences listed in Table 10 for HC sequences (heavy chain), VH sequences (variable heavy chain sequences), LC sequences (light chain), and VL sequences (variable light chain sequences) (SEQ ID NOs: 660 to 683) are amino acid molecules that are synthetic constructs.

[0324] Screening was performed in NCIH1975 (non-small cell lung cancer) or HT29 (colorectal cancer) cells using the method described in Example 8. Briefly, cells were seeded into 96-well plates and incubated overnight at 37°C and 5% CO2. The following morning, cells were treated with 50 nM of therapeutic bispecific binding agent or control antibody. 24 hours after treatment, cells were harvested using a dissociation reagent, stained with a fluorescently labeled anti-EGFR antibody, and acquired on a Cytek Northern Lights flow cytometer. The percentage of EGFR cell surface removal was calculated using untreated control samples after background was established using an isotype control.

[0325] The bispecific binders with the EgB4-binding domain demonstrated similar levels of EGFR cell surface removal as the bispecific binders with the MAv2-binding domain (Figures 4A-4B). For durigotuzumab (HER3), the bispecific binders with the MAv2-binding domain (EPI1022-1) and the bispecific binders with the EgB4-binding domain (EPI1527-1) achieved 75.1% and 78.6%, respectively. For hu15G11v5 (CD71), the bispecific binders with the MAv2-binding domain (EPI873-3) and the bispecific binders with the EgB4-binding domain (EPI1556-1) achieved 76.4% and 69.2%, respectively. This demonstrates that cell surface EGFR removal can occur using different EGFR binder arms. Furthermore, EGFR-binding agents paired with degraded receptor-binding agents in a bispecific antibody format can induce robust cell surface removal of EGFR. Example 10 The bispecific binding agent exhibits synergistic internalization activity with both the EGFR binding domain and the degrader protein binding domain.

[0326] To demonstrate that the bispecific binding agent has synergistic internalization activity with both the EGFR-binding domain and the degrader protein-binding domain, the internalization activity of a bispecific antibody having both the EGFR-binding domain and the degrader protein-binding domain was compared with that of an antibody having an RSV-binding domain (palivizumab) and a degrader protein-binding domain (Figure 5). Antibodies binding to different target proteins were tested, including CD71 (bispecific antibody: EPI1015; single-arm antibody: EPI1177), MUC1 (EPI828; EPI1123), EpCAM (EPI847; EPI1149), and CD226 (EPI835; EPI1137). Additionally, cetuximab (RG001-3), an IgG1 isotype control (RG196-1), and Mav2 / palivizumab (EGFRxRSV; EPI733-1) were tested as an EGFR-only control, a negative control, and a single-arm Mav2 control, respectively, with EGFRxRSV serving as a baseline for comparison. For the internalization assay, H1975 cells were cultured in 96-well clear culture plates at 7 × 10 3 Cells were seeded at a density of 1000 cells / well. After approximately 16 hours of incubation, the test antibody was mixed with the antibody labeling reagent at a 1:3 molar ratio of test antibody to antibody labeling reagent at pH 7.0 for 15 minutes at 37°C. The labeled antibody was dispensed onto the cells. All internalization assays were performed at a single concentration. The plate was placed in an Incucyte® Live-Cell Analysis System, where images were acquired. Internalization images were sampled at 0 minutes and at 45-minute intervals for 72 hours. Image analysis was performed using Incucyte's Base Analysis software. The background was subtracted using the "Top-Hat" background subtraction method to obtain the percentage of "Red Object Intensity." An antibody with one domain that binds to the target and another domain that does not bind to proteins on the target cells was used as a baseline (single-arm target antibody).

[0327] The bispecific antibody, which has both a target-binding domain and a degrader protein-binding domain, resulted in a higher rate of internalization than an antibody that binds only to the internalization protein (Figure 5). Furthermore, the internalization rate of the bispecific antibody was higher than the combined internalization rate of the antibody that binds only to the degrader protein and the control antibody that binds only to the target protein. These observations indicate the synergistic internalization activity of antibodies that bind to both the target protein and the degrader protein on the target cell. Example 11 Screening EGFR internalization to identify effective degrader protein binding domains for EGFR-targeting bispecific antibodies:

[0328] To further screen and validate the effective degrader protein binding domains identified in the cell surface depletion screen, an EGFR internalization assay was performed using 52 bispecific binders (Figure 6A). The 52 bispecific antibodies bound to EGFR as the target protein and 15 unique degrader proteins. For previously identified degrader proteins, multiple binding domains binding to different epitopes were tested. Cetuximab, an IgG1 isotype control, and EGFR x RSV were tested for comparison. Screening was performed in NCIH1975 (non-small cell lung cancer) cells using the method described in Example 12. Screening results identified a group of degrader proteins and specific molecular epitopes that, when paired with EGFR in a bispecific antibody format, showed improved EGFR internalization compared to palivizumab / Mav2, a bispecific binder paired with EGFR as a non-targeting control arm. In this example, cetuximab corresponds to EPI431 (commercially available cetuximab (MedChemExpress)), the RSV negative control corresponds to EPI692, the EGFR / RSV single arm corresponds to EPI733, and CD71.EGFR corresponds to EPI259. Degrader proteins identified as effective include RNF43, MST1R, CD276, EpCAM, LGR5, ITGB6, TROP2, MUC1, and CD71. Four of these hits (CD276, MUC1, CD71, and EpCAM) showed synergistic internalization activity, and three of the four were also identified in the cell surface depletion assay. This demonstrates consistency between assays and indicates that EGFR bispecific antibodies can induce synergistic internalization via identifiable degrader protein binding domains. Example 12 Whole cell lysis screen to identify effective degrader protein binding domains for EGFR-targeting bispecific antibodies.

[0329] To measure target protein degradation, whole-cell degradation of EGFR was tested using the AlphaLISA assay and Western blot. In this example, 27 bispecific binders binding to 15 unique degrader proteins were screened using this assay (Figure 6B). For previously identified degrader proteins, multiple binding domains binding to different epitopes were tested. Cetuximab, an IgG1 isotype control, and EGFR x RSV were tested for comparison. For the AlphaLISA assay, NCIH1975 cells were seeded in 384-well plates in low-serum medium. After approximately 16 hours of culture, a single concentration of antibody was added to the cells in low-serum medium and treated for 48 hours. The medium was removed, and the cells were stimulated with EGF in serum-free medium. The medium was removed, and the cells were lysed. AlphaLISA acceptor beads and biotinylated antibodies were added to the lysate and incubated at room temperature for 1 hour. AlphaLISA donor beads were added to the lysate and incubated at room temperature for 2 hours. Plates were read on a Perkin Elmer Envision to determine total EGFR levels. Screening identified a set of degrader proteins and specific molecular epitopes that, when paired with EGFR in a bispecific antibody format, demonstrated improved whole-cell degradation of EGFR compared with palivizumab x Mav2, a bispecific binder paired with a non-targeting control arm. Degrader proteins identified as effective in inducing EGFR degradation included CD276, LGR5, ITGB6, CD71, MUC1, RNF43, HER3, and EpCAM. As a result of this screening, four bispecific binders (CD276, RNF43, MUC1, and ITGB6) were prioritized for further evaluation. Eight bispecific antibodies were further evaluated for whole-cell degradation using Western blot (Figures 7A-7B). Cetuximab, an IgG1 isotype control, and palivizumab x Mav2 (RSV and EGFR binding arms) were tested for comparison. For Western blots, NCIH1975 cells were seeded at a density of 4e5 cells in 6-well tissue culture plates.After approximately 16 hours of culture, a single concentration of antibody (50 nM) was added to cells in serum-starved medium and treated for 24–48 hours. The medium was removed and cells were stimulated with EGF in serum-free medium. The medium was then removed and lysed. The prepared samples were loaded onto a 4–12% BisTris gel and transferred to a PVDF membrane. The membrane was probed with EGFR or the housekeeping gene β-actin (Figure 7A). Data were quantified using Empiria Studio, and percent degradation was normalized to β-actin and compared to the PBS control (Figure 7B). Western blot analysis showed that four of the eight bispecific antibodies demonstrated improved whole-cell degradation of EGFR compared to EGFR×RSV, a bispecific binder that pairs EGFR with a non-targeting control arm. We also found that bispecific binders with a MUC1-binding domain resulted in greater EGFR degradation than cetuximab at various antibody concentrations (Figures 8A and 8B). Taken together, these results demonstrate that EGFR bispecific antibodies can cause increased EGFR degradation when conjugated to various degrader protein binding domains, in this example, isotypes corresponding to EPI1102, cetuximab to EPI431, EGFR / CD71 to EPI1015, and EGFR / MUC1 to EPI828. Example 13 EGFR degradation and cancer cell killing using an EGFRxRNF43 bispecific binder.

[0330] To determine whether bispecific antibodies could induce targeted EGFR proteolysis and tumor cell growth suppression through EGFR ubiquitination, we developed bispecific antibodies targeting EGFR and RNF43 (Figure 9B). The expression of RNF43 and EGFR mRNA in various cell lines was examined by RNA sequencing. The assays were performed at Board Institute (MA, USA), and the data were archived as part of the Cancer Cell Line Encyclopedia (Nature. 2019 May;569(7757):503-508). mRNA expression levels were analyzed using cBioPortal (Table 11). Table 11. Expression of RNF43 and EGFR in various cell lines [Table 11]

[0331] To identify RNF43-binding domains for targeting in these studies, HEK293T cells transiently expressing GFP-tagged RNF43 were used to identify RNF43-specific binders (Figure 9A). HEK293T cells were transfected with a construct expressing GFP-tagged RNF43. Expression levels were determined by microscopic detection of GFP expression. 24 hours after transfection, cells were harvested as a suspension using a cell scraper for flow cytometry analysis. After centrifugation at 1,200 rpm for 4 minutes, the supernatant was discarded. Cells were resuspended in antibody-containing FACS buffer (PBS, 0.5% BSA, 0.05% sodium azide) for 60 minutes on ice. Cells were then washed three times with FACS buffer before incubation with the secondary staining antibody (goat anti-human IgG(H+L)-647) for 45 minutes on ice. After staining, cells were washed four times in FACS buffer and stained with live / dead dye. Flow cytometry was performed on a CyTEK Aurora flow cytometer. SSC, FSC, and RNF43+ and GFP+ profiles were analyzed using Cytobank. Antibodies that specifically bound to RNF43-GFP+ cells but not to GFP-, untransfected, or empty-plasmid GFP+ cells were selected and reconstituted in a bispecific format with an anti-EGFR binder.

[0332] Next, cancer cell lines showing EGFR and RNF43 co-expression were selected, and membrane expression was verified before degradation studies. The cancer cell lines used were HPAFII, HT29, LS180, and LS513, which show RNF43 and EGFR expression. To verify membrane expression of RNF43 on cancer cell lines, cells were grown on coverslips in 24-well plates until attached and stained in 1% BSA containing an RNF43 binder and E-cadherin antibody for 1 hour on ice, followed by incubation with secondary antibodies for 45 minutes at room temperature. Cells were briefly washed with 1x PBS and fixed with 4% paraformaldehyde for 15 minutes. Stained cells were counterstained with ProLong Gold Antifade Mountant containing the blue DNA dye DAPI and mounted on microscope slides (Figure 9C).

[0333] To characterize the EGFR degradation function of the EGFRxRNF43 bispecific antibody on tumor cells, degradation assays were performed. For these assays, HT29 and LS180 cells (Figure 10A), and HPAFII and LS513 cells (Figure 10B) were grown in 2D culture conditions and treated with antibodies at a concentration of 200 nM for 48 hours, followed by Western blot analysis (Table 12). Table 12. EGFR molecular information [Table 12]

[0334] For Western blots, cells were washed twice in ice-cold phosphate-buffered saline (PBS), lysed in 1% Triton lysis buffer (25 μM Tris [pH 7.5], 150 μM NaCl, 1% Triton X-100, 1 μM EDTA, 1 μM EGTA, 20 μM NaF, 1 μM Na2VO4, and 1 μM DTT) supplemented with a protease inhibitor cocktail (Roche), and clarified by centrifugation. Protein concentrations were determined by the Bio-Rad protein assay (BioRad). Equal amounts of protein extracts were resolved by SDS-PAGE (NuPAGE; Invitrogen), and proteins were transferred to nitrocellulose or PVDF membranes. Membranes were immunoblotted with the indicated primary antibodies, incubated with secondary antibodies, and visualized using a LI-COR Odyssey scanner. The EGFR×RNF43 bispecific antibody induced targeted EGFR degradation in all four tumor cell lines compared with cetuximab, a single-arm EGFR binder, and a single-arm RNF43 binder (Figures 10A-10B). In some cases, an EGFR×CD71 bispecific binder was used as a positive control for comparison, as previously described, which also degraded EGFR to varying degrees in these cell lines. Phosphorylated EGFR was also degraded to varying degrees in these cells, confirming EGFR×RNF43 bispecific binder-mediated pharmacological inhibition of proximal signaling events upstream of EGFR pathway signaling. LS180 tumor cells, which showed robust EGFR×RNF43 bispecific binder-mediated EGFR degradation, were further investigated in a dose-response experiment (Figure 10C). The strongest effect on EGFR and p-EGFR degradation was observed at 200 nM.

[0335] To characterize the cytolytic function of the EGFR x RNF43 bispecific antibody on tumor cell spheroid formation and growth, the viability of 3D tumor spheroids was assessed after 2 weeks of incubation with the bispecific binder (Figures 10D-10G). Briefly, cells were harvested, counted, and seeded into Ultra-Low Attachment Culture 96-well plates. 200 cells were seeded into wells of a low-attachment 96-well plate in medium containing 10% Matrigel and the indicated dose of bispecific binder. After 2 weeks of incubation, an equal volume of CellTiter-Glo® 3D Cell Viability Assay Reagent (Promega) was added and mixed by repeated pipetting to improve sphere lysis. The plate was incubated at room temperature for 30 minutes with gentle shaking on a rocker. Luminescence was measured using a microplate reader. Visual inspection by microscopy (representative images are depicted in Figure 10D) and functional evaluation by 3D CellTiter Glo assay (Figures 10E-10G) demonstrated that the EGFR×RNF43 bispecific binder inhibited tumor spheroid formation and growth compared to single-arm anti-EGFR, single-arm anti-RNF43 mAb, and afitinib. Furthermore, the EGFR×RNF43 bispecific binder reduced spheroid formation and growth in LS180 and LS513 cells to a greater extent than cetuximab, while both had similar pharmacological effects on HPAFII cells. These data demonstrate that bispecific antibodies that bind to both target and degrader proteins result in target protein degradation and reduced viability of cancer cells expressing both the target and degrader proteins. Example 14 Bispecific binding agents that bind to target and degrader proteins inhibit tumor growth and induce targeted protein degradation in mice.

[0336] The EGFRxCD71 (EPI511) bispecific binder was used to determine whether the bispecific antibody could pharmacologically inhibit tumor growth in a mouse tumor model and induce targeted EGFR proteolysis in tumors when administered systemically. The NCIH1975 cell line was grown in tissue culture flasks containing RPMI1640 medium supplemented with 10% fetal bovine serum at 37°C in a 5% CO atmosphere. Cells were harvested during exponential growth phase and 5x10 6 Whole cells were inoculated into the right anterior flank of BALB / c nude mice. Inoculated mice were monitored daily, and tumor volume and body weight were measured twice a week according to IACUC guidelines. Tumors were grown to a size of 205 mm 3 The animals were allowed to grow until they reached an approximate volume of 1000 μg / cm², at which point they were randomized into groups (n = 6 mice per group) and received the indicated antibody intraperitoneally. Bispecific antibodies were prepared in-house; cetuximab was purchased from MedChemExpress (HY-P9905); isotype control mAb was purchased from BioXcell (BP0297). Dosing continued twice weekly for 2 weeks, and animals were monitored for up to 50 days after the first dose. A subset of animals was dosed on day 0 and sacrificed 72 hours after a single dose of mAb for frozen tumor collection. A schematic diagram of the study design is shown (Figure 11A) to measure tumor growth kinetics and pharmacodynamic degradation activity of bispecific mAbs compared to standard-of-care mAbs using the NCI-H1975 xenograft tumor model. Tumor volume was calculated as V = (L × W × W) / 2. Graphical and statistical analyses were performed in Graphpad Prism using conventional one-way ANOVA / Tukey's multiple comparison test. * p<0.05, ** p<0.01, *** p<0.001 (Figures 11B-11D).

[0337] For the EGFRxCD71 bispecific antibody, significant tumor growth inhibition was observed at the 1, 10, and 30 mg / kg dose levels within 10 days of treatment initiation (Figure 11B). Furthermore, the 10 and 30 mg / kg dose levels of the EGFRxCD71 (EPI511) bispecific binder resulted in lower tumor volumes than the corresponding doses of cetuximab (Figure 11C). The 10 mg / kg group was monitored after the last (fourth) dose of mAb, and prolonged tumor suppression was seen over a 50-day course for cetuximab and the EGFRxCD71 (EPI511) bispecific antibody (Figure 11D).

[0338] To assess EGFR degradation in tumors, separate cohorts of animals were treated with a single dose of 10 mg / kg EGFRxCD71 bispecific binder or control mAb (controls included an isotype control (EPI1102) and cetuximab (EPI431)). These animals were sacrificed at 72 h, tumors were collected, and intratumoral EGFR and p-EGFR expression was measured by Western blot to monitor targeted protein degradation (Figure 11E). Briefly, tumors were collected and immediately frozen in liquid nitrogen. Protein lysates were prepared by mechanical homogenization using a tissue homogenizer in RIPA lysis buffer. Protein quantity was measured using ThermoFisher's Pierce BCA Protein Assay Kit according to the manufacturer's instructions. Equal amounts of protein per animal were loaded onto NuPAGE, 4-12% Bio-Tris Midi Gels, and then transferred to PVDF membranes. After blocking the membrane in TBST + 5% milk, primary antibody was added, followed by three washes in TBST. Secondary antibody in TBST + 5% milk was added, washed three times in TBST, and then detected using an Odyssey Infrared Imager. Separate gels were run for each animal, measuring EGFR, p-EGFR, and GAPDH. Protein quantification was performed using ImageJ, and relative protein measurements were performed by dividing the signal intensity in the EGFR lane by the signal in the GAPDH lane of the paired sample (Figure 11F). A normalization factor equal to an arbitrary value of 1 was defined using the mean value of the isotype control animals. Each gel contained the same set of isotype control animals, collectively normalized to 1, to normalize values across all gels. Graphs and statistical analysis were performed in Graphpad Prism using a conventional one-way ANOVA / Tukey's multiple comparison test. * p<0.05, ** p<0.01, *** p<0.001.

[0339] Treatment with the EGFRxCD71 bispecific antibody resulted in lower relative amounts of total EGFR relative to GAPDH in tumor cells compared to the isotype control and single-arm EGFR-targeted antibodies, indicating higher induced intratumoral EGFR proteolysis in bispecific antibody-treated samples (Figures 11E and 11F). The EGFRxCD71 bispecific antibody also resulted in less relative phospho-EGFR relative to GAPDH when compared to the control (Figures 11G and 11H). These results demonstrate that bispecific antibodies that bind to target and degrader proteins on the surface of cancer cells inhibit tumor growth and induce EGFR degradation in vivo. Example 17 Pharmacokinetic evaluation of bispecific antibodies.

[0340] To determine the pharmacokinetic properties of bispecific antibodies that bind to target cancer-associated proteins and cancer-associated degrader proteins in tumor-free mice, immunocompromised mice were randomized into groups and treated intraperitoneally with a single 10 mg / kg dose of the EGFR×CD71 (EPI511) bispecific antibody or a control antibody (Figure 12A). Briefly, cohorts of 6-8 week-old BALB / c nude mice were randomized into groups (n = 8 / group) based on body weight and then injected intraperitoneally with a single 5 μL / g volume dose of the bispecific antibody or 10 mg / kg of cetuximab. The bispecific antibody was prepared in-house, and cetuximab was purchased from MedChemExpress (HY-P9905). The initial dose was noted as time point 0. Serum samples were collected from the saphenous vein of each animal's leg and frozen according to IACUC guidelines at the following time points: pre-dose, 0.25, 4, 24, 48, 96, 168, and 240 hours (Figures 12B and 12C). The pre-dose serum collection served as a reference control. Subgroups (n = 4 per group) were used to alternate blood collection from individual animals. According to IACUC guidelines, animals were monitored daily and weighed at multiple time points per week. The serum concentration (ng / mL) of each mAb was measured using a Human Therapeutic IgG1 ELISA Kit (Cayman, #500910) according to the manufacturer's instructions. Serum human IgG1 concentrations were calculated against a standard curve of a positive control sample. Pharmacokinetic analysis was performed using WinNonlin Phoenix software (Certara, version 8.2 or later). Graphs were generated in Graphpad Prism on a logarithmic or linear scale. The dotted line on the graph indicates 10,000 ng / mL for reference.

[0341] All treatment groups had measurable human IgG in serum at levels greater than 10 μg / mL at 0.25 hours post-dose, with maximum concentrations (Cmax) achieved 4 hours post-dose (Figure 12B). By 24 hours, serum concentrations began to decline but remained above 10 μg / mL throughout the 10-day study. Serum concentrations from individual animals were closely tracked within and between groups, with the greatest variation at 0.25 hours (distribution) and 240 hours (Figure 12C). Projected pharmacokinetic profiles showed that the EGFRxCD71 bispecific antibody, EGFR single-arm control, and cetuximab had similar estimated half-lives (102.64, 122.71, and 162.73 hours, respectively) and clearance rates (0.62, 0.48, and 0.42 mL / h / kg, respectively; Table 13). Table 13. Pharmacokinetic properties of treatments in mice. [Table 13]

[0342] These data demonstrate that bispecific antibodies that bind to a target protein and a degrader protein have pharmacokinetics similar to standard of care antibodies. Further embodiments Embodiment 1: A method of degrading an EGFR protein on a target cell, comprising contacting an EGFR protein and a membrane-bound internalizing protein on the target cell with a bispecific binding agent, wherein contacting the EGFR protein and the membrane-bound internalizing protein with the bispecific binding agent results in internalization and degradation of the EGFR protein, and wherein the bispecific binding agent comprises (a) a first binding domain that specifically binds to an extracellular epitope of the membrane-bound internalizing protein; and (b) a second binding domain that specifically binds to an extracellular epitope on the EGFR protein; The method, wherein the membrane-bound internalizing protein is selected from CEACAM5, CEACAM6, HER3, MUC1, CD205, CD166, PRLR, SLC34A2, ITGB6, LRRC15, MUC16, SLC39A6, AXL, CD40, CD228, MUC5A, ITGB1, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, I-AG7, ENPP3, CD46, CD56, ROR1, GPR20, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, GPR56, CD71, RNF43, RNF128, CD276, and CDH17. Embodiment 2: The method of embodiment 1, wherein the membrane-bound internalizing protein is selected from CD205, CD166, SLC34A2, ITGB6, LRRC15, MUC16, SLC39A6, AXL, CD40, CD228, MUC5A, ITGB1, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, I-AG7, ENPP3, CD46, CD56, ROR1, GPR20, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, GPR56, and CD71. Embodiment 3: The method of embodiment 1 or embodiment 2, wherein the membrane-bound internalization protein is CEACAM5. Embodiment 4: The method of embodiment 1 or embodiment 2, wherein the membrane-bound internalization protein is CEACAM6. Embodiment 5: The method of embodiment 1 or embodiment 2, wherein the membrane-bound internalizing protein is HER3. Embodiment 6: The method of embodiment 1 or embodiment 2, wherein the membrane-bound internalization protein is MUC1. Embodiment 7: The method of embodiment 1 or embodiment 2, wherein the membrane-bound internalization protein is CD205. Embodiment 8: The method of embodiment 1 or embodiment 2, wherein the membrane-bound internalization protein is CD166. Embodiment 9: The method of embodiment 1 or embodiment 2, wherein the membrane-bound internalization protein is PRLR. Embodiment 10: The method of embodiment 1 or embodiment 2, wherein the membrane-bound internalization protein is SLC34A2. Embodiment 11: The method of embodiment 1 or embodiment 2, wherein the membrane-bound internalization protein is ITGB6. Embodiment 12: The method of embodiment 1 or embodiment 2, wherein the membrane-bound internalization protein is LRRC15. Embodiment 13: The method of embodiment 1 or embodiment 2, wherein the membrane-bound internalization protein is MUC16. Embodiment 14: The method of any one of embodiments 1 to 13, wherein the bispecific binding agent comprises an antibody or portion thereof. Embodiment 15: The method of any one of embodiments 1 to 13, wherein the bispecific binding agent comprises a bispecific antibody or a portion thereof. Embodiment 16: The method of any one of embodiments 1 to 13, wherein the bispecific binding agent comprises a knob-and-hole bispecific IgG. Embodiment 17: The method of any one of embodiments 1 to 13, wherein the bispecific binding agent does not comprise an antibody-drug conjugate. Embodiment 18: A bispecific binding agent, comprising a bispecific antibody or antibody derivative, comprising: a) a first binding domain that specifically binds to an extracellular epitope of an EGFR protein on a target cell; and b) a second binding domain that specifically binds to an extracellular epitope of a membrane-bound internalized protein on the target cell, wherein the membrane-bound internalized protein is selected from the group consisting of CD205, CD166, SLC34A2, ITGB6, LRRC15, and MUC16. 1. A bispecific binding agent selected from SLC39A6, AXL, CD40, CD228, MUC5A, ITGB1, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, I-AG7, ENPP3, CD46, CD56, ROR1, GPR20, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, GPR56, CD71, RNF43, RNF128, CD276, and CDH17. Embodiment 19: The bispecific binding agent of embodiment 18, wherein the membrane-bound internalizing protein is CD205. Embodiment 20: The bispecific binding agent of embodiment 18, wherein the membrane-bound internalizing protein is CD166. Embodiment 21: The bispecific binding agent of embodiment 18, wherein the membrane-bound internalizing protein is SLC34A2. Embodiment 22: The bispecific binding agent of embodiment 18, wherein the membrane-bound internalization protein is ITGB6. Embodiment 23: The bispecific binding agent of embodiment 18, wherein the membrane-bound internalization protein is LRRC15. Embodiment 24: The bispecific binding agent of embodiment 18, wherein the membrane-bound internalizing protein is MUC16. Embodiment 25: The bispecific binding agent of any one of embodiments 18 to 24, wherein the bispecific binding agent comprises a knob-and-hole bispecific IgG. Embodiment 26: The bispecific binding agent of any one of embodiments 18 to 25, wherein the bispecific binding agent does not comprise an antibody-drug conjugate. Embodiment 27: A pharmaceutical composition comprising the bispecific binding agent of any one of claims 18 to 26 and a pharmaceutically acceptable excipient. Embodiment 28: A method of treating cancer in a subject in need thereof, comprising administering to the subject the bispecific binding agent of any one of embodiments 18 to 26 or the pharmaceutical composition of embodiment 27. Embodiment 29: A method of arresting the proliferation of a target cell, comprising contacting the cell with the bispecific binding agent of any one of embodiments 18 to 26 or the pharmaceutical composition of embodiment 27. Embodiment 30: The method of embodiment 29, wherein the cell is a cancer cell.

Claims

1. 1. A method for degrading a target protein on the surface of a target cell, comprising: contacting an endogenous internalization receptor on the surface of the target cell and the target protein with a binding agent, wherein the binding agent: (i) a first binding domain that specifically binds to an endogenous internalizing receptor selected from the group consisting of MUC1, ITGB6, CEACAM5, and CDH17; (ii) a second binding domain that specifically binds to the target protein, wherein the target protein comprises EGFR; A method comprising:

2. 2. The method of claim 1, wherein the binding agent is a multispecific antibody, a bispecific diabody, a bispecific Fab2, a bispecific camelid antibody, a bispecific peptibody scFv-Fc, a bispecific IgG, a knob-and-hole bispecific IgG, an Fc-Fab, or a knob-and-hole bispecific Fc-Fab.

3. 3. The method of claim 2, wherein the first binding domain comprises a first binding domain variable heavy chain and a first binding domain variable light chain.

4. The method of claim 3, wherein the endogenous internalization receptor is MUC1.

5. 5. The method of claim 4, wherein the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

71.

6. 6. The method of claim 5, wherein the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

71.

7. 7. The method of claim 6, wherein the first binding domain variable heavy chain comprises SEQ ID NO:

71.

8. 8. The method of any one of claims 4 to 7, wherein the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO:

73.

9. 9. The method of claim 8, wherein the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO:

73.

10. 10. The method of claim 9, wherein the first binding domain variable light chain comprises SEQ ID NO:

73.

11. 11. The method of any one of claims 4 to 10, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 71 and 73.

12. 12. The method of claim 11, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 71 and 73.

13. 11. The method of any one of claims 4 to 10, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that does not contain any amino acids from an epitope bound by an antibody comprising SEQ ID NOs: 71 and 73.

14. 4. The method of claim 3, wherein the endogenous internalization receptor is CDH17.

15. 15. The method of claim 14, wherein the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

47.

16. 16. The method of claim 15, wherein the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

47.

17. 17. The method of claim 16, wherein the first binding domain variable heavy chain comprises SEQ ID NO:

47.

18. 18. The method of any one of claims 14 to 17, wherein the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO:

49.

19. 19. The method of claim 18, wherein the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO:

49.

20. 20. The method of claim 19, wherein the first binding domain variable light chain comprises SEQ ID NO:

49.

21. 21. The method of any one of claims 14 to 20, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 47 and 49.

22. 22. The method of claim 21, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 47 and 49.

23. 21. The method of any one of claims 14 to 20, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that does not contain any amino acids from an epitope bound by an antibody comprising SEQ ID NOs: 47 and 49.

24. The method of claim 3, wherein the endogenous internalization receptor is ITGB6.

25. 25. The method of claim 24, wherein the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

287.

26. 26. The method of claim 25, wherein the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

287.

27. 27. The method of claim 26, wherein the first binding domain variable heavy chain comprises SEQ ID NO:

287.

28. 28. The method of any one of claims 24 to 27, wherein the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO:

289.

29. 29. The method of claim 28, wherein the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO:

289.

30. 30. The method of claim 29, wherein the first binding domain variable light chain comprises SEQ ID NO:

289.

31. 31. The method of any one of claims 24 to 30, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 287 and 289.

32. 32. The method of claim 31 , wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 287 and 289.

33. 31. The method of any one of claims 24 to 30, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that does not contain any amino acids from an epitope bound by an antibody comprising SEQ ID NOs: 287 and 289.

34. The method of claim 3, wherein the endogenous internalization receptor is CEACAM5.

35. 35. The method of claim 34, wherein the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

87.

36. 36. The method of claim 35, wherein the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

87.

37. 37. The method of claim 36, wherein the first binding domain variable heavy chain comprises SEQ ID NO:

87.

38. 38. The method of any one of claims 34 to 37, wherein the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO:

89.

39. 39. The method of claim 38, wherein the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO:

89.

40. 40. The method of claim 39, wherein the first binding domain variable light chain comprises SEQ ID NO:

89.

41. 41. The method of any one of claims 34 to 40, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 87 and 89.

42. 42. The method of claim 41 , wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 87 and 89.

43. 41. The method of any one of claims 34 to 40, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that does not contain any amino acids from an epitope bound by an antibody comprising SEQ ID NOs: 87 and 89.

44. 44. The method of any one of claims 1 to 43, wherein the second binding domain comprises a second binding domain variable heavy chain.

45. 45. The method of claim 44, wherein the second binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

651.

46. 46. The method of claim 45, wherein the second binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

651.

47. 47. The method of claim 46, wherein the second binding domain variable heavy chain comprises SEQ ID NO:

651.

48. 48. The method of any one of claims 3 to 47, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 80% sequence identity to an epitope to which cetuximab binds.

49. 49. The method of claim 48, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 90% sequence identity to an epitope to which cetuximab binds.

50. 48. The method of any one of claims 3 to 47, wherein the second binding domain binds to an epitope of the target protein on the target cell that does not include any amino acids from the epitope to which cetuximab binds.

51. 48. The method of any one of claims 3 to 47, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 80% sequence identity to the epitope to which Mav2 binds.

52. 52. The method of claim 51, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 90% sequence identity to an epitope to which Mav2 binds.

53. 48. The method of any one of claims 3 to 47, wherein the second binding domain binds to an epitope of the target protein on the target cell that does not contain any amino acids derived from the epitope to which Mav2 binds.

54. 54. The method of any one of claims 1 to 53, wherein after contact, EGFR is internalized into the target cell along with the endogenous internalizing receptor and EGFR is degraded.

55. 55. The method of any one of claims 1 to 54, wherein the endogenous internalizing receptor is recycled to the target cell surface after internalization of the binding agent.

56. 55. The method of any one of claims 1 to 54, wherein the endogenous internalizing receptor is degraded.

57. 57. The method of any one of claims 1 to 56, wherein the target cell is a cancer cell.

58. 58. The method of claim 57, wherein the cancer cells are selected from the group consisting of breast cancer cells, B-cell lymphoma cells, pancreatic cancer cells, Hodgkin's lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells, non-Hodgkin's B-cell lymphoma (B-NHL) cells, melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, glioma cells, glioblastoma cells, bladder cancer cells, colorectal cancer cells, and head and neck cancer cells.

59. 59. The method of claim 57 or 58, wherein expression of EGFR on the cancer cells is decreased after contact with the multispecific binding agent compared to control cancer cells not contacted with the binding agent.

60. 60. The method of claim 59, wherein expression of EGFR on the cancer cells is reduced by 50% or more compared to expression of EGFR on control cancer cells not contacted with the binding agent.

61. 59. The method of claim 57 or 58, wherein expression of EGFR on the cancer cells is reduced by 50% or more compared to expression of EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

62. 62. The method of any one of claims 57-61, wherein cell surface ablation of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells not contacted with the binding agent.

63. 62. The method of any one of claims 57-61, wherein cell surface ablation of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

64. 64. The method of any one of claims 57 to 63, wherein the internalization of EGFR in the cancer cells is at least 20% or more compared to the internalization of EGFR in control cancer cells not contacted with the binding agent.

65. 64. The method of any one of claims 57 to 63, wherein the internalization of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

66. 66. The method of any one of claims 57-65, wherein the degradation of EGFR in the cancer cells is at least 20% or more compared to the degradation of EGFR in control cancer cells not contacted with the binding agent.

67. 66. The method of any one of claims 57 to 65, wherein cytolysis of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

68. 68. The method of any one of claims 61, 63, 65, or 67, wherein the monospecific EGFR-binding agent is cetuximab.

69. 69. The method of any one of claims 57 to 68, wherein the method increases the sensitivity of the cancer cells to a cancer therapeutic agent.

70. 70. The method of claim 69, wherein the cancer therapeutic agent is a cytotoxic agent.

71. 71. The method of any one of claims 57 to 70, wherein the proliferation of the cancer cells is reduced.

72. 72. The method of any one of claims 57 to 71, wherein the method increases the killing of cancer cells.

73. 73. The method of any one of claims 1 to 72, wherein said contacting is performed in vivo.

74. 1. A method for treating cancer in a subject, comprising: administering to the subject a binding agent, wherein the binding agent: (i) a first binding domain that specifically binds to an endogenous internalizing receptor expressed on a target cell and selected from the group consisting of MUC1, ITGB6, CEACAM5, and CDH17; (ii) a second binding domain that specifically binds to a target protein, including EGFR; A method comprising:

75. 75. The method of claim 74, wherein the endogenous internalization receptor is MUC1.

76. 75. The method of claim 74, wherein the endogenous internalization receptor is ITGB6.

77. 75. The method of claim 74, wherein the endogenous internalization receptor is CEACAM5.

78. 75. The method of claim 74, wherein the endogenous internalizing receptor is CDH17.

79. 79. The method of any one of claims 74 to 78, wherein the cancer is breast cancer, B-cell lymphoma, pancreatic cancer, Hodgkin's lymphoma, ovarian cancer, prostate cancer, mesothelioma, lung cancer, non-Hodgkin's B-cell (B-NHL) lymphoma, melanoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, neuroblastoma, glioma, glioblastoma, bladder cancer, colorectal cancer, or head and neck cancer.

80. 80. The method of any one of claims 74 to 79, wherein the tumor volume of the tumor contacted with the multispecific binding agent is reduced by 20% or more compared to the tumor volume of a tumor not contacted with the bispecific binding agent.

81. 81. The method of any one of claims 74 to 80, wherein the tumor volume of the tumor contacted with the multispecific binding agent is at least 80% or less in volume compared to the tumor volume of a tumor not contacted with the bispecific binding agent.

82. 82. The method of any one of claims 74 to 81, wherein the expression of EGFR on the cancer cells is reduced by 20% compared to EGFR expression in cancer cells not contacted with the bispecific binding agent.

83. 82. The method of any one of claims 74 to 81, wherein the expression of EGFR on the cancer cells is reduced by 20% compared to EGFR expression in cancer cells contacted with the monospecific EGFR-binding agent.

84. 84. The method of claim 83, wherein the monospecific EGFR-binding agent is cetuximab.

85. (a) a first binding domain that specifically binds to an endogenous internalizing receptor selected from the group consisting of MUC1, ITGB6, CEACAM5, or CDH17; and (b) a second binding domain that specifically binds to a target protein that is EGFR; 10. A multispecific binding agent comprising:

86. 86. The multispecific binding agent of claim 85, wherein the multispecific binding agent is a multispecific antibody, a bispecific antibody, a bispecific diabody, a bispecific Fab2, a bispecific camelid antibody, a bispecific peptibody scFv-Fc, a bispecific IgG, a knob-and-hole bispecific IgG, an Fc-Fab, or a knob-and-hole bispecific Fc-Fab.

87. 87. The method of claim 86, wherein the first binding domain comprises a first binding domain variable heavy chain and a first binding domain variable light chain.

88. 88. The multispecific binding agent of claim 87, wherein the endogenous internalizing receptor is MUC1.

89. 89. The multispecific binding agent of claim 88, wherein the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

71.

90. 90. The multispecific binding agent of claim 89, wherein the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

71.

91. 91. The multispecific binding agent of claim 90, wherein the first binding domain variable heavy chain comprises SEQ ID NO:

71.

92. 92. The multispecific binding agent of any one of claims 88 to 91, wherein the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO:

73.

93. 93. The multispecific binding agent of claim 92, wherein the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO:

73.

94. 94. The multispecific binding agent of claim 93, wherein the first binding domain variable light chain comprises SEQ ID NO:

73.

95. 95. The multispecific binding agent of any one of claims 88 to 94, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 71 and 73.

96. 96. The multispecific binding agent of claim 95, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 71 and 73.

97. 95. The multispecific binding agent of any one of claims 88 to 94, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that does not contain any amino acids from the epitope bound by an antibody comprising SEQ ID NOs: 71 and 73.

98. 88. The multispecific binding agent of claim 87, wherein the endogenous internalizing receptor is ITGB6.

99. 99. The multispecific binding agent of claim 98, wherein the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

287.

100. 100. The multispecific binding agent of claim 99, wherein the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

287.

101. 101. The multispecific binding agent of claim 100, wherein the first binding domain variable heavy chain comprises SEQ ID NO:

287.

102. 102. The multispecific binding agent of any one of claims 98 to 101, wherein the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO:

289.

103. 103. The multispecific binding agent of claim 102, wherein the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO:

289.

104. 104. The multispecific binding agent of claim 103, wherein the first binding domain variable light chain comprises SEQ ID NO:

289.

105. 105. The multispecific binding agent of any one of claims 98 to 104, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 287 and 289.

106. The multispecific binding agent of claim 105, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 287 and 289.

107. 105. The multispecific binding agent of any one of claims 98 to 104, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that does not contain any amino acids from an epitope bound by an antibody comprising SEQ ID NOs: 287 and 289.

108. 88. The multispecific binding agent of claim 87, wherein the endogenous internalizing receptor is CEACAM5.

109. 109. The multispecific binding agent of claim 108, wherein the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

87.

110. 110. The multispecific binding agent of claim 109, wherein the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

87.

111. 111. The multispecific binding agent of claim 110, wherein the first binding domain variable heavy chain comprises SEQ ID NO:

87.

112. 112. The multispecific binding agent of any one of claims 108 to 111, wherein the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO:

89.

113. 113. The multispecific binding agent of claim 112, wherein the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO:

89.

114. 114. The multispecific binding agent of claim 113, wherein the first binding domain variable light chain comprises SEQ ID NO:

89.

115. 115. The multispecific binding agent of any one of claims 108 to 114, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 87 and 89.

116. The multispecific binding agent of claim 115, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 87 and 89.

117. 115. The multispecific binding agent of any one of claims 108 to 114, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that does not contain any amino acids from the epitope bound by an antibody comprising SEQ ID NOs: 87 and 89.

118. 88. The multispecific binding agent of claim 87, wherein the endogenous internalizing receptor is CDH17.

119. 119. The multispecific binding agent of claim 118, wherein the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

47.

120. 120. The multispecific binding agent of claim 119, wherein the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

47.

121. 121. The multispecific binding agent of claim 120, wherein the first binding domain variable heavy chain comprises SEQ ID NO:

47.

122. 122. The multispecific binding agent of any one of claims 118 to 121, wherein the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO:

49.

123. 123. The multispecific binding agent of claim 122, wherein the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO:

49.

124. 124. The multispecific binding agent of claim 123, wherein the first binding domain variable light chain comprises SEQ ID NO:

49.

125. 125. The multispecific binding agent of any one of claims 118 to 124, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 47 and 49.

126. The multispecific binding agent of claim 125, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 47 and 49.

127. 125. The multispecific binding agent of any one of claims 118 to 124, wherein the first binding domain binds to an epitope of the internalizing receptor on the target cell that does not contain any amino acids from the epitope bound by an antibody comprising SEQ ID NOs: 47 and 49.

128. 128. The multispecific binding agent of any one of claims 87 to 127, wherein the second binding domain comprises a second binding domain variable heavy chain.

129. 129. The multispecific binding agent of claim 128, wherein the second binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

651.

130. 130. The multispecific binding agent of claim 129, wherein the second binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

651.

131. 131. The multispecific binding agent of claim 130, wherein the second binding domain variable heavy chain comprises SEQ ID NO:

651.

132. 132. The multispecific binding agent of any one of claims 87-131, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 80% sequence identity to an epitope to which cetuximab binds.

133. 133. The multispecific binding agent of claim 132, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 90% sequence identity to an epitope to which cetuximab binds.

134. 132. The multispecific binding agent of any one of claims 87-131, wherein the second binding domain binds to an epitope of the target protein on the target cell that does not include any amino acids from the epitope to which cetuximab binds.

135. 132. The multispecific binding agent of any one of claims 87 to 131, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 80% sequence identity to an epitope to which Mav2 binds.

136. 136. The multispecific binding agent of claim 135, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 90% sequence identity to the epitope to which Mav2 binds.

137. 132. The multispecific binding agent of any one of claims 87 to 131, wherein the second binding domain binds to an epitope of the target protein on the target cell that does not contain any amino acids from the epitope to which Mav2 binds.

138. 138. The method of any one of claims 85-137, wherein the half-life of the multispecific binding agent is within 20% of the half-life of cetuximab.

139. 139. The method of any one of claims 85 to 138, wherein the clearance rate of the multispecific binding agent is within 20-95% of the clearance rate of cetuximab.

140. 140. The method of any one of claims 85-139, wherein the Kd of the multispecific binding agent is at least two-fold less than or equal to two-fold the binding affinity of cetuximab to EGFR.

141. 141. The method of claim 140, wherein the Kd of the multispecific binding agent is at least 5-fold less than or equal to 5-fold less than the binding affinity of cetuximab to EGFR.

142. 142. The method of claim 141, wherein the Kd of the multispecific binding agent is at least 10-fold less than or equal to 10-fold less than the binding affinity of cetuximab to EGFR.

143. 143. The method of any one of claims 85-142, wherein the Kd of the binding affinity of the multispecific binding agent may be within an order of magnitude of the binding affinity of the monovalent binding agent.

144. 144. The method of any one of claims 85-143, wherein the Kd of the multispecific binding agent is within ±10% of the binding affinity of cetuximab to EGFR.

145. 145. The method of claim 144, wherein the Kd of the multispecific binding agent is within ±20% of the binding affinity of cetuximab to EGFR.

146. 146. The method of claim 145, wherein the Kd of the multispecific binding agent is within ±30% of the binding affinity of cetuximab to EGFR.

147. 147. The method of any one of claims 85 to 146, wherein the Kd of the multispecific binding agent is less than the binding affinity of cetuximab to EGFR.

148. 147. The method of any one of claims 85-146, wherein the Kd of the multispecific binding agent is greater than the binding affinity of cetuximab to EGFR.

149. 1. A method for degrading a target protein on the surface of a target cell, comprising: contacting an endogenous internalization receptor on the surface of the target cell and the target protein with a binding agent, wherein the binding agent: (i) a first binding domain that specifically binds to an endogenous internalizing receptor, including B7-H3; (ii) a second binding domain that specifically binds to the target protein, wherein the target protein comprises EGFR; A method comprising:

150. 150. The method of claim 149, wherein the binding agent is a multispecific antibody, a bispecific diabody, a bispecific Fab2, a bispecific camelid antibody, a bispecific peptibody scFv-Fc, a bispecific IgG, a knob-and-hole bispecific IgG, an Fc-Fab, or a knob-and-hole bispecific Fc-Fab.

151. 151. The method of any one of claims 149 or 150, wherein the first binding domain binds to an epitope of the endogenous internalizing receptor on the target cell, the epitope comprising at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 99 and 101.

152. The method of claim 151, wherein the first binding domain binds to an epitope of the endogenous internalizing receptor on the target cell, the epitope comprising at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 99 and 101.

153. 151. The method of any one of claims 149 or 150, wherein the first binding domain binds to an epitope of the endogenous internalizing receptor on the target cell that does not contain any amino acids derived from the epitope bound by an antibody comprising SEQ ID NOs: 99 and 101.

154. 154. The method of any one of claims 149 to 153, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 80% sequence identity to an epitope to which cetuximab binds.

155. 155. The method of claim 154, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 90% sequence identity to an epitope to which cetuximab binds.

156. 154. The method of any one of claims 149 to 153, wherein the second binding domain binds to an epitope of the target protein on the target cell that does not include any amino acids from the epitope to which cetuximab binds.

157. 154. The method of any one of claims 149 to 153, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 80% sequence identity to the epitope to which Mav2 binds.

158. 158. The method of claim 157, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 90% sequence identity to the epitope to which Mav2 binds.

159. 154. The method of any one of claims 149 to 153, wherein the second binding domain binds to an epitope of the target protein on the target cell that does not contain any amino acids derived from the epitope to which Mav2 binds.

160. 160. The method of any one of claims 149 to 159, wherein the first binding domain comprises a first binding domain variable heavy chain and a first binding domain variable light chain.

161. 161. The method of claim 160, wherein the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

99.

162. 162. The method of claim 161, wherein the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

99.

163. 163. The method of claim 162, wherein the first binding domain variable heavy chain comprises SEQ ID NO:

99.

164. 164. The method of any one of claims 160 to 163, wherein the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO:

101.

165. 165. The method of claim 164, wherein the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO:

101.

166. 166. The method of claim 165, wherein the first binding domain variable light chain comprises SEQ ID NO:

101.

167. 167. The method of any one of claims 160 to 166, wherein the second binding domain comprises a second binding domain variable heavy chain.

168. 168. The method of claim 167, wherein the second binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

655.

169. 169. The method of claim 168, wherein the second binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

655.

170. 170. The method of claim 169, wherein the second binding domain variable heavy chain comprises SEQ ID NO:

655.

171. 171. The method of any one of claims 149 to 170, wherein the endogenous internalization receptor is recycled to the target cell surface after internalization of the binding agent.

172. 171. The method of any one of claims 149 to 170, wherein the endogenous internalizing receptor is degraded.

173. 173. The method of any one of claims 149 to 172, wherein the target cell is a cancer cell.

174. 174. The method of claim 173, wherein the cancer cells are selected from the group consisting of breast cancer cells, B-cell lymphoma cells, pancreatic cancer cells, Hodgkin's lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells, non-Hodgkin's B-cell lymphoma (B-NHL) cells, melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, glioma cells, glioblastoma cells, bladder cancer cells, colorectal cancer cells, and head and neck cancer cells.

175. 175. The method of claim 173 or 174, wherein expression of EGFR on the cancer cells is decreased after contact with the multispecific binding agent compared to control cancer cells not contacted with the binding agent.

176. 176. The method of claim 175, wherein expression of EGFR on the cancer cells is reduced by 50% or more compared to expression of EGFR on control cancer cells not contacted with the binding agent.

177. 175. The method of claim 173 or 174, wherein expression of EGFR on the cancer cells is reduced by 50% or more compared to expression of EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

178. 178. The method of any one of claims 173-177, wherein cell surface ablation of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells not contacted with the binding agent.

179. 178. The method of any one of claims 173-177, wherein cell surface ablation of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

180. The method of any one of claims 173 to 179, wherein the internalization of EGFR in the cancer cells is at least 20% or more compared to the internalization of EGFR in control cancer cells not contacted with the binding agent.

181. 179. The method of any one of claims 173-179, wherein the internalization of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

182. 182. The method of any one of claims 173 to 181, wherein the degradation of EGFR in the cancer cells is at least 20% or more compared to the degradation of EGFR in control cancer cells not contacted with the binding agent.

183. 182. The method of any one of claims 173-181, wherein degradation of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

184. 184. The method of any one of claims 177, 179, 181, or 183, wherein the monospecific EGFR-binding agent is cetuximab.

185. 183. The method of any one of claims 173 to 182, wherein the method increases the sensitivity of the cancer cells to a cancer therapeutic agent.

186. 186. The method of claim 185, wherein the cancer therapeutic agent is a cytotoxic agent.

187. 187. The method of any one of claims 173 to 186, wherein the proliferation of the cancer cells is reduced.

188. 188. The method of any one of claims 173 to 187, wherein the method increases the killing of cancer cells.

189. 189. The method of any one of claims 173 to 188, wherein said contacting is carried out in vivo.

190. 1. A method for treating cancer in a subject, comprising: administering to the subject a binding agent, wherein the binding agent: (i) a first binding domain that is expressed on a target cell and specifically binds to an endogenous internalizing receptor, which is B7-H3; (ii) a second binding domain that specifically binds to a target protein, including EGFR; A method comprising:

191. 191. The method of claim 190, wherein the cancer is breast cancer, B-cell lymphoma, pancreatic cancer, Hodgkin's lymphoma, ovarian cancer, prostate cancer, mesothelioma, lung cancer, non-Hodgkin's B-cell (B-NHL) lymphoma, melanoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, neuroblastoma, glioma, glioblastoma, bladder cancer, colorectal cancer, or head and neck cancer.

192. 192. The method of claim 190 or 191, wherein the tumor volume of a tumor contacted with the multispecific binding agent is reduced by 20% or more compared to the tumor volume of a tumor not contacted with the bispecific binding agent.

193. 193. The method of any one of claims 190 to 192, wherein the tumor volume of the tumor contacted with the multispecific binding agent is less than 80% or less compared to the tumor volume of a tumor not contacted with the bispecific binding agent.

194. 194. The method of any one of claims 190 to 193, wherein expression of EGFR on the cancer cells is reduced by 20% compared to EGFR expression in cancer cells not contacted with the bispecific binding agent.

195. 194. The method of any one of claims 190 to 193, wherein the expression of EGFR on the cancer cells is reduced by 20% compared to EGFR expression in cancer cells contacted with the monospecific EGFR-binding agent.

196. 196. The method of claim 195, wherein the monospecific EGFR binding agent is cetuximab.

197. (a) a first binding domain that specifically binds to an endogenous internalizing receptor that is B7-H3; and (b) a second binding domain that specifically binds to a target protein that is EGFR; 10. A multispecific binding agent comprising:

198. 200. The multispecific binding agent of claim 197, wherein the multispecific binding agent is a multispecific antibody, a bispecific antibody, a bispecific diabody, a bispecific Fab2, a bispecific camelid antibody, a bispecific peptibody scFv-Fc, a bispecific IgG, a knob-and-hole bispecific IgG, an Fc-Fab, or a knob-and-hole bispecific Fc-Fab.

199. The multispecific binding agent of claim 197 or 198, wherein the first binding domain binds to an epitope of the endogenous internalizing receptor on the target cell, the epitope comprising at least 80% sequence identity to the epitope bound by an antibody comprising SEQ ID NOs: 99 and 101.

200. The multispecific binding agent of claim 199, wherein the first binding domain binds to an epitope of the endogenous internalizing receptor on the target cell, the epitope comprising at least 90% sequence identity to the epitope bound by an antibody comprising SEQ ID NOs: 99 and 101.

201. The multispecific binding agent of claim 197 or 198, wherein the first binding domain binds to an epitope of the endogenous internalizing receptor on the target cell that does not contain any amino acids derived from the epitope bound by an antibody comprising SEQ ID NOs: 99 and 101.

202. 202. The multispecific binding agent of any one of claims 197-201, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 80% sequence identity to an epitope to which cetuximab binds.

203. 203. The multispecific binding agent of claim 202, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 90% sequence identity to an epitope to which cetuximab binds.

204. 202. The multispecific binding agent of any one of claims 197-201, wherein the second binding domain binds to an epitope of the target protein on the target cell that does not include any amino acids from the epitope to which cetuximab binds.

205. 202. The multispecific binding agent of any one of claims 197 to 201, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 80% sequence identity to the epitope to which Mav2 binds.

206. 206. The multispecific binding agent of claim 205, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 90% sequence identity to the epitope to which Mav2 binds.

207. 202. The multispecific binding agent of any one of claims 197 to 201, wherein the second binding domain binds to an epitope of the target protein on the target cell that does not contain any amino acids derived from the epitope to which Mav2 binds.

208. 208. The multispecific binding agent of any one of claims 198-207, wherein the first binding domain comprises a first binding domain variable heavy chain and a first binding domain variable light chain.

209. 209. The multispecific binding agent of claim 208, wherein said first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

99.

210. 210. The multispecific binding agent of claim 209, wherein the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

99.

211. 211. The multispecific binding agent of claim 210, wherein the first binding domain variable heavy chain comprises SEQ ID NO:

99.

212. 212. The multispecific binding agent of any one of claims 208 to 211, wherein the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO:

101.

213. 213. The multispecific binding agent of claim 212, wherein the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO:

101.

214. 214. The multispecific binding agent of claim 213, wherein the first binding domain variable light chain comprises SEQ ID NO:

101.

215. 215. The multispecific binding agent of any one of claims 208 to 214, wherein the second binding domain comprises a second binding domain variable heavy chain.

216. 216. The multispecific binding agent of claim 215, wherein the second binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

655.

217. 217. The multispecific binding agent of claim 216, wherein the second binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

655.

218. 218. The multispecific binding agent of claim 217, wherein the second binding domain variable heavy chain comprises SEQ ID NO:

655.

219. 219. The method of any one of claims 197-218, wherein the half-life of the multispecific binding agent is within 20% of the half-life of cetuximab.

220. 220. The method of any one of claims 197 to 219, wherein the clearance rate of the multispecific binding agent is within 20-95% of the clearance rate of cetuximab.

221. 221. The method of any one of claims 174 to 220, wherein the Kd of the multispecific binding agent is at least two-fold less than or equal to two-fold the binding affinity of cetuximab to EGFR.

222. 222. The method of claim 221, wherein the Kd of the multispecific binding agent is at least 5-fold less than or equal to 5-fold the binding affinity of cetuximab to EGFR.

223. 223. The method of claim 222, wherein the Kd of the multispecific binding agent is at least 10-fold less than or equal to 10-fold less than the binding affinity of cetuximab to EGFR.

224. 224. The method of any one of claims 174 to 223, wherein the Kd of the binding affinity of the multispecific binding agent may be within an order of magnitude of the binding affinity of the monovalent binding agent.

225. 225. The method of any one of claims 174 to 224, wherein the Kd of the multispecific binding agent is within ±10% of the binding affinity of cetuximab to EGFR.

226. 226. The method of claim 225, wherein the Kd of the multispecific binding agent is within ±20% of the binding affinity of cetuximab to EGFR.

227. 227. The method of claim 226, wherein the Kd of the multispecific binding agent is within ±30% of the binding affinity of cetuximab to EGFR.

228. 228. The method of any one of claims 174 to 227, wherein the Kd of the multispecific binding agent is less than the binding affinity of cetuximab to EGFR.

229. 228. The method of any one of claims 174 to 227, wherein the Kd of the multispecific binding agent is greater than the binding affinity of cetuximab to EGFR.

230. 1. A method for degrading a target protein on the surface of a target cell, comprising: contacting the E3 ligase and the target protein on the surface of the target cell with a binding agent, wherein the binding agent (i) a first binding domain that specifically binds to an E3 ligase that is RNF43; (ii) a second binding domain that specifically binds to the target protein, wherein the target protein is EGFR; A method comprising:

231. 231. The method of claim 230, wherein the binding agent is a multispecific antibody, a bispecific diabody, a bispecific Fab2, a bispecific camelid antibody, a bispecific peptibody scFv-Fc, a bispecific IgG, a knob-and-hole bispecific IgG, an Fc-Fab, or a knob-and-hole bispecific Fc-Fab.

232. The method of claim 230 or 231, wherein the first binding domain binds to an epitope of an endogenous internalizing receptor on the target cell, the epitope comprising at least 80% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 331 and 333.

233. The method of claim 232, wherein the first binding domain binds to an epitope of the endogenous internalizing receptor on the target cell, the epitope comprising at least 90% sequence identity to an epitope bound by an antibody comprising SEQ ID NOs: 331 and 333.

234. The method of claim 230 or 231, wherein the first binding domain binds to an epitope of an endogenous internalizing receptor on the target cell that does not contain any amino acids derived from the epitope bound by an antibody comprising SEQ ID NOs: 331 and 333.

235. 235. The method of any one of claims 230 to 234, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 80% sequence identity to an epitope to which cetuximab binds.

236. 236. The method of claim 235, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 90% sequence identity to an epitope to which cetuximab binds.

237. 235. The method of any one of claims 230 to 234, wherein the second binding domain binds to an epitope of the target protein on the target cell that does not include any amino acids from the epitope to which cetuximab binds.

238. 235. The method of any one of claims 230 to 234, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 80% sequence identity to the epitope to which Mav2 binds.

239. 239. The method of claim 238, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 90% sequence identity to the epitope to which Mav2 binds.

240. 235. The method of any one of claims 230 to 234, wherein the second binding domain binds to an epitope of the target protein on the target cell that does not contain any amino acids derived from the epitope to which Mav2 binds.

241. 241. The method of any one of claims 231 to 240, wherein the first binding domain comprises a first binding domain variable heavy chain and a first binding domain variable light chain.

242. 242. The method of claim 241, wherein the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

331.

243. 243. The method of claim 242, wherein the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

331.

244. 244. The method of claim 243, wherein the first binding domain variable heavy chain comprises SEQ ID NO:

331.

245. 245. The method of any one of claims 241 to 244, wherein the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO:

333.

246. 246. The method of claim 245, wherein the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO:

333.

247. 247. The method of claim 246, wherein the first binding domain variable light chain comprises SEQ ID NO:

333.

248. 248. The method of any one of claims 241 to 247, wherein the second binding domain comprises a second binding domain variable heavy chain.

249. 249. The method of claim 248, wherein the second binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

655.

250. 250. The method of claim 249, wherein the second binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

655.

251. 251. The method of claim 250, wherein the second binding domain variable heavy chain comprises SEQ ID NO:

655.

252. 252. The method of any one of claims 230 to 251, wherein the E3 ligase is degraded.

253. 253. The method of any one of claims 230 to 252, wherein the target cell is a cancer cell.

254. 254. The method of claim 253, wherein the cancer cells are selected from the group consisting of breast cancer cells, B-cell lymphoma cells, pancreatic cancer cells, Hodgkin's lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells, non-Hodgkin's B-cell lymphoma (B-NHL) cells, melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, glioma cells, glioblastoma cells, bladder cancer cells, colorectal cancer cells, and head and neck cancer cells.

255. 255. The method of claim 253 or 254, wherein expression of EGFR on the cancer cells is decreased after contact with the bispecific binding agent compared to control cancer cells not contacted with the bispecific binding agent.

256. 256. The method of claim 255, wherein the expression of EGFR on the cancer cells is reduced by 50% or more compared to the expression of EGFR on control cancer cells not contacted with the binding agent.

257. 255. The method of claim 253 or 254, wherein expression of EGFR on the cancer cells is reduced by 50% or more compared to expression of EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

258. 258. The method of any one of claims 253 to 257, wherein cell surface ablation of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells not contacted with the binding agent.

259. 258. The method of any one of claims 253-257, wherein cell surface ablation of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

260. The method of any one of claims 253 to 259, wherein the internalization of EGFR in the cancer cells is at least 20% or more compared to the internalization of EGFR in control cancer cells not contacted with the binding agent.

261. 260. The method of any one of claims 253-259, wherein internalization of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

262. 262. The method of any one of claims 253 to 261, wherein the degradation of EGFR in the cancer cells is at least 20% or more compared to the degradation of EGFR in control cancer cells not contacted with the binding agent.

263. 262. The method of any one of claims 253-261, wherein cytolysis of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

264. 264. The method of any one of claims 257, 259, 261, or 263, wherein the monospecific EGFR-binding agent is cetuximab.

265. 263. The method of any one of claims 253 to 262, wherein the method increases the sensitivity of the cancer cells to a cancer therapeutic agent.

266. 266. The method of claim 265, wherein the cancer therapeutic agent is a cytotoxic agent.

267. 267. The method of any one of claims 253 to 266, wherein the proliferation of the cancer cells is reduced.

268. 268. The method of any one of claims 253 to 267, wherein the method increases the killing of cancer cells.

269. 269. The method of any one of claims 253 to 268, wherein said contacting is carried out in vivo.

270. 1. A method for treating cancer in a subject, comprising: administering to the subject a binding agent, wherein the binding agent: (i) a first binding domain that specifically binds to an E3 ligase that is RNF43; (ii) a second binding domain that specifically binds to a target protein, including EGFR; A method comprising:

271. 271. The method of claim 270, wherein the first binding domain comprises a first binding domain variable heavy chain and a first binding domain variable light chain.

272. 272. The method of claim 271, wherein the first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

331.

273. 273. The method of claim 272, wherein the first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

331.

274. 274. The method of claim 273, wherein the first binding domain variable heavy chain comprises SEQ ID NO:

331.

275. 275. The method of any one of claims 271 to 274, wherein the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO:

333.

276. 276. The method of claim 275, wherein the first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO:

333.

277. 277. The method of claim 276, wherein the first binding domain variable light chain comprises SEQ ID NO:

333.

278. 278. The method of any one of claims 271 to 277, wherein the second binding domain comprises a second binding domain variable heavy chain.

279. 279. The method of claim 278, wherein the second binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

655.

280. 280. The method of claim 279, wherein the second binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

655.

281. 281. The method of claim 280, wherein the second binding domain variable heavy chain comprises SEQ ID NO:

655.

282. 282. The method of any one of claims 270 to 281, wherein the cancer is breast cancer, B-cell lymphoma, pancreatic cancer, Hodgkin's lymphoma, ovarian cancer, prostate cancer, mesothelioma, lung cancer, non-Hodgkin's B-cell (B-NHL) lymphoma, melanoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, neuroblastoma, glioma, glioblastoma, bladder cancer, colorectal cancer, or head and neck cancer.

283. (a) a first binding domain that specifically binds to an E3 ligase that is RNF43; and (b) a second binding domain that specifically binds to a target protein that is EGFR; 10. A multispecific binding agent comprising:

284. 284. The multispecific binding agent of claim 283, wherein the multispecific binding agent is a multispecific antibody, a bispecific antibody, a bispecific diabody, a bispecific Fab2, a bispecific camelid antibody, a bispecific peptibody scFv-Fc, a bispecific IgG, a knob-and-hole bispecific IgG, an Fc-Fab, or a knob-and-hole bispecific Fc-Fab.

285. The multispecific binding agent of claim 283 or 284, wherein the first binding domain binds to an epitope of RNF43 on the target cell that comprises at least 80% sequence identity to the epitope bound by an antibody comprising SEQ ID NOs: 331 and 333.

286. The multispecific binding agent of claim 285, wherein the first binding domain binds to an epitope of RNF43 on the target cell that comprises at least 90% sequence identity to the epitope bound by an antibody comprising SEQ ID NOs: 331 and 333.

287. The multispecific binding agent of claim 283 or 284, wherein the first binding domain binds to an epitope of RNF43 on the target cell that does not contain any amino acids derived from the epitope bound by an antibody comprising SEQ ID NOs: 331 and 333.

288. 288. The multispecific binding agent of any one of claims 283-287, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 80% sequence identity to an epitope to which cetuximab binds.

289. 289. The multispecific binding agent of claim 288, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 90% sequence identity to the epitope to which cetuximab binds.

290. 288. The multispecific binding agent of claims 283-287, wherein the second binding domain binds to an epitope of the target protein on the target cell that does not contain any amino acids from the epitope to which cetuximab binds.

291. 288. The multispecific binding agent of any one of claims 283 to 287, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 80% sequence identity to the epitope to which Mav2 binds.

292. 292. The multispecific binding agent of claim 291, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 90% sequence identity to the epitope to which Mav2 binds.

293. 288. The multispecific binding agent of claims 283 to 287, wherein the second binding domain binds to an epitope of the target protein on the target cell that does not contain any amino acids derived from the epitope to which Mav2 binds.

294. 294. The multispecific binding agent of any one of claims 284 to 293, wherein the first binding domain comprises a first binding domain variable heavy chain and a first binding domain variable light chain.

295. 295. The multispecific binding agent of claim 294, wherein said first binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

331.

296. 300. The multispecific binding agent of claim 295, wherein said first binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

331.

297. 300. The multispecific binding agent of claim 296, wherein said first binding domain variable heavy chain comprises SEQ ID NO:

331.

298. 298. The multispecific binding agent of any one of claims 294 to 297, wherein the first binding domain variable light chain comprises at least 80% sequence identity to SEQ ID NO:

333.

299. 300. The multispecific binding agent of claim 298, wherein said first binding domain variable light chain comprises at least 90% sequence identity to SEQ ID NO:

333.

300. 300. The multispecific binding agent of claim 299, wherein said first binding domain variable light chain comprises SEQ ID NO:

333.

301. 301. The multispecific binding agent of any one of claims 294-300, wherein the second binding domain comprises a second binding domain variable heavy chain.

302. 302. The multispecific binding agent of claim 301, wherein said second binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

655.

303. 303. The multispecific binding agent of claim 302, wherein said second binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

655.

304. 304. The multispecific binding agent of claim 303, wherein said second binding domain variable heavy chain comprises SEQ ID NO:

655.

305. 1. A method for degrading a target protein on the surface of a target cell, comprising: contacting an endogenous internalization receptor on the surface of the target cell and the target protein with a binding agent, wherein the binding agent: (i) a first binding domain that specifically binds to an endogenous internalizing receptor selected from the group consisting of LGR5, HER3, LY75, MST1R, MSLN, EpCAM, TNFRSF10B, and CD71; (ii) a second binding domain that specifically binds to the target protein, wherein the target protein comprises EGFR; A method comprising:

306. 306. The method of claim 305, wherein the binding agent is a multispecific antibody, a bispecific diabody, a bispecific Fab2, a bispecific camelid antibody, a bispecific peptibody scFv-Fc, a bispecific IgG, a knob-and-hole bispecific IgG, an Fc-Fab, or a knob-and-hole bispecific Fc-Fab.

307. The method of claim 305 or 306, wherein the first binding domain binds to an epitope of the endogenous internalizing receptor on the target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising any one of the variable heavy chain sequences or any one of the variable light chain sequences listed in Table 1 or Table 2.

308. The method of claim 307, wherein the first binding domain binds to an epitope of the endogenous internalizing receptor on the target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising any one of the variable heavy chain sequences or any one of the variable light chain sequences listed in Table 1 or Table 2.

309. The method of claim 305 or 306, wherein the first binding domain binds to an epitope of the endogenous internalizing receptor on the target cell that does not contain any amino acids from an epitope bound by an antibody comprising any one of the variable heavy chain sequences or any one of the variable light chain sequences listed in Table 1 or Table 2.

310. 310. The method of any one of claims 305 to 309, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 80% sequence identity to an epitope to which cetuximab binds.

311. 311. The method of claim 310, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 90% sequence identity to the epitope to which cetuximab binds.

312. 310. The method of any one of claims 305-309, wherein the second binding domain binds to an epitope of the target protein on the target cell that does not include any amino acids from the epitope to which cetuximab binds.

313. 310. The method of any one of claims 305 to 309, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 80% sequence identity to the epitope to which Mav2 binds.

314. 314. The method of claim 313, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 90% sequence identity to the epitope to which Mav2 binds.

315. 310. The method of any one of claims 305 to 309, wherein the second binding domain binds to an epitope of the target protein on the target cell that does not contain any amino acids derived from the epitope to which Mav2 binds.

316. 316. The method of any one of claims 305 to 315, wherein the first binding domain comprises a first binding domain variable heavy chain and a first binding domain variable light chain.

317. 317. The method of claim 316, wherein said first binding domain variable heavy chain comprises at least 80% sequence identity to any one of the variable heavy chain sequences listed in Table 1.

318. 318. The method of claim 317, wherein said first binding domain variable heavy chain comprises at least 90% sequence identity to any one of the variable heavy chain sequences listed in Table 1.

319. 319. The method of claim 318, wherein said first binding domain variable heavy chain comprises any one of the variable heavy chain sequences listed in Table 1.

320. 320. The method of any one of claims 316 to 319, wherein said first binding domain variable light chain comprises at least 80% sequence identity to any one of the variable light chain sequences listed in Table 1.

321. 321. The method of claim 320, wherein said first binding domain variable light chain comprises at least 90% sequence identity to any one of the variable light chain sequences listed in Table 1.

322. 322. The method of claim 321, wherein said first binding domain variable light chain comprises any one of the variable light chain sequences listed in Table 1.

323. 323. The method of any one of claims 316 to 322, wherein the second binding domain comprises a second binding domain variable heavy chain.

324. 324. The method of claim 323, wherein the second binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

655.

325. 325. The method of claim 324, wherein the second binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

655.

326. 326. The method of claim 325, wherein the second binding domain variable heavy chain comprises SEQ ID NO:

655.

327. 327. The method of any one of claims 305 to 326, wherein the endogenous internalization receptor is recycled to the target cell surface after internalization of the binding agent.

328. 327. The method of any one of claims 305 to 326, wherein the endogenous internalizing receptor is degraded.

329. 329. The method of any one of claims 305 to 328, wherein the target cell is a cancer cell.

330. 330. The method of claim 329, wherein the cancer cells are selected from the group consisting of breast cancer cells, B-cell lymphoma cells, pancreatic cancer cells, Hodgkin's lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells, non-Hodgkin's B-cell lymphoma (B-NHL) cells, melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, glioma cells, glioblastoma cells, bladder cancer cells, colorectal cancer cells, and head and neck cancer cells.

331. 331. The method of claim 329 or 330, wherein expression of EGFR on the cancer cells is decreased after contact with the multispecific binding agent compared to control cancer cells not contacted with the binding agent.

332. 332. The method of claim 331, wherein the expression of EGFR on the cancer cells is reduced by 50% or more compared to the expression of EGFR on control cancer cells not contacted with the binding agent.

333. 331. The method of claim 329 or 330, wherein expression of EGFR on the cancer cells is reduced by 50% or more compared to expression of EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

334. 334. The method of any one of claims 329-333, wherein cell surface ablation of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells not contacted with the binding agent.

335. 334. The method of any one of claims 329-333, wherein cell surface ablation of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

336. The method of any one of claims 329 to 335, wherein the internalization of EGFR in the cancer cells is at least 20% or more compared to the internalization of EGFR in control cancer cells not contacted with the binding agent.

337. 336. The method of any one of claims 329-335, wherein internalization of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

338. The method of any one of claims 329 to 337, wherein the degradation of EGFR in the cancer cells is at least 20% or more compared to the degradation of EGFR in control cancer cells not contacted with the binding agent.

339. 338. The method of any one of claims 329-337, wherein cytolysis of EGFR on the cancer cells is at least 20% or more compared to EGFR on control cancer cells contacted with the monospecific EGFR-binding agent.

340. 339. The method of any one of claims 333, 335, 337, or 339, wherein the monospecific EGFR-binding agent is cetuximab.

341. 339. The method of any one of claims 329 to 338, wherein the method increases the sensitivity of the cancer cells to a cancer therapeutic agent.

342. 342. The method of claim 341, wherein the cancer therapeutic agent is a cytotoxic agent.

343. 343. The method of any one of claims 329 to 342, wherein the proliferation of the cancer cells is reduced.

344. 344. The method of any one of claims 329 to 343, wherein the method increases the killing of cancer cells.

345. 345. The method of any one of claims 329 to 344, wherein said contacting is carried out in vivo.

346. 1. A method for treating cancer in a subject, comprising: administering to the subject a binding agent, wherein the binding agent: (i) a first binding domain that specifically binds to an endogenous internalizing receptor expressed on a target cell and selected from the group consisting of LGR5, HER3, LY75, MST1R, MSLN, EpCAM, TNFRSF10B, and CD71; (ii) a second binding domain that specifically binds to a target protein, including EGFR; A method comprising:

347. 347. The method of claim 346, wherein the cancer is breast cancer, B-cell lymphoma, pancreatic cancer, Hodgkin's lymphoma, ovarian cancer, prostate cancer, mesothelioma, lung cancer, non-Hodgkin's B-cell (B-NHL) lymphoma, melanoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, neuroblastoma, glioma, glioblastoma, bladder cancer, colorectal cancer, or head and neck cancer.

348. 348. The method of claim 346 or 347, wherein the tumor volume of a tumor contacted with the multispecific binding agent is reduced by at least 20% or more compared to the tumor volume of a tumor not contacted with the bispecific binding agent.

349. 349. The method of any one of claims 346 to 348, wherein the tumor volume of the tumor contacted with the multispecific binding agent is less than 80% or less compared to the tumor volume of a tumor not contacted with the bispecific binding agent.

350. 350. The method of any one of claims 346 to 349, wherein expression of EGFR on said cancer cells is reduced by at least 20% compared to EGFR expression in cancer cells not contacted with said bispecific binding agent.

351. 308. The method of any one of claims 304 to 307, wherein the expression of EGFR on the cancer cells is reduced by 20% compared to EGFR expression in cancer cells contacted with the monospecific EGFR-binding agent.

352. 352. The method of claim 351, wherein the monospecific EGFR binding agent is cetuximab.

353. (c) a first binding domain that specifically binds to an endogenous internalizing receptor selected from the group consisting of LGR5, HER3, LY75, MST1R, MSLN, EpCAM, TNFRSF10B, and CD71; and (d) a second binding domain that specifically binds to a target protein that is EGFR.

10. A multispecific binding agent comprising:

354. 354. The multispecific binding agent of claim 353, wherein the multispecific binding agent is a multispecific antibody, a bispecific antibody, a bispecific diabody, a bispecific Fab2, a bispecific camelid antibody, a bispecific peptibody scFv-Fc, a bispecific IgG, a knob-and-hole bispecific IgG, an Fc-Fab, or a knob-and-hole bispecific Fc-Fab.

355. The multispecific binding agent of claim 353 or 354, wherein the first binding domain binds to an epitope of the endogenous internalizing receptor on the target cell that comprises at least 80% sequence identity to an epitope bound by an antibody comprising any one of the variable heavy chain sequences or any one of the variable light chain sequences listed in Table 1 or Table 2.

356. The multispecific binding agent of claim 355, wherein said first binding domain binds to an epitope of said endogenous internalizing receptor on said target cell that comprises at least 90% sequence identity to an epitope bound by an antibody comprising any one of the variable heavy chain sequences or any one of the variable light chain sequences listed in Table 1 or Table 2.

357. The multispecific binding agent of claim 353 or 354, wherein the first binding domain binds to an epitope of the endogenous internalizing receptor on the target cell that does not contain any amino acids derived from an epitope bound by an antibody comprising any one of the variable heavy chain sequences or any one of the variable light chain sequences listed in Table 1 or Table 2.

358. 358. The multispecific binding agent of any one of claims 353-357, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 80% sequence identity to an epitope to which cetuximab binds.

359. 359. The multispecific binding agent of claim 358, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 90% sequence identity to the epitope to which cetuximab binds.

360. 358. The multispecific binding agent of any one of claims 353-357, wherein the second binding domain binds to an epitope of the target protein on the target cell that does not include any amino acids from the epitope to which cetuximab binds.

361. 358. The multispecific binding agent of any one of claims 353 to 357, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 80% sequence identity to the epitope to which Mav2 binds.

362. The multispecific binding agent of claim 361, wherein the second binding domain binds to an epitope of the target protein on the target cell that comprises at least 90% sequence identity to the epitope to which Mav2 binds.

363. 358. The multispecific binding agent of any one of claims 353 to 357, wherein the second binding domain binds to an epitope of the target protein on the target cell that does not contain any amino acids from the epitope to which Mav2 binds.

364. 364. The multispecific binding agent of any one of claims 353-363, wherein said first binding domain comprises a first binding domain variable heavy chain and a first binding domain variable light chain.

365. 365. The multispecific binding agent of claim 364, wherein said first binding domain variable heavy chain comprises at least 80% sequence identity to any one of the variable heavy chain sequences listed in Table 1.

366. 366. The multispecific binding agent of claim 365, wherein said first binding domain variable heavy chain comprises at least 90% sequence identity to any one of the variable heavy chain sequences listed in Table 1.

367. 367. The multispecific binding agent of claim 366, wherein said first binding domain variable heavy chain comprises any one of the variable heavy chain sequences listed in Table 1.

368. 368. The multispecific binding agent of any one of claims 364-367, wherein said first binding domain variable light chain comprises at least 80% sequence identity to any one of the variable light chain sequences listed in Table 1.

369. 369. The multispecific binding agent of claim 368, wherein said first binding domain variable light chain comprises at least 90% sequence identity to any one of the variable light chain sequences listed in Table 1.

370. 370. The multispecific binding agent of claim 369, wherein said first binding domain variable light chain comprises any one of the variable light chain sequences listed in Table 1.

371. 371. The multispecific binding agent of any one of claims 364-370, wherein the second binding domain comprises a second binding domain variable heavy chain.

372. 372. The multispecific binding agent of claim 371, wherein said second binding domain variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:

655.

373. 373. The multispecific binding agent of claim 372, wherein said second binding domain variable heavy chain comprises at least 90% sequence identity to SEQ ID NO:

655.

374. 374. The multispecific binding agent of claim 373, wherein said second binding domain variable heavy chain comprises SEQ ID NO:

655.

375. 375. The method of any one of claims 353-374, wherein the half-life of the multispecific binding agent is within 20% of the half-life of cetuximab.

376. 376. The method of any one of claims 353 to 375, wherein the clearance rate of the multispecific binding agent is within 20-95% of the clearance rate of cetuximab.

377. The method of any one of claims 353 to 376, wherein the Kd of the multispecific binding agent is at least two-fold less than or equal to two-fold the binding affinity of cetuximab to EGFR.

378. The method of claim 377, wherein the Kd of the multispecific binding agent is at least 5-fold less than or equal to 5-fold the binding affinity of cetuximab to EGFR.

379. The method of claim 378, wherein the Kd of the multispecific binding agent is at least 10-fold less than or equal to 10-fold less than the binding affinity of cetuximab to EGFR.

380. 379. The method of any one of claims 353 to 379, wherein the Kd of the binding affinity of the multispecific binding agent may be within an order of magnitude of the binding affinity of the monovalent binding agent.

381. 381. The method of any one of claims 353 to 380, wherein the Kd of the multispecific binding agent is within ±10% of the binding affinity of cetuximab to EGFR.

382. The method of claim 381, wherein the Kd of the multispecific binding agent is within ±20% of the binding affinity of cetuximab to EGFR.

383. The method of claim 382, wherein the Kd of the multispecific binding agent is within ±30% of the binding affinity of cetuximab to EGFR.

384. 384. The method of any one of claims 353 to 383, wherein the Kd of the multispecific binding agent is less than the binding affinity of cetuximab to EGFR.

385. 384. The method of any one of claims 353 to 383, wherein the Kd of the multispecific binding agent is greater than the binding affinity of cetuximab to EGFR.

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