Compositions for Targeted Lysosomal Degradation and Methods of Use Thereof

Abstract

The present disclosure provides a composition of a degrader based on a bispecific binding molecule that degrades a target protein, such as a protein on a cancer cell, via the lysosomal pathway. The bispecific binding molecule specifically binds to the target protein of interest and neuropilin-1 (NRP1). The present disclosure also provides methods of using the degrader based on the bispecific binding molecule, such as for enhancing the therapeutic effect of cancer treatment.

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 369,948, filed Jul. 30, 2022, and U.S. Provisional Patent Application No. 63 / 423,454, filed Nov. 7, 2022, the entire contents of both applications are hereby incorporated by reference herein.

Background Art

[0002] Background Proteolysis controls many aspects of cellular homeostasis. The endogenous proteolytic machinery has been reprogrammed to remove various intracellular substrates by an approach called targeted proteolysis. Targeted proteolysis (TPD) has emerged as a promising therapeutic strategy that is more advantageous than conventional inhibition. Unlike inhibitors, degraders enable catalytic and sustained knockdown of protein levels. Proteolysis-targeting chimeras (PROTACs) consist of two sites that bind to a target and a ubiquitin ligase (E3) separated by a flexible linker. However, PROTACs are limited to targeting intracellular proteins due to the action of their intracellular mechanisms.

[0003] To remove disease-causing proteins, lysosomal degradation platforms such as chimeras targeting cytokine receptors (KineTac) using E3 / USP and chimeras targeting lysosomes (LYTAC) have been developed. The KineTac platform is a genetically encoded bispecific antibody consisting of a cytokine arm that binds to a homologous cytokine receptor and a target binding moiety that binds to the protein of interest. However, KineTac is limited in its application to cytokine receptors on T cells and can degrade the target protein if the target protein is also present on T cells. The LYTAC platform, consisting of an antibody-glycan conjugate, degrades cell surface and extracellular proteins by repeatedly trafficking the target protein to lysosomes for degradation. Therefore, an additional approach for lysosomal degradation platforms is needed in the art.

Summary of the Invention

[0004] Summary The present disclosure relates to a novel and unique bispecific binding molecule consisting of a fully recombinant bispecific binding domain that utilizes neuropilin-1 (NRP1)-mediated internalization to target various therapeutically relevant target proteins, particularly cell surface receptors such as receptor tyrosine kinases (RTKs), for lysosomal degradation. Accordingly, the present disclosure provides a degrading agent based on a bispecific binding molecule that degrades target proteins such as target proteins on cancer cells. The bispecific binding molecule specifically binds to the target protein of interest and neuropilin-1 (NRP1). The present disclosure further relates to methods of using NRP1-dependent bispecific binding molecules to induce degradation of target proteins via the lysosomal degradation pathway. The present disclosure provides methods of inhibiting tumor growth using bispecific binding molecules, such as methods of enhancing the therapeutic effect of cancer treatment.

[0005] The present disclosure relates to bispecific binding molecules comprising a target protein binding domain and a neuropilin-1 (NRP1) binding domain that is an antibody or an NRP-1 binding fragment thereof. In some embodiments, it relates to bispecific binding molecules comprising a target protein binding domain that specifically binds to its cognate protein receptor and an NRP1 binding domain that binds to its cognate receptor. In some embodiments, the target protein and NRP1 are membrane-bound. In some embodiments, binding of the bispecific binding molecule to NRP1 results in internalization of the target protein bound to the bispecific binding molecule. In some embodiments, binding of the bispecific binding molecule to NRP1 subsequently induces lysosomal degradation of the target protein.

[0006] In certain embodiments, the target protein binding domain of the bispecific molecule binds to a receptor tyrosine kinase (RTK). Thus, in one aspect, the present disclosure provides (a) a target protein binding domain that specifically binds to a receptor tyrosine kinase (RTK); and (b) a neuropilin-1 (NRP1) binding domain that binds to NRP1, comprising an antibody or an NRP-1 binding fragment thereof of a bispecific binding molecule, wherein binding of the bispecific binding molecule to the target protein and NRP1 results in lysosomal degradation of the target protein in target cells.

[0007] In one embodiment, the RTK is EGFR. In another embodiment, the RTK is not the epidermal growth factor receptor (EGFR) (i.e., the target protein is an RTK, but the RTK is not EGFR).

[0008] In other embodiments, the RTK is selected from the EGFR family of receptors, the HER family of receptors, the insulin growth factor receptor (IGFR), the Met receptor tyrosine kinase (MET), the platelet-derived growth factor receptor (PDGFR), the fibroblast growth factor receptor (FGFR), and the vascular endothelial growth factor (VEGFR).

[0009] In one aspect, the receptor tyrosine kinase is cMET. In one aspect, the receptor tyrosine kinase is HER2. In one aspect, the receptor tyrosine kinase is IGF1R.

[0010] In one aspect, the target cell is a cancer cell. In certain aspects, the cancer cell is selected from the group consisting of lung cancer, breast cancer, colorectal cancer, head and neck cancer, esophageal gastric cancer, liver cancer, glioblastoma, prostate cancer, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, and pancreatic cancer. In one aspect, the cancer cell is a non-small cell lung cancer (NSCLC) cell.

[0011] In certain aspects, the target protein binding domain and the NRP1 binding domain are each independently selected from the group consisting of IgG, half antibody, single domain antibody, nanobody, Fab, monospecific Fab2, Fc, scFv, minibody, IgNAR, V-NAR, hcIgG, VHH domain, camel antibody, and peptibody.

[0012] In one aspect, the NRP1 binding domain is (i) an antibody heavy chain variable (VH) domain comprising CDR1, CDR2, and CDR3 regions (HCDR1, HCDR2, and HCDR3, respectively), wherein HCDR1 consists of the sequence shown in SEQ ID NO: 79, HCDR2 consists of the sequence shown in SEQ ID NO: 80, and HCDR3 consists of the sequence shown in any one of SEQ ID NOs: 81-84; and (ii) an antibody light chain variable (VL) domain comprising CDR1, CDR2, and CDR3 regions (LCDR1, LCDR2, and LCDR3, respectively), wherein LCDR1 consists of the sequence shown in any one of SEQ ID NOs: 85-87, LCDR2 consists of the sequence shown in SEQ ID NO: 88, and LCDR3 consists of the sequence shown in SEQ ID NO: 89 including.

[0013] In one aspect, (i) HCDR1 consists of the sequence shown in SEQ ID NO: 79, HCDR2 consists of the sequence shown in SEQ ID NO: 80, and HCDR3 consists of any one of the sequences shown in SEQ ID NO: 84; and (ii) LCDR1 consists of the sequence shown in SEQ ID NO: 85, LCDR2 consists of the sequence shown in SEQ ID NO: 88, and LCDR3 consists of the sequence shown in SEQ ID NO: 89.

[0014] In another aspect, the present disclosure relates to a nucleic acid encoding the bispecific binding molecule of the invention, an expression vector containing the nucleic acid of the invention, and a cell capable of protein expression containing the nucleic acid of the invention (e.g., an expression vector).

[0015] In another aspect, the present disclosure relates to the use of the bispecific binding molecule of the invention in the manufacture of a medicament for treating cancer in a subject.

[0016] In another embodiment, the present disclosure relates to a method of inducing lysosomal degradation of a target protein in a cell, the method comprising contacting the cell with the bispecific binding molecule of the invention such that lysosomal degradation of the target protein is induced in the cell. In one embodiment, the cell has one or more mutations in the target protein and / or overexpresses the target protein. In one embodiment, the cell is resistant or refractory to an inhibitor of the target protein.

[0017] In one embodiment, the target protein is EGFR. In one embodiment, the target protein is an RTK other than EGFR. In one embodiment, the target protein is cMET. In one embodiment, the target protein is HER2. In one embodiment, the target protein is IGF1R.

[0018] In another aspect, the present disclosure relates to a method of inhibiting tumor growth in a subject having a tumor, the method comprising administering to the subject a bispecific binding molecule of the invention such that tumor growth in the subject is inhibited. In certain embodiments, the tumor comprises one or more mutations in a target protein and / or overexpresses the target protein. In certain embodiments, the tumor is resistant or refractory to the responsiveness to an inhibitor of the target protein.

[0019] In one embodiment, the target protein is EGFR. In one embodiment, the target protein is an RTK other than EGFR. In one embodiment, the target protein is cMET. In one embodiment, the target protein is HER2. In one embodiment, the target protein is IGF1R.

[0020] In certain embodiments, the bispecific binding molecule is administered intravenously, intraperitoneally, intrathecally, intraventricularly, or into the brain parenchyma.

[0021] The present disclosure relates to a bispecific binding molecule comprising a target protein binding domain that specifically binds to a target protein and a neuropilin-1 (NRP1) binding domain that binds to NRP1. In some embodiments, the target protein and NRP1 are membrane-bound. In certain embodiments, the target protein is a cell surface receptor protein. In certain embodiments, the target protein is a transmembrane protein.

[0022] In some embodiments, the target protein comprises a receptor tyrosine kinase (RTK). In some embodiments, the tyrosine kinase comprises epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), receptor tyrosine kinase Met (MET), and receptor vascular endothelial growth factor (VEGFR).

[0023] In some embodiments, the receptor tyrosine kinases include EGFR / HER1 / Erb1, HER2 / ErbB2, HER3 / ErbB3, HER4 / ErbB4, FGFR1, FGFR2, FGFR3, FGFR4, MET, RON, PDGFR, PDGFRα, PDGFRβ, CSF-1R, Kit, FLT-3, VEGFR1, VEGFR2, and VEGFR3.

[0024] In some embodiments, the target protein includes EGFR. In some embodiments, EGFR is selected from the group consisting of EGFR / HER1 / Erb1, HER2 / ErbB2, HER3 / ErbB3, and HER4 / ErbB4. In some embodiments, the target protein is EGFR.

[0025] In some embodiments, the target protein includes FGFR. In some embodiments, FGFR1 is selected from the group consisting of FGFR1, FGFR2, FGFR3, and FGFR4.

[0026] In one embodiment, the target protein includes the receptor tyrosine kinase MET. In some embodiments, the receptor tyrosine kinase MET is MET or macrophage-stimulating protein receptor (MST1R / RON).

[0027] In some embodiments, the target protein consists of PDGFR. In some embodiments, PDGFR is selected from the group consisting of PDGFR, PDGFRα, PDGFRβ, CSF-1R, Kit, and FLT-3.

[0028] In some embodiments, the target protein includes VEGFR. In some embodiments, VEGFR is selected from the group consisting of VEGFR1, VEGFR2, and VEGFR3.

[0029] In some embodiments, the receptor tyrosine kinase is selected from the group consisting of EGFR / HER1 / Erb1, HER2 / ErbB2, HER3 / ErbB3, HER4 / ErbB4, VEGFR1, VEGFR2, and VEGFR3.

[0030] In some embodiments, the target protein includes a receptor serine / threonine kinase (RSTK), a G protein-coupled receptor (GPCR), an immune checkpoint receptor, and an ion channel receptor.

[0031] In some embodiments, the target protein consists of a receptor serine / threonine kinase (RSTK). In some embodiments, the RSTK includes ACVRL1, ACVR1, ACVR1B, ACVR1C, BMPR1A, BMPR1B, TGFBR1, ACVR2A, ACVR2B, AMHR2, BMPR2, and TGFBR2.

[0032] In some embodiments, the target protein includes a G protein-coupled receptor (GPCR). In some embodiments, the GPCR is selected from the group consisting of CXCR4, CCR5, FFAR2, GLP2R, 5-HT1A receptor, 5-HT2A receptor, 5-HT4 receptor, 5-HT5A receptor, M1 receptor, M2 receptor, A1 receptor, A2A receptor, α1A-adrenergic receptor, α2A-adrenergic receptor, β1-adrenergic receptor, β3-adrenergic receptor, AT1 receptor, BB1 receptor, B1 receptor, CB1 receptor, CB2 receptor, chemerin receptor 1, CCR1, CX3CR1, ACKR3, CCK1 receptor, GPR3, GPR12, GPR17, GPR32, and GPR35.

[0033] In some embodiments, the target protein comprises an immune checkpoint receptor. In some embodiments, the immune checkpoint receptor is selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, NOX2, PD-1, TIM-3, VISTA, SIGEC7, and PD-L1.

[0034] In some embodiments, the target protein comprises an ion channel receptor. In some embodiments, the ion channel receptor is selected from the group consisting of KCa1.1, KCa2.1, CatSper1, TPC1, CNGA1, HCN1, Kir1.1, Kir3.2, RyR1, TRPA1, TRPC3, TRPM1, TRPP1, TRPV1, K2P1.1, K2P10.1, Cav1.1, Cav2.1, Kv1.1, Kv1.8, Kv11.2, Hv1, Nav1.1, and Nav1.2.

[0035] In some embodiments, the target protein comprises a membrane-associated target protein, and the target protein binding domain of the bispecific binding molecule binds to a cell surface receptor. In some embodiments, the target protein binding domain of the bispecific binding molecule binds to an extracellular epitope of the membrane-associated target protein. In one embodiment, the target cells include tumor cells. In one embodiment, the target cells are cancer cells. In some embodiments, the cancer cells are selected from the group consisting of lung cancer, breast cancer, colorectal cancer, head and neck cancer, esophageal gastric cancer, liver cancer, glioblastoma, prostate cancer, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, and pancreatic cancer. In one embodiment, the target cells include immune cells.

[0036] In some embodiments, the target protein binding domain and the NRP1 binding domain are each independently selected from the group consisting of IgG, half antibody, single domain antibody, nanobody, Fab, monospecific Fab2, Fc, scFv, minibody, IgNAR, V-NAR, hcIgG, VHH domain, camel antibody, and peptibody.

[0037] In some embodiments, the target protein binding domain and the NRP1 binding domain together form a bispecific binding molecule, a bispecific diabody, a bispecific Fab2, a bispecific camelid antibody, or a bispecific peptibody scFv-Fc, a bispecific IgG, a knob-in-hole bispecific IgG, an Fc-Fab, and a knob-in-hole bispecific Fc-Fab, a cytokine-IgG fusion, a cytokine-Fab fusion, and a cytokine-Fc-scFv fusion. In some embodiments, the target protein binding domain comprises an Fc-Fab and the NRP1 binding domain comprises an Fc-fusion. In another embodiment, the bispecific binding molecule is immunoglobulin G1 (IgG1) or a variant thereof. In another embodiment, the IgG1 is human IgG1 or a variant thereof.

[0038] In some embodiments, the bispecific binding molecule comprises two identical heavy chain polypeptides, where the first heavy chain is fused to a first single-chain variable fragment (scFv) by a peptide linker to produce a first heavy chain fusion polypeptide, and the second heavy chain is fused to a second scFv by a peptide linker to produce a second heavy chain fusion polypeptide, where the first and second scFvs are identical; and two identical light chains comprising a first light chain and a second light chain, where each heavy chain fusion polypeptide sequence is SEQ ID NO: 1-11 or 39, and the light chain polypeptide sequence is SEQ ID NO: 12. In some embodiments, the heavy chain fusion polypeptide sequence is SEQ ID NO: 11 and the light chain polypeptide is SEQ ID NO: 12. In some embodiments, the peptide linker comprises the amino acid sequence (GGGGS)n set forth in SEQ ID NO: 13, where n is an integer from 1 to 20, each independently. In some embodiments, the present disclosure further provides a nucleotide sequence encoding each heavy chain fusion polypeptide set forth in any of SEQ ID NOs: 15-24 or 40, and the light chain polypeptide set forth in SEQ ID NO: 25.

[0039] In another aspect, the bispecific binding molecule comprises: (a) two identical heavy chain polypeptides comprising making a first heavy chain fusion polypeptide by fusing a first heavy chain to a first single-chain variable fragment (scFv) via a peptide linker, and making a second heavy chain fusion polypeptide by fusing a second heavy chain to a second scFv via a peptide linker, wherein the first and second scFvs are identical; and (b) two identical light chains comprising a first light chain and a second light chain, wherein (i) the heavy chain fusion polypeptide comprises a variable heavy chain (VH), a constant heavy chain 1 (CH1), CH2, CH3, and a short-chain variable fragment (scFv) comprising an NRP1 binding domain at the C-terminus of CH3, and (ii) a light chain polypeptide comprising a variable light chain (VL) and a constant light chain (CL), wherein the VL and VH of the heavy chain fusion polypeptide and the light chain polypeptide comprise an scFv comprising a target protein binding domain that binds to a target protein and an NRP1 binding domain that binds to NRP1.

[0040] In some aspects, the NRP1 binding domain of the bispecific binding molecule is (i) an antibody heavy chain variable (VH) domain comprising CDR1, CDR2 and CDR3 regions (HCDR1, HCDR2 and HCDR3, respectively), wherein HCDR1 consists of the sequence shown in SEQ ID NO: 79, HCDR2 consists of the sequence shown in SEQ ID NO: 80, and HCDR3 consists of the sequence shown in any one of SEQ ID NOs: 81-84; and (ii) an antibody light chain variable (VL) domain comprising CDR1, CDR2 and CDR3 regions (LCDR1, LCDR2 and LCDR3, respectively), wherein LCDR1 consists of the sequence shown in any one of SEQ ID NOs: 85-87, LCDR2 consists of the sequence shown in SEQ ID NO: 88, and LCDR3 consists of the sequence shown in SEQ ID NO: 89 comprising.

[0041] In another aspect, (iii) HCDR1 consists of the sequence shown in SEQ ID NO: 79, HCDR2 consists of the sequence shown in SEQ ID NO: 80, and HCDR3 consists of any one of the sequences shown in SEQ ID NO: 84; and, (iv) LCDR1 consists of the sequence shown in SEQ ID NO: 85, LCDR2 consists of the sequence shown in SEQ ID NO: 88, and LCDR3 consists of the sequence shown in SEQ ID NO: 89.

[0042] In some embodiments, the binding affinity (K D ) of the bispecific binding molecule to the target protein is <0.1 nM. In some embodiments, the binding affinity (K D ) of the bispecific binding molecule to NRP1 is in the range of 0.1 nM to 100 nM. In some embodiments, the binding affinity (K D ) of the binding antibody to the target protein is at least 2-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, or 100-fold greater than the binding affinity (K D ) of the bispecific binding molecule to NRP1.

[0043] In some embodiments, the bispecific binding molecule (bispecific antibody) is part of an antibody-drug conjugate (ADC), where the bispecific binding molecule is bound to a drug via a linker. Non-limiting examples of agents suitable for use in ADC compounds are described herein.

[0044] In some embodiments, the EGFRxNRP1 bispecific antibody inhibits VEGF-mediated VEGFR2 phosphorylation in HUVEC cells. In some embodiments, the binding of the bispecific binding molecule to the target protein and NRP1 induces potent degradation of EGFR T790M / L858 in vitro. In one embodiment, the degradation of the target protein results in inhibition of target cell proliferation. In some embodiments, the EGFRxNRP1 bispecific antibody decreases cell viability in vitro.

[0045] In some embodiments, the bispecific binding molecule reduces tumor volume in an osimertinib-sensitive xenograft mouse model. In some embodiments, the bispecific binding molecule reduces tumor volume in an osimertinib-resistant or osimertinib-refractory xenograft mouse model. In some embodiments, treatment with the bispecific binding molecule enhances the anti-tumor effect.

[0046] In some embodiments, a nucleic acid encoding the bispecific binding molecule described in Table 2 is used. In some embodiments, the nucleic acid is operably linked to a promoter.

[0047] An expression vector comprising a nucleic acid of the bispecific binding molecule is provided. In some embodiments, the vector further comprises a promoter, where the promoter is operably linked to the nucleic acid.

[0048] The present disclosure provides a cell capable of protein expression comprising a nucleic acid of the bispecific binding molecule. In one embodiment, the recombinant cell comprises a cancer cell.

[0049] In another embodiment, the present disclosure provides a method for producing a bispecific binding molecule, where the method comprises: (a) providing a cell capable of protein synthesis comprising a nucleic acid described herein; and (b) inducing the expression of the bispecific binding molecule.

[0050] The present disclosure also provides a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a bispecific binding molecule, a nucleic acid, a vector or a recombinant cell, and a pharmaceutically acceptable carrier.

[0051] The present disclosure also provides the use of a bispecific binding molecule in the manufacture of a medicament for treating cancer in a subject.

[0052] The present disclosure provides a method of treating cancer in a subject. In some embodiments, the method comprises administering to the subject in need thereof a therapeutically effective amount of a bispecific binding molecule, nucleic acid, vector, recombinant cell, or pharmaceutical composition provided herein. In some embodiments, the bispecific binding molecule is administered intraperitoneally.

[0053] The present disclosure further provides a method of preventing cancer in a subject. In some embodiments, a method of inhibiting the growth of tumor cells is provided.

[0054] In some embodiments, a kit is provided that includes one or more unit doses of a pharmaceutical composition and instructions for one or more unit doses of the pharmaceutical composition to a subject in need thereof.

[0055] In some embodiments, the disease includes a neoplastic disease, an inflammatory disease, a metabolic disease, or a neurological disease.

[0056] In certain embodiments, the neoplastic disease includes lung cancer, breast cancer, colorectal cancer, head and neck cancer, esophageal gastric cancer, liver cancer, glioblastoma, prostate cancer, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, and pancreatic cancer. In one embodiment, the lung cancer is non-small cell lung cancer (NSCLC).

[0057] Other features and advantages of the present invention may be apparent from the following detailed description and claims.

Brief Description of the Drawings

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[0061] **Detailed Description of the Invention** **Definitions** Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Accordingly, the following terms are intended to have the following meanings: The singular forms "a", "an", and "the" include references to the plural unless the context clearly dictates otherwise.

[0062] As used herein, the term "human EGFR" or "EGFR" means the human epidermal growth factor receptor protein (UniProKB / Swiss-Pro number P00533), including variants, isoforms, and species homologs of EGFR that are naturally expressed by cells including tumor cells or expressed on cells transfected with the EGFR gene or cDNA.

[0063] As used herein, the term "human NRP1" or "NRP1" means the human neuropilin-1 protein (UniProKB / Swiss-Pro number O14786), including variants, isoforms, and species homologs of NRP1 that are naturally expressed by cells including tumor cells or expressed on cells transfected with the NRP1 gene or cDNA.

[0064] As used herein, the term "receptor tyrosine kinase" (plural) or "RTK" means a protein that is a receptor (i.e., binds a ligand) and phosphorylates tyrosine residues.

[0065] As used herein, the term "non-receptor tyrosine kinase" (plural) or "non-RTK" means a protein that is not an RTK, i.e., a protein that is not a receptor and / or does not phosphorylate tyrosine residues.

[0066] As used herein, "administering" or "administration" of a bispecific binding molecule or polypeptide thereof described herein means delivery to a subject of a polypeptide or composition of the invention described herein, or a prodrug or other pharmaceutically acceptable derivative thereof, using, for example, any suitable formulation or route of administration described herein.

[0067] The term "and / or" when used in a list of two or more items means that any one of the listed items can be employed alone or any combination of two or more of the listed items can be employed.

[0068] As used herein, "treatment", "treat" or "treating", when used with respect to a disease or medical condition, means that at least one improvement in symptoms associated with the medical condition from which the subject suffers is achieved, where improvement is used in a broad sense and means at least one decrease in a parameter associated with the medical condition being treated, such as the magnitude of a symptom. The term "treatment" or "treat" means an approach for obtaining a therapeutic effect. Therapeutic benefit is determined by whether a tumor shrinks, stays the same size, or has an extended progression-free survival compared to a placebo. Thus, treatment includes prevention (i.e., reducing the risk of onset of clinical symptoms, including not causing clinical symptoms to develop, e.g., preventing progression of a disease) and inhibition (i.e., preventing the appearance or further appearance of clinical symptoms, e.g., reducing or completely suppressing an active disease).

[0069] As used herein, the term "subject" can mean any animal having cancer, such as mammals including laboratory animals, livestock, pets, etc. In one aspect, the animal is a primate, preferably a human. The terms "subject" and "subject" as used herein are used interchangeably. The terms "subject" and "subject" refer to an animal (e.g., birds such as chickens, quails or turkeys, or mammals), specifically, non - primate animals (e.g., cows, pigs, horses, sheep, rabbits, guinea pigs, rats, cats, dogs and mice) and primate animals (e.g., monkeys, chimpanzees and humans), more specifically a human, i.e., a "mammal". In one aspect, the subject is a non - human animal such as livestock (e.g., horses, cows, pigs or sheep), pets (e.g., dogs, cats, guinea pigs or rabbits). In a preferred aspect, the subject is a "human".

[0070] As used herein, the term "fusion" means integrating two molecules having the same or different functions or structures, and the methods of fusion include physical, chemical or biological methods that can bind a peptide to a protein, a small molecule drug, a nanoparticle, or a liposome. Preferably, the fusion may be mediated by a linker peptide, for example, the linker peptide may be fused to the C - terminus of a fragment of the variable region of the antibody light chain (Fc). Alternatively, two molecules are fused by integrating multiple domains within a polypeptide sequence.

[0071] As used herein, the term "linker" or "flexible linker" is a molecule or peptide that connects two polypeptide subunits. The linker peptide sequence may include the amino acid sequence subunit (GGGGS)n, where n defines the number of repeats of the subunit. The number of repeats of the subunit determines the flexibility of the linker peptide. A flexible peptide linker increases the flexibility between two binding domains.

[0072] As used herein, "effective amount" means an amount sufficient to elicit a desired anti-cancer response. In the present invention, the desired biological response is to inhibit cell proliferation. The exact amount of the bispecific binding molecule administered to a subject will vary depending on the method of administration, the type and severity of the cancer, and the characteristics of the subject, such as general health status, age, gender, weight, and drug resistance. One of ordinary skill in the art can determine the appropriate dosage according to these and other factors. When used in combination with other anti-cancer agents, for example, in combination with chemotherapy, the "effective amount" of the second agent will vary depending on the type of agent used. Appropriate dosages are known for approved drugs and can be adjusted by one of ordinary skill in the art according to the condition of the subject, the type of cancer being treated, and the amount of the polypeptide administered herein. When the amount is not specified, an effective amount should be assumed. For example, the bispecific antibodies described herein can be administered to a subject at a dosage of about 0.01 to 100 mg / kg body weight / day at intervals of once a week or once every other week.

[0073] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein and mean a polymer of amino acid residues.

[0074] As used herein, the term "variant" refers to a sequence that has changed from a wild-type, conventional, or primary sequence. The variation can take the form of amino acid or nucleotide deletions, substitutions, or insertions. A variant can include one or more combinations of sequence variations.

[0075] The terms "reduce" or "decrease" or other forms such as "decrease" generally mean a decrease in an event or property (e.g., one or more symptoms, or the binding of one protein to another protein). This is usually relative to some standard or expected value, that is, in other words, relative, but it is understood that it is not necessarily necessary to refer to a standard or relative value.

[0076] As used in the context of "binding affinity", the term "affinity" means a reduction in the affinity of one molecule for another. For example, in some embodiments, a protein, domain, or motif can specifically bind to a particular target, such as a peptide, polypeptide, protein, carbohydrate, sugar, polysaccharide, glycosaminoglycan, or an epitope thereof, with a predetermined affinity. The term "affinity" means the strength of the sum of non-covalent interaction between a single binding domain of a molecule and its binding target or partner (e.g., antigen). The affinity of a molecule for its target can be represented by the dissociation constant (K D ), which is the ratio of the dissociation rate constant and the association rate constant (k off and k on ) respectively. The strength or affinity of a binding interaction can be represented by the dissociation constant (K D ), where a smaller K D indicates a higher affinity. The binding properties (affinity) of a selected polypeptide can be quantified using methods well known in the art. One such method is to measure the rate of formation and dissociation of the antigen-binding domain / antigen complex, which depends on the concentration of the complex partner, the affinity of the interaction, and the geometric parameters that equally affect the rates in both directions. Thus, both the "binding constant" (K on ) and the "dissociation constant" (K off ) can be determined by calculation of concentration and the actual association and dissociation rates (see Nature 361:186-87 (1993)).

[0077] The ratio of K off / K on allows cancellation of all parameters not related to affinity and is equal to the dissociation constant K D (generally see Davies et al. (1990) Annual Rev Biochem 59:439-473). In some embodiments, the recombinant polypeptide of the invention can specifically bind to an epitope when the equilibrium binding constant (K D ) is ≦ 1 μM. In some embodiments, the recombinant polypeptide of the invention has an equilibrium binding constant (K D) can specifically bind to the epitope when it is ≤100 nM. In some embodiments, the recombinant polypeptide of the present invention has an equilibrium binding constant (K D ) can specifically bind to the epitope when it is ≤10 nM. In some embodiments, the recombinant polypeptide of the present invention has an equilibrium binding constant (K D ) can specifically bind to the epitope when it is ≤100 pM to about 1 pM. In some embodiments, K D is 10 -5 M or less (e.g., 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -8 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13 M or less, 10 -14 M or less, 10 -15 M or less, or 10 -16 M or less).

[0078] Thus, equivalent affinities may include different rate constants as long as the ratio of the rate constants is the same. Thus, in some embodiments, "decrease in binding" means a decrease in affinity for each interaction. Conversely, "increase in binding" means an increase in binding affinity for each interaction.

[0079] As used herein, the term "isotype" means the class of immunoglobulin encoded by a heavy chain constant region gene (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM).

[0080] As used herein, the term "bispecific binding molecule" means an antibody having two different antigen-binding regions defined by different antibody sequences.

[0081] The "EGFRxNRP1 bispecific antibody" or "anti-EGFRxNRP1 bispecific antibody" is a bispecific binding molecule that includes an antigen-binding domain that specifically binds to the antigen EGFR1 and an antigen-binding domain that specifically binds to NRP1. The same nomenclature is used throughout for other bispecific binding molecules that bind to targets other than EGFR, such as "HER2xNRP1 bispecific antibody" or "cMETxNRP1 bispecific antibody", to represent a bispecific binding molecule that includes one antigen-binding domain that specifically binds to HER2 or cMET, respectively, and the other antigen-binding domain that specifically binds to NRP1.

[0082] As used herein, the term "heavy chain" can be interpreted to include a heavy chain variable region domain (VH) that includes an amino acid sequence having a variable region sequence sufficient to confer antigen specificity, and a full-length heavy chain that includes three heavy chain constant region domains CH1, CH2, and CH3, or a fragment thereof. Also, as used herein, the term "light chain" can be interpreted to include a light chain variable region domain (VL) that includes an amino acid sequence having a variable region sequence sufficient to confer antigen specificity and a light chain constant region domain (CL), and a full-length light chain that includes a fragment thereof. As used herein, the term "Fab" means the region that binds to an antigen. Fab consists of one variable heavy and light chain, and one constant heavy and light chain.

[0083] As used herein, the term "percent identity" between two sequences (e.g., amino acid or nucleotide sequences) means the percentage of positions (out of a possible 100%) that are identical when the sequences are optimally aligned and compared (with appropriate insertions or deletions made, if necessary, to obtain the optimal alignment). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for the optimal alignment of the two sequences and the length of each gap (e.g., percent identity = number of identical positions / total number of positions x 100). Comparison of sequences and determination of percent identity between two sequences can be carried out using mathematical algorithms, as described in the following non-limiting examples. Methods and algorithms for determining homology between two protein sequences are well established in the art.

[0084] For example, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol. (48):444-453) algorithm. This algorithm is incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com) and uses either the Blossum 62 matrix or the PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. Further, an amino acid sequence of a protein can be used as a "query sequence" for performing a search against a public database, for example, to identify related sequences. Such a search can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Mol. Biol. 215:403-10. To obtain an amino acid sequence homologous to the protein molecule of the present invention, a BLAST protein search can be performed using the XBLAST program, score = 50, word length = 3. To obtain a gapped alignment for comparison, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of each program (for example, XBLAST and NBLAST) can be used.

[0085] As used herein, the term "monoclonal antibody" means a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" means an antibody having a single binding specificity and having variable and constant regions derived from immunoglobulin sequences of the human germ line.

[0086] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to mean a cell into which an expression vector has been introduced, such as an expression vector encoding an antibody of the present invention. Examples of recombinant host cells include transfectedomas such as CHO cells, CHO-S cells, HEK cells, HEK293 cells, HEK-293F cells, Expi293F cells, PER.C6 cells or NSO cells, and lymphocyte cells.

[0087] "Treatment" means administering an effective amount of a therapeutically active bispecific binding molecule of the present invention for the purpose of alleviating, ameliorating, preventing or eradicating (curing) a symptom or disease state.

[0088] The term "effective amount" or "therapeutically effective amount" means an amount effective for the dosage and period required to achieve the desired therapeutic result. The therapeutically effective amount of a bispecific binding molecule can vary depending on factors such as the disease state of the subject, age, sex, weight, and the ability of the bispecific binding molecule to induce the desired response in the subject. A therapeutically effective amount is also an amount where the toxicity or adverse effects of the antibody or antibody portion do not outweigh the therapeutically beneficial effects.

[0089] As used herein, the terms "in combination" or "co-administered" can be used interchangeably to mean the use of one or more therapies (e.g., one or more prophylactic and / or therapeutic agents). The use of the terms does not limit the order in which the therapeutic agents (e.g., prophylactic and / or therapeutic agents) are administered to the subject.

[0090] As used herein, the term "synergistic" means a combination of a polypeptide of the present invention with another therapy (e.g., a prophylactic or therapeutic agent), and this combination is more effective than the additive effects of those therapies.

[0091] The term "pharmaceutically acceptable salt" means salts of the active bispecific antibody prepared using relatively non-toxic acids or bases depending on the specific substituents found in the bispecific antibodies described herein.

[0092] As used herein, the term "parenteral" includes, but is not limited to, subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intramedullary, intraliver, topical and intracranial injection or infusion techniques.

[0093] The term "carrier" means a vehicle used in formulating a composition and can be composed of a plurality of excipients.

[0094] As used herein, the term "excipient" means a pharmacologically inactive, natural or synthetic component or substance that is formulated either concomitantly with (e.g., in combination with) or subsequent to the active ingredient of the present invention. In some embodiments, an excipient can be any additive, adjuvant, binder, bulking agent, carrier, coating agent, diluent, disintegrant, filler, lubricant, preservative, vehicle, or combination thereof into which the recombinant polypeptide of the present invention can be administered, or is useful in preparing the composition of the present invention. Excipients include materials known in the art that are non-toxic and do not interact with other components of the composition. In some embodiments, an excipient can be formulated with the recombinant polypeptide (and thus is often referred to as a bulking agent, filler, or diluent) for the purpose of bulking up the composition when preparing the composition. In other embodiments, an excipient can be used to impart an enhancing effect on the active ingredient in the final formulation, such as promoting absorption and / or solubility. In yet other embodiments, an excipient can be used to provide stability or prevent contamination (e.g., microbial contamination). In other embodiments, an excipient can be used to impart physical properties to the composition (e.g., a composition in the physical form of dry granules or dry flowing powder). Excipients include both one excipient and two or more excipients. Suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences, by E.W. Martin, the entire contents of which are incorporated herein by reference.

[0095] Unless the context otherwise requires, as used herein, the terms "comprising", "including", or variations thereof such as "comprise" or "include" are to be understood to mean the inclusion of the stated step, element, or integer, or group of steps, elements, or integers, but not the exclusion of other steps, elements, or integers, or group of elements, steps, or integers.

[0096] All patent applications, patents, and publications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Also, all patent applications, patents, and publications cited herein are incorporated by reference in their entirety herein, except for definitions, disclaimers of subject matter, or where the incorporated material is inconsistent with the explicit disclosure herein (in which case the description of the present disclosure controls).

[0097] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be included within the scope of the appended claims. Further, all numerical values are approximations and are provided for illustrative purposes only.

[0098] Bispecific antibody (EGFRxNRP Ab) having an EGFR binding domain and an NRP1 binding domain As described above, the present invention relates to a bispecific binding molecule that specifically binds to a target protein and neuropilin-1 (NRP1).

[0099] Bispecific antibodies have many advantages compared to monoclonal antibodies. First, bispecific binding molecules have two effective targets in one molecule, so the production efficiency is high. For example, bispecific binding molecules can target multiple immune cell receptors (CD3, CD16, CD47) or immune checkpoint proteins (PD1, LAG-3, CTLA-4), or both. For example, bispecific antibodies can target tumor-related essential receptors such as EGFR, and immune cell receptors or immune checkpoint regulators such as neuropilin 1 (NRP1). Bispecific antibodies for anti-cancer drug treatment are designed to enhance the anti-cancer effect.

[0100] EGFR binding domain Epidermal growth factor receptor (EGFR) is one of the most frequently mutated oncogenes in solid cancers. Increased EGFR signaling promotes proliferation and cell survival in many cancer types, such as breast cancer, prostate cancer, non-small cell lung cancer (NSCLC), esophageal gastric cancer, liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, pancreatic cancer, colon cancer, and rectal cancer. Increased EGFR signaling can occur due to overexpression of EGFR, mutations in EGFR or signaling factors within the EGFR signaling pathway that result in constitutive activation, and / or increased levels of EGFR cognate ligands such as EGF, tumor necrosis factor α (TGF-α), amphiregulin (AREG), epigen, β-cellulin, heparin-binding EGF (HB-EGF), and epiregulin. EGFR signaling activates downstream signaling cascades including the RAS-RAF-MEK-ERK and PI3K-Akt-mTOR axes, leading to cancer cell proliferation and survival. Unfortunately, anti-EGFR agents such as tyrosine kinase inhibitors, monoclonal antibodies, and radiotherapy are only effective in some cancer types, such as metastatic colorectal cancer, non-small cell lung cancer (NSCLC), and advanced head and neck cancer. Furthermore, only some subjects have improved survival with treatment, and the initial effect usually ends due to drug resistance. Therefore, new rational designs are needed to improve the treatment effect.

[0101] Aspects of the disclosure further provide a method of treating cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a bispecific binding molecule. In some aspects, the disclosure provides the use of a bispecific binding molecule described herein in the manufacture of a medicament for treating cancer.

[0102] In certain aspects of the bispecific binding molecule, the disclosure provides a method of treating EGFR-overexpressing cancer in a subject in need thereof, comprising administering a therapeutically effective amount of the bispecific binding molecule and / or a pharmaceutically acceptable carrier or diluent provided herein. In certain aspects, the EGFR-overexpressing cancer is non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, colorectal cancer, squamous cell carcinoma of the head and neck, gastroesophageal junction adenocarcinoma, hepatocellular carcinoma, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, renal cancer, and pancreatic cancer, or other solid cancer tissues that overexpress an EGFR or EGFR kinase domain mutation. In some aspects of the method of treatment, the method further comprises administering to the subject an agent that increases cellular EGFR expression.

[0103] In certain aspects of the treatment method, the EGFR-overexpressing cancer is resistant to treatment with an anti-EGFR monoclonal antibody. In certain aspects of the treatment method, the anti-EGFR monoclonal antibody is amivantamab, cetuximab, depatuxizumab, depatuxizumab mafodotin, durvalumab, futuximab, GC1118, imigatamab, matuzumab, necitumumab, nimotuzumab, panitumumab, zalutumumab, or HumMR1. In some aspects of the treatment method, the subject is human. In some aspects, the EGFR overexpression is resistant or refractory to an EGFR tyrosine kinase inhibitor. In some aspects, the EGFR-overexpressing cancer is refractory to osimertinib.

[0104] An antibody is a protein that binds to an antigen with high specificity and high affinity and can neutralize the activity of the antigen. Anti-EGFR antibodies block ligand-activated EGFR signaling, induce receptor endocytosis, and induce EGFR degradation by the proteasome. When a mutation occurs in the EGFR-binding domain of an antibody, the subject may acquire resistance to anti-EGFR antibodies during the treatment period. Cetuximab, a mouse / human chimeric monoclonal antibody, and panitumumab, a fully humanized monoclonal antibody, have different binding domains on EGFR, so panitumumab is effective even after resistance to cetuximab has occurred, and vice versa. Therefore, a subject who has acquired resistance to a certain anti-EGFR antibody can be administered another anti-EGFR antibody that targets a different EGFR-binding domain.

[0105] Panitumumab is an IgG2 anti-EGFR humanized monoclonal antibody that binds to EGFR with approximately 8-fold higher affinity than cetuximab, an IgG1 anti-EGFR chimeric human / mouse monoclonal antibody (Garcia-Foncillas et al. 2019). Before inducing endocytosis, antibody binding can induce an immune response. The binding of panitumumab induces antibody-dependent cell-mediated cytotoxicity (ADCC) and initiates antibody-dependent cell phagocytosis (ADCP) through the activation of neutrophils and monocytes (Schneider-Merk et al. 2010). The IgG1 Fc domain of cetuximab binds to the FcγRIIIA (CD16) receptor on natural killer (NK) cells and induces ADCC. Activated NK cells secrete perforin and granzyme to lyse cancer cells and release immunostimulatory molecules such as interferon-γ (IFN-γ), TNF-α, chemokines, and granulocyte macrophage colony-stimulating factor (GM-CSF). The secretion of cytokines by NK cells stimulates the maturation of dendritic cells, cross-talk of NK cells, and co-expression of CD137. The expression of CD137 recruits anti-EGFR CD8 + T cells and increases the killing of EGFR-expressing cancer cells. Mature dendritic cells further activate NK cells and cytotoxic CD8 +It presents tumor antigens to T cells. The IgG1 Fc region also binds to Fc receptors on macrophages or plasmacytoid dendritic effector cells, or to the first subunit of the C1 complement complex (C1q), initiating antibody-dependent cell-mediated phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC) by the cells, respectively. Similar to cetuximab, anti-EGFR antibodies such as cetuximab, necitumumab, and nimotuzumab also have an IgG1 Fc region for ADCC induction.

[0106] NRP1 binding domain Cancer cells overexpress angiogenesis-promoting factors that promote the rapid growth of new blood vessels. There are abnormalities in the blood vessels surrounding the tumor, with capillaries constricting and the overall blood flow to the tumor decreasing. The decrease in blood flow increases the tumor interstitial fluid pressure and reduces the drug flow from the blood vessels to the tumor (Milosevic et al., 1999). Unlike normal tissues, tumors have few lymphatic vessels, which also contributes to abnormal angiogenesis and high tumor interstitial pressure. Inhibition of angiogenesis targeting vascular endothelial growth factor-A (VEGF165) can normalize angiogenesis and vascular fluid pressure and increase the accumulation of therapeutic drugs at the tumor site (Marcucci et al. 2013). However, anti-VEGF antibodies such as bevacizumab have side effects and are effective only for a narrow range of exposures (Kamba and McDonald, 2007).

[0107] Neuropilin-1 (NRP1) receptor and neuropilin-2 (NRP2) receptor are multifunctional single-pass transmembrane glycoproteins that play important roles in angiogenesis and lymphangiogenesis, respectively. The C-terminal regions of the VEGF ligand family and Sema3 ligands that bind to NRP1 and NRP2 bind to the arginine-binding pocket of the b1 domain of NRP1 and NRP2 (Parker et al., 2012). Binding to the arginine-binding pocket occurs through a motif of R / K-x-x-R / K (R = arginine, K = lysine, x = any amino acid), which is commonly present in the C-terminal regions of NRP-binding ligands and is known as "C-end rule" (CendR) (Teesalu et al. 2009). Proteins or peptides containing the C-end rule sequence can bind to NRP through the C-terminal arginine (Arg) residue or lysine (Lys) residue (Zanuy et al, 2013).

[0108] Silencing NRP2 significantly increases EGFR expression in lung cancer cells and gastric cancer cells, so selectively targeting NRP1 is important for anti-EGFR cancer therapy (Rizzolio et al., 2017). Furthermore, NRP1 is overexpressed in many cancer cells such as colon cancer, melanoma, glioblastoma, lung cancer, prostate cancer and pancreatic ductal adenocarcinoma, and plays an important role in cancer progression (Graziani and Lacal, 2015). In addition to being overexpressed in various cancer types, NRP1 is also overexpressed in tumor-associated endothelial cells. NRP1 is vascular endothelial growth factor (VEGF 165) It functions as a coreceptor for ligands involved in angiogenesis, such as class 3 semaphorin ligands, integrin β1, TGF-β, HGF, FGF, PDGF, and galectin-1. NRP1 interacts with receptor tyrosine kinases (RTKs) such as VEGFR1 and VEGFR2 and contributes to VEGFR signaling that promotes angiogenesis. NRP1 also binds to secreted class 3 semaphorin ligands (Sema3A, Sema3B, Sema3C, Sema3D, Sema3E, Sema3F, Sema3G) and functions as a coreceptor for plexin family receptors that regulate angiogenesis.

[0109] Blocking NRP1 ligand binding with an anti-NPR1 specific binding domain reduces the expression of the endothelial adhesion molecule VE-cadherin, and as a result, increases the extravasation of anti-EGFR antibodies from blood vessels. Increased extravasation reduces the interstitial pressure of the tumor and allows drugs to flow more freely from blood vessels into the tumor environment.

[0110] The epithelial barrier around solid tumors consists of an intercellular space densely filled with stromal epithelial cells linked by cell adhesion factors, which hinders the penetration of therapeutic drugs into the tumor. Overexpression of E-cadherin contributes to cell-cell adhesion. Since a substance that reduces E-cadherin was found in a virus (adenovirus-3), an example has been reported where only the part (JO-1) of the proteins constituting the virus that has the activity to reduce cellular E-cadherin at tight junctions was co-administered with an antibody, enhancing the anti-cancer effect of the antibody (Beyer et al. 2011). Targeting NRP1 reduces the expression of the epithelial barrier adhesion molecules E-cadherin and integrin β₁ subunit, which are overexpressed in many solid cancers, and increases the binding of the extracellular matrix, resulting in reduced drug permeability. Furthermore, the integrin β₁ subunit is involved in the activation of cell proliferation via growth factor receptors.

[0111] In summary, targeting NRP1 can reduce VEGF165 binding and decrease the expression of VE-cadherin, E-cadherin, and integrin β1, and as a result, angiogenesis and tumor interstitial pressure can be reduced, and the extravasation and tumor penetration of anti-EGFR antibodies can be increased.

[0112] Cancer cells produce immune checkpoint molecules that inhibit the immune response. NRP1 is an immune checkpoint molecule, and its expression is increased in tumor-associated endothelial cells and cancer cells. The expression of NRP1 induces immunosuppression by enhancing Treg activity and reducing tumor-specific CD8 + T cell responses (Chuckran et al. 2020). Thus, the NRP1 binding domain not only increases the extravasation and penetration of drugs but also functions as an immune checkpoint inhibitor. In yet another aspect, the present disclosure provides a bispecific binding molecule that targets NRP1, reduces the immunosuppression of cancer-mediated checkpoint molecules, permits innate immunity at the tumor site, and enhances the potential for a tumor response.

[0113] The anti-NRP1 antibody MNRP1685A, also known as besencumab, competitively binds to VEGF165 on NRP1 and has the function of affecting angiogenesis, cell survival, migration, adhesion, and invasion by inhibiting VEGF signaling via VEGFR2 (Pan Q et al. 2007). However, subjects administered besencumab in a phase I trial for advanced solid cancers endured intolerable side effects such as gastrointestinal bleeding, fungemia, duodenal obstruction, thrombocytopenia, proteinuria, alopecia, dysphonia, fatigue, and nausea, and further trials were aborted (Weekes et al., 2014, Patnaik et al., 2014).

[0114] NRP1 exhibits activity as a homodimer or heterodimer, and monomeric peptides such as the cleavable transmembrane peptide iRGD have a weak ability to control the biological activity of NRP1 (Sugahara et al. 2010). Therefore, a peptide that selectively binds to NRP1 as a homodimer and regulates its biological activity is preferred. Unfortunately, although the Fc heavy chain fusion-type NRP-binding peptide A22p is presented as a double peptide (homodimer), A22p binds to both NRP1 and NRP2 (Shin et al. 2014). Thus, it is important to identify an effective homodimeric and non-toxic NRP1-selective targeting molecule.

[0115] The NRP1-binding domain includes an antibody or an NRP1-binding fragment thereof. For example, an NRP1 antibody known in the art or all or part of the anti-NRP1 antibody provided herein can be used. Non-limiting examples of the anti-NRP1 mAb heavy chain polypeptide sequences are set forth in SEQ ID NOs: 41-47, which include the N-terminal NPR1-binding domain. In some embodiments, the monoclonal antibody heavy chain polypeptide includes an N-terminal NRP1-binding domain that includes a variable heavy chain (VH) and a constant heavy chain 1 (CH1), and an Fc domain that includes a constant heavy chain 2 (CH2) and a constant heavy chain 3 (CH3). In some embodiments, the monoclonal antibody includes a light chain polypeptide sequence set forth in any of SEQ ID NOs: 48-54. Thus, the heavy chains of SEQ ID NOs: 41-47 can pair with the light chains of SEQ ID NOs: 48-54, respectively. The polynucleotide sequences encoding the heavy chain and light chain polypeptides are set forth in SEQ ID NOs: 55-61 and 62-68, respectively.

[0116] Bispecific antibody that binds to EGFR and NRP1 The present disclosure provides a bispecific antibody comprising a heavy chain fusion polypeptide having an N-terminal EGFR binding domain at the N-terminus of the heavy chain polypeptide and an NRP1 binding domain at the C-terminus. The heavy chain fusion polypeptide of the bispecific binding molecule comprises an N-terminal EGFR binding region comprising a variable heavy chain (VH) and a constant heavy chain 1 (CH1), a constant heavy chain 2 (CH2), an Fc domain comprising a constant heavy chain 3 (CH3), and a short-chain variable fragment (scFv) comprising an NRP1 binding region fused to the C-terminus of CH3. The scFv comprising the NRP1 binding domain is formed by fusing a variable heavy chain (VH) and a variable light chain (VL) with a flexible peptide linker.

[0117] A schematic diagram of a representative bispecific antibody structure of the present disclosure is shown in FIG. 1.

[0118] Exemplary bsAb heavy chain polypeptide sequences are set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 39, and the polynucleotide sequences encoding the heavy chain antibody polypeptide sequences of the bispecific antibodies are set forth in SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 40, respectively. In some embodiments, the bispecific antibody further comprises a light chain polypeptide sequence comprising a variable light chain and a constant light chain set forth in SEQ ID NO: 12 that pairs with the heavy chain polypeptide. The polynucleotide sequence encoding the light chain polypeptide of SEQ ID NO: 12 is set forth in SEQ ID NO: 25. In some embodiments, the present disclosure provides polynucleotide sequences used to encode GGGGS subunits, and these sequences are set forth in SEQ ID NOs: 26-38. Table 1 shows the polypeptide sequences of the bispecific antibodies, and Table 2 shows the polynucleotide sequences encoding the polypeptides. Example 1 describes a method for preparing the bispecific antibody.

[0119] In some embodiments, the bispecific antibody comprises (or consists of) an anti-EGFR binding arm comprising the VH amino acid sequence set forth in SEQ ID NO: 69 and the VL amino acid sequence set forth in SEQ ID NO: 70. In some embodiments, the bispecific antibody comprises (or consists of) an anti-EGFR binding arm comprising a heavy chain CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences set forth in SEQ ID NOs: 71, 72, and 73, respectively. In some embodiments, the bispecific antibody comprises (or consists of) an anti-EGFR binding arm comprising a heavy chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 73. In some embodiments, the bispecific antibody comprises (or consists of) an anti-EGFR binding arm comprising a light chain CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences set forth in SEQ ID NOs: 74, 75, and 76, respectively. In some embodiments, the bispecific antibody comprises (or consists of) an anti-EGFR binding arm comprising a light chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 76. In some embodiments, the anti-EGFR binding arm comprises (or consists of) one or more sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.8% sequence identity to any of the above VH, VL, HCDR or LCDR sequences.

[0120] In some embodiments, the bispecific antibody comprises (or consists of) an anti-NRP1 binding arm that comprises the VH amino acid sequence set forth in SEQ ID NO: 77 and the VL amino acid sequence set forth in SEQ ID NO: 78. In some embodiments, the bispecific antibody comprises (or consists of) an anti-NRP1 binding arm that comprises a heavy chain CDR1 region, a CDR2 region, and a CDR3 region that comprise the amino acid sequences set forth in SEQ ID NOs: 79, 80, and 81, respectively. In some embodiments, the bispecific antibody comprises (or consists of) an anti-NRP1 binding arm that comprises a heavy chain CDR3 region that comprises the amino acid sequence set forth in SEQ ID NO: 81. In some embodiments, the bispecific antibody comprises (or consists of) an anti-NRP1 binding arm that comprises a light chain CDR1 region, a CDR2 region, and a CDR3 region that comprise the amino acid sequences set forth in SEQ ID NOs: 82, 83, and 84, respectively. In some embodiments, the bispecific antibody comprises (or consists of) an anti-NRP1 binding arm that comprises a light chain CDR3 region that comprises the amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the anti-NRP1 binding arm comprises (or consists of) one or more sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.8% sequence identity to any of the above VH, VL, HCDR, or LCDR sequences.

[0121] Accordingly, in one embodiment, the invention provides (a) two identical heavy chain polypeptides, wherein the first heavy chain polypeptide is fused to a first scFv by a peptide linker to produce a first heavy chain fusion polypeptide, and the second heavy chain is fused to a second scFv by a peptide linker to produce a second heavy chain fusion polypeptide, wherein the first and second scFvs are identical and bind to NRP1; and (b) two identical light chains comprising a first light chain and a second light chain, A composition comprising a bispecific binding molecule, wherein the VL and VH of the heavy chain fusion polypeptide and the light chain polypeptide comprise a target protein binding domain that binds to a target protein, and the scFv comprises an NRP1 binding domain that binds to NRP1.

[0122] In some embodiments of the bispecific binding molecule, the heavy chain fusion polypeptide pairs with the light chain polypeptide. In some embodiments, the heavy chain polypeptide comprises: (a) two identical heavy chain polypeptides, wherein the first heavy chain polypeptide is fused to a first scFv by a peptide linker to produce a first heavy chain fusion polypeptide, and the second heavy chain is fused to a second scFv by a peptide linker to produce a second heavy chain fusion polypeptide, wherein the first and second scFvs are identical and bind to NRP1; and (b) two identical light chains comprising a first light chain and a second light chain, wherein the VL and VH of the heavy chain fusion polypeptide and the light chain polypeptide comprise a target protein binding domain that binds to a target protein, and the scFv comprises an NRP1 binding domain that binds to NRP1.

[0123] Thereby, bispecific antibodies with different binding affinities for EGFR and NRP1 are provided. In one embodiment, it is preferred that the binding affinity for NRP1 is lower than the binding affinity for EGFR. In some embodiments,

[0124] Therefore, the present invention provides (a) two identical heavy chain polypeptides, wherein the first heavy chain polypeptide is fused to a first scFv by a peptide linker to produce a first heavy chain fusion polypeptide, and the second heavy chain is fused to a second scFv by a peptide linker to produce a second heavy chain fusion polypeptide, wherein the first and second scFvs are identical and bind to NRP1; and (b) two identical light chains comprising a first light chain and a second light chain, Regarding bispecific binding molecules comprising, wherein the VL and VH of the heavy chain fusion polypeptide and the light chain polypeptide comprise target protein binding domains that bind to a target protein, and the scFv comprises an NRP1 binding domain that binds to NRP1.

[0125] In some embodiments of the bispecific binding molecule, the heavy chain fusion polypeptide pairs with the light chain polypeptide. In some embodiments, the heavy chain polypeptide comprises: (a) two identical heavy chain polypeptides, wherein the first heavy chain polypeptide is fused to the first scFv by a peptide linker to produce a first heavy chain fusion polypeptide, and the second heavy chain is fused to the second scFv by a peptide linker to produce a second heavy chain fusion polypeptide, wherein the first and second scFvs are identical and bind to NRP1; and (b) two identical light chains comprising a first light chain and a second light chain, wherein the VL and VH of the heavy chain fusion polypeptide and the light chain polypeptide comprise target protein binding domains that bind to a target protein, and the scFv comprises an NRP1 binding domain that binds to NRP1.

[0126] In some embodiments, the present disclosure provides polynucleotide sequences used to encode GGGGS subunits, and these sequences are set forth in SEQ ID NOs: 26-38. Table 1 shows the polypeptide sequences of the bispecific binding molecules, and Table 2 shows the polynucleotide sequences encoding the polypeptides. Example 1 describes a method for producing the bispecific binding molecule.

[0127] In some embodiments, the EGFR binding domain of the bispecific binding molecule has an EGFR binding affinity (K D ) in the sub-nanomolar range (<0.1 nM). In some embodiments, the NRP1 binding domain of the bispecific binding molecule has an NRP1 affinity (K D) It has. Thus, in one aspect, the present disclosure provides a bispecific antibody having an affinity for EGFR in the sub-nanomolar range and an NRP1 binding domain having an affinity for NRP1 in the sub-nanomolar range, as shown in Table 2 of Example 2. Figures 2A-2K show the binding affinity curves of bispecific binding molecules to immobilized huEGFR. In some embodiments, the binding affinity of the bispecific binding molecule for EGFR is <0.1 nM. In some embodiments, the binding affinity of the bispecific binding molecule for EGFR is <0.01 nM. Figures 3A-3K show the binding affinity curves of the double binding molecules to immobilized huNRP1. In some embodiments, the binding affinity of the bispecific binding molecule for NRP1 ranges from 0.01 nM to 1000 nM. In some embodiments, the binding affinity of the bispecific binding molecule for NRP1 ranges from 0.1 nM to 100 nM.

[0128] One aspect of the present disclosure provides a bispecific antibody having an asymmetric binding affinity for EGFR and NRP1. In some embodiments, the K of the EGFR binding domain for EGFR D is at least 2-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 100-fold, or more than 100-fold greater than the K of the NRP1 binding domain for NRP1 D . The asymmetric affinity reduces the cytotoxicity targeting NRP1 while providing better induction to cancer cells expressing EGFR.

[0129] Inhibiting the binding of VEGF to NRP1 using the NRP1 binding domain of the bispecific binding molecule can inhibit the activation of VEGFR2 as measured by phosphorylation of VEGFR2. However, the bispecific antibody having the heavy chain of SEQ ID NO: 1 has a K of 69.7 nM for NRP1 Dhas, but does not inhibit VEGFR phosphorylation (Figure 4). In some embodiments, the bispecific binding molecule inhibits VEGFR2 phosphorylation to prevent angiogenesis (see Table 3 and Figures 5A - 5B and 11A - 11B). In some embodiments, the inventors have discovered that the dissociation constant (K D ) of the NRP1 binding domain does not reflect the ability of the bispecific binding molecule to inhibit VEGFR2 phosphorylation. For example, the bispecific binding molecule having the heavy chain SEQ ID NO: 6 inhibits 50% of VEGFR2 phosphorylation at 10,130 nM (IC50), while the KD of NRP1 is 3.76 nM, whereas the bispecific binding molecule having the heavy chain SEQ ID NO: 4 inhibits 50% of VEGFR2 phosphorylation at 410 nM (IC50), while the KD of NRP1 is 8.8 nM. Thus, in some embodiments, the present disclosure provides bispecific antibodies in which the NRP1 binding domain affinity and biological function are unbalanced with respect to the inhibition of VEGFR2 phosphorylation.

[0130] In some embodiments, the bispecific antibody inhibits cancer cell proliferation. In some embodiments, the Fc domain of the bispecific binding molecule comprising constant heavy chains 2 and 3 (CH2 and CH3) is of the IgG1 or IgG2 subclass. In some embodiments, the present disclosure provides that a bispecific binding molecule having an EGFR binding domain and an NRP1 binding domain of an scFv potently inhibits the cell proliferation of cancer cell line H1975 (see Table 4 and Figures 12A and 12B). This result indicates that the scFv is important for the function of the bispecific binding molecule as a degrader. For NRP1, Example 4 describes the method used to measure the IC 50 of the bispecific binding molecule for cell proliferation inhibition in the H1975 cancer cell line.

[0131] Further bispecific antibody having binding affinity for NRP1 and receptor tyrosine kinase (RTK) In addition to the above-described EGFRxNRP1 bispecific antibody construct, in other aspects, the present disclosure provides additional bispecific antibody constructs having binding affinity for receptor tyrosine kinases (RTKs) other than NRP1 and EGFR. Non-limiting examples include the following representative constructs.

[0132] cMETxNRP1 bispecific construct In one aspect, the RTK is a cMET family member. In one aspect, the RTK is cMET. Receptor degradation via the cMETxNRP1 bispecific antibody construct is described in detail in Example 8.

[0133] In certain aspects, the cMETxNRP1 bispecific antibody comprises an anti-NRP1 binding arm comprising heavy chain CDR1, CDR2, and CDR3 regions comprising (or consisting of) the amino acid sequences set forth in SEQ ID NOs: 79, 80, and 81, respectively. In one aspect, the bispecific antibody comprises an anti-NRP1 binding arm comprising a heavy chain CDR3 region comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 81. In one aspect, the bispecific antibody comprises an anti-NRP1 binding arm comprising light chain CDR1, CDR2, and CDR3 regions comprising (or consisting of) the amino acid sequences set forth in SEQ ID NOs: 82, 83, and 84, respectively. In one aspect, the bispecific antibody comprises an anti-NRP1 binding arm comprising a light chain CDR3 region comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 84.

[0134] In one aspect, the cMET binding arm comprises the sequences of anti-cMET monoclonal antibodies (mAbs) known and available in the art, such as the heavy and light chain CDRs (1-3) of the mAb or the VH / VL polypeptide of the mAb. A number of anti-cMET mAbs have been described in the art, for example, in U.S. Pat. Nos. 7,476,724, 8,673,302 and 9,068,011, U.S. Patent Publications US2013 / 0216527, US2019 / 0248907 and US2021 / 0087278, and PCT Publication WO2010 / 064089, and the entire contents of each of these (including the anti-cMET antibody sequences) are expressly incorporated herein by reference. Non-limiting examples of anti-cMET mAbs known in the art include emibetuzumab (also known as LY2875358 in the art) (e.g., Rosen et al. (2017) Clin. Cancer Res. 23:1910-1919; Yan et al. (2018) Invest. New Drug 36:536-544); onartuzumab (e.g., Merchant et al. (2013) Proc. Natl. Acad. Sci. USA 110:e2987-e2996; Spigel et al. (2018) J. Clin. Oncol. 35:412-420) and telisotuzumab (e.g., Camidge et al. (2018) Annals Oncol. 29:496-497; Camidge et al. (2022) JTO Clin. Res. Rep. 3:100262), and the entire contents of each of these references are expressly incorporated herein by reference.

[0135] HER2xNRP1 bispecific construct In one aspect, the RTK is a HER family member. In one aspect, the RTK is HER2. Receptor degradation by the HER2xNRP1 bispecific antibody construct is described in detail in Example 9.

[0136] In certain embodiments, the HER2xNRP1 bispecific antibody comprises an anti-NRP1 binding arm that comprises a heavy chain CDR1 region, a CDR2 region, and a CDR3 region that comprises (or consists of) the amino acid sequences set forth in SEQ ID NOs: 79, 80, and 81, respectively. In one embodiment, the bispecific antibody comprises an anti-NRP1 binding arm that comprises a heavy chain CDR3 region that comprises (or consists of) the amino acid sequence set forth in SEQ ID NO: 81. In certain embodiments, the bispecific antibody comprises an anti-NRP1 binding arm that comprises a light chain CDR1 region, a CDR2 region, and a CDR3 region that comprises (or consists of) the amino acid sequences set forth in SEQ ID NOs: 82, 83, and 84, respectively. In one embodiment, the bispecific antibody comprises an anti-NRP1 binding arm that comprises a light chain CDR3 region that comprises (or consists of) the amino acid sequence set forth in SEQ ID NO: 84.

[0137] In certain embodiments, the HER2 binding arm comprises the sequences of anti-HER2 monoclonal antibodies (mAbs) known and available in the art, such as the heavy and light chain CDRs (1-3) of the mAb or the VH / VL polypeptides of the mAb. Numerous anti-HER2 mAbs have been described in the art, such as in U.S. Patent No. 10,377,825, U.S. Patent Publications US2009 / 0226466, US2010 / 0047230, US2011 / 0313137, US2012 / 0309942, US2015 / 0322162, US2017 / 0066829, and US2018 / 06201692, as well as PCT Publication WO2013 / 075382, the entire contents of each of which (including the anti-cHER2 antibody sequences) are expressly incorporated herein by reference. Non-limiting examples of anti-HER2 mAbs known in the art include trastuzumab (e.g., Romond et al. (2005) New Engl. J. Med. 353:1673-1684; Hudis (2007) New Engl. J. Med. 357:39-51) and pertuzumab (e.g., Baselga et al. (2010) J. Clin. Oncol. 28:1138-1144; Swain et al. (2015) New Engl. J. Med. 372:724-734), the entire contents of which are expressly incorporated herein by reference. In one embodiment, the HER2xNRP1 bispecific construct comprises a heavy chain comprising (or consisting of) the amino acid sequence shown in SEQ ID NO: 90 and a light chain comprising (or consisting of) the amino acid sequence shown in SEQ ID NO: 91, which are encoded by the nucleic acid sequences of SEQ ID NOs: 92 and 93, respectively.

[0138] IGF1RxNRP1 bispecific construct In one embodiment, the RTK is a member of the IGF receptor family. In one embodiment, the RTK is IGF1R. Receptor degradation via the IGF1RxNRP1 bispecific antibody construct is described in detail in Example 10.

[0139] In certain embodiments, the IGF1RxNRP1 bispecific antibody comprises an anti-NRP1 binding arm that comprises a heavy chain CDR1 region, a CDR2 region, and a CDR3 region that comprise (or consist of) the amino acid sequences set forth in SEQ ID NOs: 79, 80, and 81, respectively. In one embodiment, the bispecific antibody comprises an anti-NRP1 binding arm that comprises a heavy chain CDR3 region that comprises (or consists of) the amino acid sequence set forth in SEQ ID NO: 81. In certain embodiments, the bispecific antibody comprises an anti-NRP1 binding arm that comprises a light chain CDR1 region, a CDR2 region, and a CDR3 region that comprise (or consist of) the amino acid sequences set forth in SEQ ID NOs: 82, 83, and 84, respectively. In one embodiment, the bispecific antibody comprises an anti-NRP1 binding arm that comprises a light chain CDR3 region that comprises (or consists of) the amino acid sequence set forth in SEQ ID NO: 84.

[0140] In certain embodiments, the IGF1R binding arm comprises the sequences of anti-IGF1R monoclonal antibodies (mAbs) known and available in the art, such as the heavy and light chain CDRs (1-3) of the mAb or the VH / VL polypeptides of the mAb. For example, U.S. Pat. Nos. 10,106,614, 10,112,998, and 10,519,245, U.S. Patent Publications US2010 / 0143340 and US2014 / 0079665, and PCT Publication WO2011 / 057064, etc. describe a number of anti-IGF1R mAbs, and the entire contents of these documents (including anti-IGF1R antibody sequences) are expressly incorporated herein by reference. Non-limiting examples of anti-IGF1R mAbs known in the art include ganitumab (also known to those skilled in the art as AMG479) (e.g., Moody et al. (2004) J. Endocrinol. 221:145-155; Tap et al. (2012) J. Clin. Oncol. 30:1849-1856), figitumumab (e.g., Molife et al. (2010) Brit. J. Cancer 103:332-339; Langer et al. (2014) J. Clin. Oncol. 32:2059-2066), cixutumumab (e.g., Rathkopf et al. (2011) J. Clin. Oncol. 29:e15081; Schwartz et al. (2013) Lancet 14:371-382), and daratumumab (e.g., Scartozzi et al. (2010) Curr. Opin. Mor. Therap. 12:361-371; Atzori et al. (2011) Clin. Cancer Res. 17:6304-6312), and the entire contents of these documents are expressly incorporated herein by reference.

[0141] Tumor growth inhibitory effect of bispecific binding molecules in xenograft mouse models In some aspects of the present disclosure, treatment with bispecific antibodies reduces tumor growth. In some aspects, a bispecific binding molecule having a heavy chain SEQ ID NO: 11 and a light chain SEQ ID NO: 12 significantly reduces the tumor growth rate. In some aspects, the addition of an scFv containing an NRP1 binding domain significantly increases the efficacy of tumor growth inhibition. Example 5 describes a method and results of treating H1975 xenograft mouse models with bispecific antibodies to inhibit tumor growth. Table 5 shows the percentage of inhibited tumor growth, and FIGS. 13A-13F show tumor growth over several days after transplantation.

[0142] Biological effects of bispecific binding molecules In summary, the present disclosure provides a bispecific antibody that binds an EGFR binding domain and an NRP1 binding domain to promote homing to EGFR-high expressing tumors by increasing EGFR affinity. This bispecific binding molecule has multiple biological effects that reduce cancer cell growth and survival, such as suppressing abnormal angiogenesis and increasing extravasation and penetration by reducing the expression of VE-cadherin, E-cadherin, and integrin β1. NRP1 targeting further reduces EGFR surface pooling, enhances EGFR downregulation, and inhibits NRP1 checkpoint immunosuppression. The combination of these features results in a bispecific binding molecule with a potent ability to suppress EGFR-mediated cancer cell growth and survival.

[0143] FcRn binding for transcytosis recycling Since the size of the antibody is ~150 kD, it has a long half-life in serum and the therapeutic effect persists for a long time. Furthermore, the antibody IgG Fc portion consisting of the heavy chain constant regions 2 and 3 (CH2 and CH3) binds to the neonatal Fc receptor (FcRn) and the Fcγ receptor (FcγR) on the cell surface, is endocytosed, recycled to the serum by transcytosis, and further increases the serum half-life of the antibody. Cancers with low expression of FcRn or FcγR are associated with poor prognosis (Pyzik et al., 2019), so recycling via FcRn or FcγR plays an important role in maintaining the serum concentration of the antibody. IgG1 antibodies have a more efficient recycling process of FcRn and FcγR than IgG2 antibodies. Thus, including the IgG1 Fc domain in therapeutic antibodies helps maintain the therapeutic level of the drug and reduce the number of administrations, and a shortened half-life is ideal for diagnostic tests or toxicity control.

[0144] In some embodiments, the polypeptide of the present invention comprises an immunoglobulin domain, and this immunoglobulin domain comprises a polypeptide comprising an immunoglobulin domain comprising an Fc domain selected from the IgG1 subclass.

[0145] Expression construct As used herein, the term "vector" or "expression vector" means a means for expressing a target gene in a host cell. For example, vectors may include viral vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, and the like. Recombinant vectors can be prepared by manipulating plasmids commonly used in the art (such as pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), phages (such as λgt4AB, λ-Charon, λAZ1, M13, etc.), or viruses (such as CMV, SV40, etc.). In some embodiments, the expression vector contains nucleic acids encoding the heavy chain fusion polypeptide and the light chain polypeptide of a bispecific binding molecule, wherein the sequence of the nucleic acid encoding the heavy chain fusion polypeptide is any one of SEQ ID NOs: 15 to 24, and the sequence of the nucleic acid encoding the light chain polypeptide is SEQ ID NO: 25.

[0146] In another aspect, the present disclosure provides an isolated polynucleotide encoding a bispecific binding molecule heavy and light chain amino acid sequence. The polynucleotide encoding in a recombinant vector may be operably linked to a promoter. As used herein, the term "operably linked" means a functional linkage between a nucleotide expression control sequence (such as a promoter sequence) and a second nucleotide sequence. Thus, the regulatory sequence can control the transcription and / or translation of the second nucleotide sequence. The recombinant vector may generally be constructed as a cloning vector or as an expression vector. As the expression vector, a vector generally used in the art for expressing foreign proteins derived from plants, animals or microorganisms can be used. The recombinant vector can be constructed by various methods known in the art. The recombinant vector can be constructed using eukaryotic cells having an origin of replication such as fl origin of replication, SV40 origin of replication, pMB1 origin of replication, adenovirus origin of replication, AAV origin of replication, CMV origin of replication, BBV origin of replication, etc. as a host, but is not limited thereto.

[0147] Furthermore, a promoter derived from the genome of mammalian cells (e.g., the metallothionein promoter) or a promoter derived from a virus of mammalian cells (e.g., the adenovirus anaphase promoter, the vaccinia virus 7.5K promoter, the SV40 promoter, the cytomegalovirus (CMV) promoter, or the thymidine kinase (TK) promoter of herpes simplex virus (HSV)) can be used, and the promoter generally has a polyadenylation sequence as a transcription termination sequence. The vector can express not only a peptide domain that specifically binds to NRP1 according to the present disclosure, but also an antibody having a peptide fused thereto, and a linker peptide. In the case of an antibody fused with a peptide, the vector can use both a vector system that expresses the peptide and the antibody or a fragment thereof in one vector, and a vector system that expresses the peptide and the antibody or a fragment thereof in separate vectors. In the latter case, the two vectors can be introduced into the host cell by co-transformation and targeted transformation. Thus, in another aspect, the present disclosure includes a vector containing the nucleic acid set forth in SEQ ID NOs: 14-38. Example 1 describes the characteristics of vectors that can be utilized to produce bispecific antibodies.

[0148] Another aspect of the present disclosure provides a host cell transformed with a recombinant vector. As the host cell, any type of host cell known in the art can be used. Examples of prokaryotic cells include strains belonging to the genus Escherichia coli such as Escherichia coli JM109 strain, Escherichia coli BL21 strain, Escherichia coli RR1 strain, Escherichia coli LE392 strain, Escherichia coli B strain, Escherichia coli X1776 strain, Escherichia coli W3110 strain, strains belonging to the genus Bacillus such as Bacillus subtilis, Bacillus thuringiensis, Salmonella typhimurium strain, Serratia marces strain, and enteric flora and strains such as various Pseudomonas genera. Transformation of prokaryotic cells is useful for large-scale plasmid cloning. Prokaryotic host cells may lack essential post-translational modifications required for antibody assembly and structure, and thus eukaryotic host cells, such as yeast (Saccharomyces cerevisiae), insect cells, plant cells, mammalian cells, such as SP2 / 0, CHO (Chinese hamster ovary) K1, CHO DG44, CHO-S, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RN, and MDCK cell lines are preferred. In some embodiments, the host cell comprises nucleic acids encoding a heavy chain fusion polypeptide and a light chain polypeptide of a bispecific binding molecule in an expression vector, wherein the sequence of the nucleic acid encoding the heavy chain fusion polypeptide is any one of SEQ ID NOs: 15 to 24, and the sequence of the nucleic acid encoding the light chain polypeptide is SEQ ID NO: 25.

[0149] Another aspect of the present disclosure provides a method for preparing a peptide that specifically binds to NRP1, the method comprising culturing the above-described host cell. The polynucleotide and the recombinant vector containing the polynucleotide can be inserted into the host cell using insertion methods well known in the art. For example, when the host cell is a prokaryotic cell, introduction can be carried out according to the CaC12 method, the electroporation method, etc., and when the host cell is a eukaryotic cell, the vector can be introduced into the host cell by various methods such as the microinjection method, the calcium phosphate precipitation method, the electroporation method, the transformation method via liposomes, and the gene bombardment method, but the introduction method is not limited thereto. The method for selecting the transformed host cell can be easily carried out according to methods well known in the art using the phenotype expressed by the selected label. For example, when the selected label is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic. Its acceptable salts, and pharmaceutically acceptable carriers, diluents, adjuvants, or vehicles. Pharmaceutically acceptable carriers include, for example, pharmaceutically diluents, excipients, or carriers appropriately selected for the intended dosage form and conforming to conventional pharmaceutical practices.

[0150] Antibody-drug conjugate In another aspect, the present disclosure provides an antibody-drug conjugate (ADC) comprising a bispecific antibody (proteolytic product) of the present disclosure conjugated to a drug or other functional compound, also referred to as a payload. ADC technology is well established in the art (e.g., described in Strohl and Strohl (eds) "Ch. 15: Antibody-Drug Conjugates" in Therapeutic Antibody Engineering (2012); Tumey (2020) Methods Mol.Biol.2078:1-22), and numerous ADC therapies have been approved by the FDA.

[0151] In an ADC, the payload is conjugated to the bispecific antibody via a linker and / or using site - specific conjugation chemistry established in the art.

[0152] For the use of linkers in ADCs, there is a review in, for example, Nareshkumar et al. (2015) Pharm. Res. 32:3526 - 3540. In one aspect, the linker is a cleavable linker. In another aspect, the linker is a non - cleavable linker. Suitable linkers based on chemical motifs such as disulfide, hydrazone, peptide, thioether, etc. are available in the art.

[0153] For the use of various site - specific conjugation chemistries in ADCs, there is a review in, for example, Zhou et al. (2017) Biomedicines 5:64. These methods bind the payload to specifically defined sites in the antibody portion of an ADC (i.e., the bispecific antibody of the present disclosure) that includes cysteine, glutamine, non - natural amino acids, short peptide tags, and sugar chains.

[0154] In one aspect, the payload used in an ADC is toxic to cells (i.e., exhibits cytotoxicity). In another aspect, the payload used in an ADC is non - toxic to cells (i.e., does not exhibit cytotoxicity).

[0155] In certain embodiments, the payload used in an ADC functions as a cytotoxic or cytolytic agent. In certain embodiments, the payload used in an ADC functions to enhance one or more pharmacokinetic properties of the molecule, such as serum half - life. In certain embodiments, the payload used in an ADC functions to target the molecule to a desired microenvironment (e.g., a target cell type or tissue). In certain embodiments, the payload used in an ADC confers one or more immunomodulatory properties to the molecule. In certain embodiments, the payload used in an ADC confers one or more enzymatic properties to the molecule.

[0156] In certain embodiments, the payload used in the ADC is a cytotoxic or cytolytic agent, and non-limiting examples thereof include small molecule drugs (e.g., chemotherapeutic agents), protein toxins, bacterial toxins, cytolytic proteins / peptides, and radionuclides.

[0157] In certain embodiments, the payload conjugated to the bispecific antibody functions by a mechanism selected from the group consisting of folate receptor targeting, microtubule inhibition, DNA cleavage, and TOP1 inhibition.

[0158] In certain embodiments, the payload conjugated to the bispecific antibody belongs to a class of payloads selected from the group consisting of maytansinoids (e.g., maytansinoid DM4), MMAE / auristatin, and camptothecin.

[0159] In certain embodiments, the payload conjugated to the bispecific antibody is selected from the group consisting of vedotin, emtansine, givituximab, talirine, ozogamicin, pasudotox, deruxtecan, mafodotin, solabubtansine, and tesirine.

[0160] In certain embodiments, the payload conjugated to the bispecific molecule is a radionuclide, non-limiting examples of which include 90 Y and 111 In.

[0161] In certain embodiments, the payload conjugated to the bispecific molecule is a protein toxin, such as Pseudomonas endotoxin or diphtheria toxin.

[0162] In certain embodiments, the payload that binds to the bispecific molecule is an immunomodulatory peptide, such as Fas ligand (FasL).

[0163] In certain embodiments, the payload conjugated to the bispecific molecule is a biologically active peptide, such as to extend the pharmacological half-life (e.g., GLP1) or to target specific cells. For example, calcitonin has been used as a target for ADCs to osteoclasts (Newa et al. (2011) Res. 28:1131-1143).

[0164] In some embodiments, the payload conjugated to the bispecific molecule is an enzyme.

[0165] In certain embodiments, the payload conjugated to the bispecific molecule is an oligonucleotide.

[0166] Pharmaceutical composition Embodiments of the disclosure further include a composition. According to some embodiments, the composition of the disclosure includes the bispecific binding molecule of the disclosure. For example, the bispecific binding molecule can be any of the bispecific binding molecules described in the above section on bispecific antibodies herein, the description of which is incorporated herein by reference for the sake of brevity and not repeated herein.

[0167] The polypeptides described herein can be formulated into pharmaceutical compositions further comprising a pharmaceutically acceptable carrier, diluent, adjuvant, or vehicle. In one embodiment, the disclosure provides a pharmaceutical composition comprising the disclosed polypeptide, and a pharmaceutically acceptable carrier, diluent, adjuvant, or vehicle. In one embodiment, the invention is a pharmaceutical composition comprising the bispecific antibody described herein or a pharmaceutically effective amount thereof.

[0168] A pharmaceutically acceptable carrier or excipient can include an inert component that does not overly inhibit the biological activity of the polypeptide. The pharmaceutically acceptable carrier is desirably biocompatible, e.g., non-toxic, non-inflammatory, non-immunogenic, or free of other undesirable reactions or side effects upon administration to a subject. Standard formulation techniques can be used.

[0169] The pharmaceutically acceptable carriers, adjuvants, or vehicles used herein include any or all solvents, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, etc. that are suitable for the particular desired dosage form. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutically acceptable compositions and known techniques for their preparation. The use thereof is contemplated to be within the scope of the invention, except when any conventional carrier medium is incompatible with the polypeptides described herein, such as by producing an undesirable biological effect or interacting in a detrimental manner with other components of the pharmaceutically acceptable composition. As used herein, the term "side effect" includes undesirable side effects and adverse effects of treatment.

[0170] Materials that can function as pharmaceutically acceptable carriers for antibodies enhance conformational stability, reduce protein dynamics, inhibit aggregation, protect proteins that adsorb to the liquid-air interface, and include cyclodextrin hydrogels, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffering substances (such as Tween 80, phosphate, glycine, sorbic acid, potassium sorbate, etc.), partial glyceride mixtures of vegetable saturated fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, etc.), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylate, wax, polyethylene - polyoxypropylene block polymer, methylcellulose, hydroxypropylmethylcellulose, lanolin, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; glycols such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide, aluminum hydroxide; alginic acid; pyrogen - free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solution, and other non - toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening agents, flavoring agents and perfuming agents, preservatives and antioxidants may also be present in the composition according to the judgment of the formulator.

[0171] In some embodiments, the compositions of the invention include pharmaceutically acceptable salts. When the polypeptides of the invention include relatively acidic functional groups, the base addition salts can be obtained by contacting the neutral form of such polypeptides with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium salts, potassium salts, calcium salts, ammonium salts, organic amino salts, or magnesium salts, or similar salts. When the polypeptides of the invention include relatively basic functional groups, the acid addition salts can be obtained by contacting the neutral form of such polypeptides with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid, or phosphorous acid, as well as salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, oxalic acid, methanesulfonic acid, etc. Also included are salts of amino acids such as alginate, and salts of organic acids such as glucuronic acid or galacturonic acid (see, e.g., Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain polypeptides of the present disclosure include both basic and acidic functional groups that allow the polypeptide to be converted into either a basic or acid addition salt.

[0172] Accordingly, the disclosed polypeptides can exist as salts with pharmaceutically acceptable acids and the like. The present invention includes such salts. Non-limiting examples of such salts include hydrochloride, hydrobromide, phosphate, sulfate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, propionate, tartrate (e.g., (+)-tartrate, (-)-tartrate, or mixtures thereof including racemic mixtures), succinate, benzoate, and salts with amino acids such as glutamate, and quaternary ammonium salts (e.g., methyl iodide, ethyl iodide, etc.). These salts can be prepared by methods known to those skilled in the art.

[0173] The neutral form of the polypeptide is preferably regenerated by contacting the salt with a base or an acid and isolating the parent polypeptide by conventional methods. The parent form of the polypeptide may differ from the various salt forms in certain physical properties such as solubility in polar solvents.

[0174] Certain polypeptides of the present invention can exist not only in the unsolvated form but also in solvated forms including the hydrated form. Generally, the solvated forms are equivalent to the unsolvated form and are encompassed within the scope of the present invention. Certain polypeptides of the present invention can exist in multiple crystalline or amorphous forms. Generally, all physical forms are equivalent for the intended uses of the present invention and are intended to be within the scope of the present invention.

[0175] In some embodiments, the subcutaneous formulation can include recombinant human PH20 hyaluronidase (rHuPH20) to facilitate dispersion of the antibody from the injection site.

[0176] In some embodiments of the bispecific binding molecule, the pharmaceutical composition includes a therapeutically effective amount of the bispecific binding molecule and a pharmaceutically acceptable carrier. In some embodiments of the bispecific binding molecule, the pharmaceutical composition further includes a therapeutically effective amount of the bispecific binding molecule and one or more therapeutic agents for chemotherapy.

[0177] In some embodiments, the kit comprises one or more unit doses of a pharmaceutical composition comprising a bispecific binding molecule described herein and a pharmaceutically acceptable carrier, and instructions for one or more unit doses of the pharmaceutical composition to a subject in need thereof.

[0178] Administration method The compositions of the invention can be administered to a subject in need of cancer treatment. The terms “administer” or “administering” mean the act of providing a composition of the invention, e.g., a polypeptide or a pharmaceutically acceptable salt thereof, to a subject in need of cancer treatment.

[0179] As used herein, “intermittent administration” includes administering a drug for a period of time (which can be considered the “first administration”), followed by a period during which the composition is not taken or is taken at a low maintenance dose (which can be considered the “off period”), and then a period during which the composition is administered again (which can be considered the “second administration”). Generally, in the second stage of administration, the dosage level of the drug is the same as that administered during the first period of administration, but can be increased or decreased if medically necessary.

[0180] In some embodiments, the compositions of the invention can be administered to a subject by oral administration, administration as a suppository, topical contact administration, intravenous administration, parenteral administration, intraperitoneal administration, intramuscular administration, intrathecal administration, intranasal administration or subcutaneous administration, or by implantation of a sustained release device, e.g., a mini osmotic pump. Thus, administration can be by any route including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, etc. Other delivery modes include, but are not limited to, the use of liposomal formulations, intravenous infusion, or administration via an implanted reservoir. Specifically, the composition is administered orally, intraperitoneally or intravenously.

[0181] In some embodiments, the bispecific binding molecule composition is administered intravenously (IV) or orally systemically for intestinal cancers such as gastric cancer or intestinal cancer (Tashima et al., 2021). Formulations for delivery can be optimized by conventional methods well known in the art. Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, elixirs, etc. In addition to the active polypeptide, liquid dosage forms can include, for example, water or other solvents, inert diluents commonly used in the art such as solubilizing and emulsifying agents, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, sorbitan fatty acid esters, and mixtures thereof. In addition to the inert diluent, oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, flavoring fragrances, etc.

[0182] In some embodiments, the bispecific binding molecule composition is administered by subcutaneous injection. Injectable bispecific binding molecule formulations can be sterilized, for example, by filtration through a bacterial retention filter or by incorporating a sterilizing agent dispersed in sterile water or other sterile injectable medium prior to use. To extend the effect of the polypeptides described herein, it is often desirable to slow the absorption of the polypeptides by subcutaneous or intramuscular injection. This can be achieved by using a poorly water-soluble crystalline or amorphous liquid suspension. The absorption rate of the polypeptide depends on the dissolution rate, which in turn depends on the crystal size and crystal form. Alternatively, delayed absorption of parenterally administered polypeptides is achieved by dissolving or suspending the polypeptide in an oil vehicle. Injectable depot formulations are made by forming microcapsule matrices of the polypeptide in biodegradable polymers such as polylactic acid-polyglycolide. The release rate of the polypeptide can be controlled by the ratio of the polypeptide to the polymer and the nature of the specific polymer employed. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injection formulations are also prepared by encapsulating the polypeptide in liposomes or microemulsions that are compatible with body tissues.

[0183] The sterile injectable forms of the compositions described herein may be aqueous suspensions or oily suspensions. These suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable formulations may also be sterile injectable solutions or suspensions in a non-toxic parenterally acceptable diluent or solvent, such as, for example, a solution in 1,3-butanediol. Vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils have conventionally been used as a solvent or suspending medium. For this purpose, any bland fixed oil containing synthetic monoglycerides or diglycerides can be employed. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, and pharmaceutically acceptable natural oils such as olive oil or castor oil, especially polyoxyethylated ones, are also useful. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersing agents such as carboxymethyl cellulose, or similar dispersing agents commonly used in the formulation of pharmaceutically acceptable dosage forms containing emulsifying and suspending agents. Other commonly used surfactants, such as Tweens, Spans, and other emulsifying agents or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable dosage forms, can also be used for the purposes of the formulation.

[0184] In some embodiments, the formulation comprises excipients, buffers, tonicity agents, preservatives, surfactants, and preferably agents such as zinc. The formulation may also include excipients or agents for polypeptide stabilization such as buffers, reducing agents, bulk proteins, carbohydrates, etc. Examples of bulk proteins useful for formulating at least one polypeptide composition include albumin, protamine, etc. Common carbohydrates useful for formulating at least one polypeptide include sucrose, mannitol, lactose, trehalose, glucose, etc. The bispecific binding molecule formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of at least one polypeptide by atomization of the solution when forming an aerosol. A variety of conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, polyoxyethylene sorbitan fatty acid esters. The amount incorporated is generally in the range of about 0.001 wt% to 4 wt% of the formulation. Particularly preferred surfactants for the purposes of the present invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, etc. Additional agents known in the art for formulating polypeptides such as antibody proteins can also be included in the formulation.

[0185] Target protein of the subject Genomic studies have revealed the presence of various types of mutations in genes encoding receptor tyrosine kinases (RTKs) such as EGFR, HER2 / Erb2, and MET. For example, EGFR mutations in non-small cell lung cancer (NSCLC), MET exon 14 skipping mutations in NSCLC, RET mutations in medullary thyroid cancer, ALK translocations and ROS1 translocations in NSCLC, amplification or overexpression of HER2 in breast cancer, etc. have been reported.

[0186] The present invention provides a bispecific binding molecule that binds to EGFR and then degrades EGFR via the lysosomal degradation pathway. Twenty receptor tyrosine kinases including the EGFR family, FGFR family, PDGFR family, MET family, and VEGFR family are known to be co-receptors for NRP1 (see Critchley et al, Cells (2018) 7(3):22).

[0187] In some embodiments, the target proteins of the bispecific binding molecules herein include, but are not limited to, receptor tyrosine kinases (RTKs) and receptor serine / threonine kinases (RSTKs), oncogenic receptors such as the fibroblast growth factor receptor (FGFR) family, platelet-derived growth factor receptor (PDGFR) family, tyrosine kinase MET family, and vascular endothelial growth factor (VEGFR) family.

[0188] In some embodiments, the target protein is an RTK, provided that the target protein is not EGFR.

[0189] In some embodiments, the receptor tyrosine kinase receptors include HER1 / ErbB1, HER2 / ErbB2, HER3 / ErbB3, and HER4 / ErbB4, FGFR1, FGFR2, FGFR3, and FGFR4, Met, and Ron, the PDGFR family PDGFRα, PDGFRβ, CSF-1R, Kit, and FLT-3, and VEGFR1, VEGFR2, and VEGFR3.

[0190] In some embodiments, the target proteins of the bispecific binding molecules herein include, but are not limited to, serine / threonine kinase receptors, G protein-coupled receptors, immune checkpoint receptors, and ion channel receptors.

[0191] In some embodiments, the receptor serine / threonine kinases are ACVR1 / ALK1, ACVR2 / ALK2, ACVR1B / ALK4, ACVR1C / ALK7, ACVRL1, BMPR1A, BMPR1B, TGFBR1, ActR2, ActR2B, MISR2, BMPR2, TGFBR2, and TGFBR3. In some embodiments, the G protein-coupled receptors (GPCRs) are CXCR4, EBI2, CCR7, ADRB2, BAI2, FZD6, CD97, GPR153, FZD4, FZD2, F2R, ADORA2B.CD97, OPN3, GPR125, GPR126, GABBR1, CNR2, GPR92, PAR1, LPAR1, SSTR1, GPRC5B, GPRC5B, GPCR68, OXTR, LPHN2, FZD7, GABBR1, GPR125 and EDG3.

[0192] In some embodiments, the G protein-coupled receptor is selected from the group consisting of GPCR, CCR, and CXCR.

[0193] In some embodiments, the immune checkpoint receptor is selected from the group consisting of CTLA4, PD-L1, PD-1, and integrin.

[0194] In some embodiments, the ion channel receptor is a voltage-dependent receptor.

[0195] In some embodiments of the bispecific binding molecule, the target proteins of interest herein include, but are not limited to, disease-related proteins

[0196] Kit Embodiments of the disclosure further include a kit. In certain embodiments, the kit finds use in the practice of methods of the disclosure, such as administering the pharmaceutical composition of the invention to a subject to enhance anti-tumor immunity in the subject, administering the pharmaceutical composition of the invention to a subject to enhance or suppress an immune response in the subject, and the like.

[0197] Accordingly, in certain aspects, the kits of the present disclosure include one or more unit doses of the pharmaceutical composition of the present invention and instructions for administering the pharmaceutical composition to a subject in need thereof. The pharmaceutical composition included in the kit can be any of the bispecific antibodies of the present invention, for example, any of the bispecific binding molecules described above herein, which will not be repeated herein for the sake of brevity.

[0198] The kits of the present disclosure can include an amount of the composition present in unit dosage forms, such as ampoules, or multiple dosage forms. Thus, in certain aspects, the kit can include one or more (e.g., two or more) unit doses (e.g., ampoules) of the composition comprising the bispecific binding molecule of the present invention. As used herein, the term "unit dose" means a physically discrete unit suitable as a unit dose for human and animal subjects, each unit containing a predetermined amount of the composition calculated to be sufficient to produce the desired effect. The unit dose will vary depending on various factors in the individual, such as the particular bispecific binding molecule used, the effect to be achieved, the pharmacodynamics associated with the bispecific binding molecule, etc. In yet other aspects, the kit can include a single multi-dose of the composition. In certain aspects, the kits of the present disclosure include instructions for administering one or more unit doses of the pharmaceutical composition to a subject in need of enhanced anti-tumor immunity. According to some aspects, the kits of the present disclosure include instructions for administering one or more unit doses of the pharmaceutical composition to a subject in need of enhancing or suppressing an immune response.

[0199] Combination therapy In some aspects, the effective amount of the bispecific antibody can be achieved in the methods or pharmaceutical compositions of the present invention using the polypeptide or a pharmaceutically acceptable salt or solvate (e.g., hydrate) thereof alone or in combination with an additional suitable chemotherapeutic agent, such as oxaliplatin, irinotecan, or FOLFOX. When "combination therapy" is used, the effective amount can be achieved using a first amount of the polypeptide, or a pharmaceutically acceptable salt or solvate (e.g., hydrate) thereof, and a second amount of the additional suitable chemotherapeutic agent.

[0200] Co - administration involves administering the first and second amounts of the polypeptide and the chemotherapeutic agent essentially simultaneously, for example, in a single pharmaceutical composition, such as a capsule having a certain ratio of the first and second amounts, or in multiple separate capsules for each. Further, such co - administration also encompasses using each polypeptide sequentially in either order.

[0201] When co - administering the first amount of the polypeptide and the second amount of the additional therapeutic agent separately, the polypeptide is administered at a time sufficiently close to exert the desired therapeutic effect. For example, the period between each administration that can bring about the desired therapeutic effect can range from several minutes to several hours and can be determined considering the characteristics of each agent such as efficacy, solubility, bioavailability, plasma half - life, kinetic profile, etc. For example, the polypeptide and the second therapeutic agent can be administered in either order within about 24 hours of each other, within about 16 hours of each other, within about 8 hours of each other, within about 4 hours of each other, within about 1 hour of each other, or within about 30 minutes of each other.

[0202] More specifically, the bispecific binding molecule of the present invention can be administered before (e.g., 5 minutes before, 15 minutes before, 30 minutes before, 45 minutes before, 1 hour before, 2 hours before, 4 hours before, 6 hours before, 12 hours before, 24 hours before, 48 hours before, 72 hours before, 96 hours before, 1 week before, 2 weeks before, 3 weeks before, 4 weeks before, 5 weeks before, 6 weeks before, 8 weeks before or 12 weeks before), concomitantly with, or after (e.g., 5 minutes after, 15 minutes after, 30 minutes after, 45 minutes after, 1 hour after, 2 hours after, 4 hours after, 6 hours after, 12 hours after, 24 hours after, 48 hours after, 72 hours after, 96 hours after, 1 week after, 2 weeks after, 3 weeks after, 4 weeks after, 5 weeks after, 6 weeks after, 8 weeks after or 12 weeks after) the administration of the second anti - cancer agent to the subject.

[0203] The method of co - administering the first amount of the bispecific binding molecule and the second amount of the additional therapeutic agent can result in an enhanced or synergistic therapeutic effect, where it is understood that the effect of the combination is greater than the additive effect that would occur if the first amount of the polypeptide and the second amount of the additional therapeutic agent were administered separately.

[0204] The synergistic effect of a combination of therapies (e.g., a combination of prophylactic or therapeutic agents) enables the use of lower doses of one or more therapies and / or a reduction in the frequency of administration of the therapy to a subject. The ability to utilize lower doses of a therapeutic agent (e.g., a prophylactic or therapeutic agent) and / or the ability to administer the therapeutic agent less frequently can reduce the toxicity associated with administration of the therapeutic agent to a subject without reducing the effectiveness of the therapeutic agent in the treatment of cancer. Further, the synergistic effect may improve the effectiveness of the agent in the prevention, management, and treatment of disorders. Finally, the synergistic effect of a combination of therapies (e.g., a combination of prophylactic and therapeutic agents) can avoid or reduce side effects or undesirable side effects that occur when either therapy is used alone.

[0205] When the combination therapy using the bispecific binding molecule of the present invention is combined with other anti-cancer agents, both therapeutic agents can be administered such that the period between each administration is longer (e.g., several days, weeks, or months).

[0206] The presence or absence of a synergistic effect can be determined using an appropriate method for evaluating drug interactions. Appropriate methods include, for example, the Sigmoid-Emax equation (Holford, N.H.G. and Scheiner, L.B., Clin. Pharmacokinet. 6: 429-453 (1981)), the Loewe additivity equation (Loewe, S and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114: 313-326 (1926)), and the median effect equation (Chou, T.C. and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). Applying the equations mentioned above to experimental data and creating the corresponding graphs can be useful for evaluating the drug combination effect. The graphs corresponding to the above equations are a concentration-effect curve, an isobologram curve, and a combination index curve, respectively.

[0207] Method Aspects of the present disclosure include methods of using the bispecific antibodies of the present disclosure. Such methods are useful in a variety of aspects including in vitro and / or in vivo research and / or clinical applications.

[0208] In one aspect, a method of enhancing anti-tumor immunity in a subject in need thereof is provided. Such a method includes administering to an individual a pharmaceutical composition of the invention in an effective amount, such as a pharmaceutical composition comprising a bispecific binding molecule of the invention comprising a target protein binding domain and an NRP1 binding domain. In certain embodiments, the method is for enhancing antibody-dependent lysosomal degradation of a target protein and / or cytotoxicity against cancer cells in an individual.

[0209] In one aspect, a method of inhibiting or suppressing the growth of tumor cells in a subject in need thereof is provided. Such a method includes administering to an individual a pharmaceutical composition of the invention in an effective amount, such as a pharmaceutical composition comprising a bispecific binding molecule of the invention comprising a target protein binding domain and an NRP1 binding domain.

[0210] The subject in need thereof may have a cell growth disorder. "Cell growth disorder" means a disorder in which unwanted cell growth of one or more subsets of cells in a multicellular organism occurs, resulting in harm to the organism, such as pain, or a decrease in lifespan. Cell growth disorders include, but are not limited to, cancer, precancerous conditions, benign tumors, vascular proliferative disorders (e.g., arthritis, restenosis, etc.), fibrotic disorders (e.g., cirrhosis, arteriosclerosis, etc.), psoriasis, epidermal cysts and dermal cysts, lipomas, adenomas, capillary hemangiomas and cutaneous hemangiomas, lymphangiomas, nevus lesions, teratomas, renal tumors, fibromatosis, osteogenic tumors, dysplastic tumors, mesangial cell proliferative disorders, and the like.

[0211] In some embodiments, the subject has cancer. The methods of the present invention can be used for treating a variety of cancers. As used herein, the term "tumor" means all neoplastic cell proliferation and growth, and all precancerous and cancerous cells and tissues, whether malignant or benign. The terms "cancer" and "cancerous" mean or refer to a physiological state in mammals that is generally characterized by unregulated cell proliferation / growth. Examples of cancers that can be treated using the methods of the present invention include, but are not limited to, carcinomas, lymphomas, blastomas, and sarcomas. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, cholangiocarcinoma, bladder cancer, liver tumor, breast cancer, colorectal cancer, endometrial cancer or corpus cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, various head and neck cancers, etc. In certain embodiments, the subject has a cancer selected from solid tumors, recurrent glioblastoma multiforme (GBM), non-small cell lung cancer, metastatic melanoma, melanoma, peritoneal cancer, epithelial ovarian cancer, glioblastoma multiforme (GBM), metastatic colorectal cancer, colorectal cancer, pancreatic ductal adenocarcinoma, squamous cell carcinoma, esophageal cancer, gastric cancer, neuroblastoma, fallopian tube cancer, bladder cancer, metastatic breast cancer, pancreatic cancer, soft tissue sarcoma, recurrent head and neck cancer, squamous cell carcinoma, head and neck cancer, anaplastic astrocytoma, malignant pleural mesothelioma, breast cancer, squamous cell non-small cell lung cancer, rhabdomyosarcoma, metastatic renal cell carcinoma, basal cell carcinoma (basal cell epithelioma), and gliosarcoma. In one aspect, the subject has a cancer selected from melanoma, Hodgkin lymphoma, renal cell carcinoma (RCC), bladder cancer, non-small cell lung cancer (NSCLC), and head and neck squamous cell carcinoma (HNSCC).

[0212] The bispecific antibodies of the present invention can be administered via a route of administration selected from oral administration (e.g., tablets, capsules, solutions, etc.), parenteral administration (e.g., intravenous injection, arterial injection, subcutaneous injection, intramuscular injection, epidural injection, etc.), topical administration, intranasal administration, or intratumoral administration.

[0213] The bispecific antibodies of the present invention can be administered in a therapeutically effective amount in a pharmaceutical composition. "Therapeutically effective amount" means an amount sufficient to bring about a desired result, for example, an amount sufficient to bring about a beneficial or desired therapeutic (including prophylactic) result such as a reduction in the symptoms of cancer and / or immune disorders as compared to a control. With respect to cancer, in some embodiments, a therapeutically effective amount is sufficient to slow tumor growth, reduce tumor size, and / or the like. The effective amount can be administered in one or multiple doses.

[0214] Aspects of the present disclosure include methods for treating a subject's cancer and / or immune disorder. Treatment means at least an improvement in one or more symptoms associated with the subject's cancer and / or immune disorder, where improvement is used in a broad sense to mean at least a decrease in a parameter associated with the cancer and / or immune disorder being treated, such as the magnitude of the symptoms. Thus, treatment includes situations where the cancer and / or immune disorder, or at least one or more symptoms associated therewith, are completely inhibited such that the subject no longer suffers from the cancer and / or immune disorder, or at least the symptoms characterizing the cancer and / or immune disorder, for example, the onset is prevented or stopped, or terminated.

[0215] The present invention is further illustrated by the following examples, which should not be construed as being more limiting. The content of the drawings and all documents, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

Examples

[0216] Examples The examples in this specification are not intended to limit the present invention and should not be used for that purpose.

[0217] Example 1. Preparation of bispecific antibody The designed protein was prepared by codon-optimized gene synthesis and inserted into pcDNA3.4 as an expression vector using NotI and HindIII restriction enzymes. Table 1 shows the sequences of the heavy-chain fusion polypeptides of the bispecific binding molecules used herein, described in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 39, which bind to the sequences of the heavy-chain polypeptide described in SEQ ID NO: 1 and the light-chain polypeptide described in SEQ ID NO: 12. Table 2 shows the sequences of the polynucleotides encoding the heavy-chain polypeptide described in SEQ ID NO: 14 and the polynucleotides encoding the heavy-chain polypeptides of the bispecific antibodies described in SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 40. The sequence of the polynucleotide encoding the light-chain polypeptide that binds to the heavy-chain polypeptide of the bispecific antibody is described in SEQ ID NO: 25. The sequences shown in SEQ ID NOs: 26-38 describe the polynucleotide sequences used to encode the GGGGS subunits of the peptide linker. The constructed expression vector contains a signal peptide for optimized transcription, and a Kozak sequence may be included in the 5' untranslated region.

[0218] To obtain the amount of plasmid construct required for transfection, the plasmid construct was transformed into One Shot (商標) Top10 E. coli competent cells and cultured overnight. The constructed plasmid was obtained using the PureLink (商標) HiPure Expi plasmid Megaprep kit.

[0219] The fusion protein was transiently expressed in the CHO-S system (Thermo Fisher Scientific Inc.). The proteins were expressed individually according to the manufacturer's instructions. Briefly, a total of 0.8 μg of plasmid DNA with a light-chain to heavy-chain ratio of 1:1 was added per 1 mL of CHO-S culture medium, using OPTIPRO (商標) SFM and ExpiFectamine (商標) to prepare the mixture. This mixture was added to a live cell density of 6×10 6 cells / mL with a viability of 98% or higher It was added to the cells. Cell culture was carried out overnight with shaking at 125 RPM using a 19 mm orbit in a Nalgene single-use PETG Erlenmeyer flask under the conditions of 37 °C, 80% humidity, and 8% CO₂. (商標) The next day, the culture was enhanced (ExpiCHO enhancer; Thermo Fisher Scientific Inc), the feed was added (ExpiCHO feed; Thermo Fisher Scientific Inc), and it was transferred to an environment of 32 °C, 80% humidity, and 5% CO₂ and shaken at 125 rpm with a 19 mm orbit. The second medium addition was performed on the 5th day, and the culture was returned to 32 °C until harvest on the 12th day. The collection was centrifuged at 4000 × g for 20 minutes. The clarified supernatant was sterilized using an asymmetric polyethersulfone (PES) 0.22 μM filter assembly (Nalgene). The filtrate was stored at 4 °C until purification the next day. (商標) The antibody sterile supernatant was all purified using MabSelect prismA resin (GE Healthcare Life Sciences) on an AKTApure (GE Healthcare Life Sciences). The resin was equilibrated with a buffer of 50 mM sodium phosphate, 150 mM NaCl, and pH 7.0. Then, the antibody supernatant was loaded onto the column. The resin was washed with a buffer of 50 mM sodium phosphate, 150 mM NaCl, and pH 7.0 until the baseline of the chromatography returned to the column equilibrium level. Then, elution was performed using 100 mM sodium acetate, 20% glycerol, and pH 3.0, and the fractions were collected. The fractions were immediately neutralized with 1 M Tris and pH 9. The fractions containing the dominant absorbance at a wavelength of 280 nm were collected into an Amicon 10-kDa ultrafiltration device for buffer exchange. The storage buffer (phosphate-buffered saline) was centrifuged semi-diluted 7 times with an Amicon concentrator to remove the elution buffer. After being subjected to SEC, it was stored at 4 °C. (商標) It was centrifuged at 4000×g for 20 minutes. The clarified supernatant was sterilized using an asymmetric polyethersulfone (PES) 0.22 μM filter assembly (Nalgene). The filtrate was stored at 4 °C until purification the next day.

[0220] All antibody sterile supernatants were purified using MabSelect prismA resin (GE Healthcare Life Sciences) on an AKTApure (GE Healthcare Life Sciences). (商標) The resin was equilibrated with a buffer of 50 mM sodium phosphate, 150 mM NaCl, and pH 7.0. Then, the antibody supernatant was loaded onto the column. The resin was washed with a buffer of 50 mM sodium phosphate, 150 mM NaCl, and pH 7.0 until the baseline of the chromatography returned to the column equilibrium level. Then, elution was performed using 100 mM sodium acetate, 20% glycerol, and pH 3.0, and the fractions were collected. The fractions were immediately neutralized with 1 M Tris and pH 9. The fractions containing the dominant absorbance at a wavelength of 280 nm were collected into an Amicon 10-kDa ultrafiltration device for buffer exchange. The storage buffer (phosphate-buffered saline) was centrifuged semi-diluted 7 times with an Amicon concentrator to remove the elution buffer. After being subjected to SEC, it was stored at 4 °C.

[0221] Cation exchange chromatography was used for antibody purification. The cation exchange chromatography column (Capto S ImpAct) was washed with 1M NaOH and rinsed with MQ. Equilibration was carried out with 50 mM NaAc pH 5.5 (starting buffer) and 50 mM NaAc pH 5.5, 1M NaCl (elution buffer). The Protein A purified antibody was loaded at a resin concentration of 1-2 g antibody / mL. Subsequently, the column was washed with 50 mM NaAc pH 5.5. Then, the antibody product was eluted using a gradient of 5% - 60% elution buffer at 25 column volumes. Each peak of CEX purification was collected separately and concentrated by centrifuging at 4000 xg using an Amicon (登録商標) Ultra-15 centrifugal filter unit, followed by buffer exchange into PBS.

[0222] Size exclusion chromatography (SEC) analysis was performed on an Agilent Infinity 1260 II Quaternary Pump high performance liquid chromatography (HPLC) system equipped with a diode array UV detector WR. 20 μg of antibody substance was injected into an XBridge Protein BEH SEC Column, 200 Å, 2.5 μm, 4.6 mm X 150 mm column. The mobile phase was 100 mM phosphoric acid, 300 mM sodium chloride, pH 7.0, 50 °C, and the flow rate was 0.3 mL / min. The antibody substance was detected at wavelengths of 220 nm, 280 nm, 330 nm, a sampling rate of 1 Hz, and a measurement time of 10 minutes.

[0223]

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

[0224]

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

[0225] Example 2. Bispecific binding molecule that binds to EGFR and NRP1 The BLI binding assay was performed using the Octet red 96 system to evaluate the binding of bispecific antibodies to recombinant huEGFR and huNRP1. Briefly, commercially available biotinylated huEGFR and huNRP1 were immobilized on streptavidin (Sa) biosensors, bound to the prepared constructs, and characterized. Using BLI technology, binding to the constructs was evaluated in both kinetics and binding affinity (equilibrium binding constant, KD), and evaluated in a bivalent format. These studies were conducted to evaluate whether tumor-associated essential receptor-targeting antibodies (TAER-TAB) bind to cancer targets (EGFR and NRP1) and the affinity for the targets. The constructs binding to huEGFR and huNRP1 as well as the determination of KD were performed using the Octet Red 96 system.

[0226] huEGFR and huNRP1 (in their respective immobilization columns) were immobilized on Sa biosensors at a concentration of 0.2 μg / ml in 2X Kinetic buffer (loading signal range was 0.2 - 0.4 nanometers), and the respective loading time was 180 seconds. Baseline post-loading was performed for 60 seconds. The binding of the constructs was started at 100 nM (10 nM for high-affinity binding), then two consecutive 1:1 dilutions were performed, and the fourth well was run with buffer (blank) only. Dissociation of the constructs continued in the baseline wells. A reference sensor was created by applying the construct to the blank AMC biosensor or streptavidin biosensor surface. The binding and dissociation steps were each 600 seconds. The data was analyzed using Octet analysis software that applied 1:1 and 2:1 model fits reporting the dissociation constant K D (M).

[0227] All EGFRxNRP1 bispecific antibody constructs containing Construct 1 (SEQ ID NOs: 1 and 12) and Construct 11 (SEQ ID NOs: 11 and 12) have a binding affinity in the tens of picomolar range for EGFR (see Table 3). With respect to binding to NRP1, EGFRxNRP1 bispecific antibody constructs 2, 3, 5, 7, 9, 10, and 11 showed sub-nanomolar affinities, while Construct 1 bound to NRP1 in the tens of nanomolar range. Figures 2A - 2K show the binding affinity curves of the bispecific antibodies to immobilized huEGFR. Figures 3A - 3K show the binding affinity curves of the EGFRxNRP1 antibodies to immobilized huNRP1.

[0228]

Table 3

[0229] Example 3. Inhibitory effect on VEGFR2 signal transduction in HUVEC cells Inhibition of VEGFR2 signaling by the EGFRxNRP1 bispecific antibody was tested by Western blotting using anti-phospho-VEGFR2 to probe electrophoresed cell lysates in HUVEC cells. HUVEC cells were cultured overnight in 6-well plates in EBM-2 medium supplemented with EGM-2 singlequots. The next day, the cells were incubated for 4 hours in 2 ml of F-12 K medium supplemented with 0.1 mg / ml heparin, endothelial cell growth supplement, and 10% FBS. To observe the dose-dependent response to the EGFRxNRP1 antibody, the EGFRxNRP1 bispecific antibody serially diluted 6-fold from a maximum concentration of 1 μM to a minimum concentration of 0.005 μM was treated with serum-free medium for 30 minutes, followed by administration of 2.2 ng / ml of VEGF165 or a control for 10 minutes. Cells were collected with a cell scraper and cell lysates were prepared by incubating on ice for 20 minutes using 100 μl of NP-40 lysis buffer supplemented with protease / phosphatase inhibitors. After centrifugation at 13,000 rpm for 10 minutes, the supernatant was collected in a new tube, protein quantification was performed using the BCA protein assay, and after mixing with NuPAGE LDS sample buffer and LDS sample reducing buffer, it was denatured at 70°C for 10 minutes.

[0230] 4 μl of PageRuler Plus Prestained Protein Ladder was added to 10 μg of cell lysate, added to a 4-12% Bis-Tris gel, and reacted at 200 V for 45 minutes. The gel was washed with distilled water and transferred to a membrane using the IBlot2 Drying Blotting System. The membrane was blocked with 5% milk in 1xTBST for 1 hour at room temperature and incubated overnight at 4°C with anti-phospho-VEGFR2 (Y1175) in 5% milk in 1xTBST. The next day, the membrane was washed 3 times with TBST for 10 minutes each, incubated with the secondary antibody in 5% milk for 1 hour at room temperature. The membrane was washed 3 times with 1xTBST for 10 minutes each. Then, the membrane was incubated with SuperSignal Femto Chemiluminescent Substrate for 1-2 minutes and imaged with Amersham ImageQuant 800. Then, the blot was stripped with Restore Plus Western Blot stripping buffer for 15 minutes at room temperature on a rocker, washed 3 times with 1xTBST, blocked with milk, and the procedure of incubating with the primary anti-VEGFR2 antibody was repeated.

[0231] The intensities of the Western blot bands of phosphorylated VEGFR2 (Y1175) and VEGFR2 were analyzed using ImageJ software. The obtained band density of phosphorylated VEGFR2 (Y1175) was normalized by VEGFR2, and the inhibition of VEGFR2 phosphorylation by the EGFRxNRP1 bispecific antibody construct 11 was calculated as a ratio to the VEGF165 control cell lysate (positive control). The IC50 of VEGFR2 phosphorylation by the EGFRxNRP1 bispecific antibody was determined using Prism9 software (see Figures 11A - 11B). The Western blot in Figure 3 shows that incubation of HUVEC cells with the bispecific binding molecule of the EGFRxNRP1 bispecific antibody construct 1 (SEQ ID NOs: 1 and 12) did not inhibit VEGFR phosphorylation mediated by VEGF165. Figures 5 - 11 show Western blots and IC50 plots of the inhibition of VEGFR2 phosphorylation after incubation with the bispecific antibody. The IC50 of the bispecific antibody is shown in Table 4.

[0232] [Table 4]

[0233] Example 4: Effect of EGFRxNRP1 bispecific antibody on cell viability Using the H1975 lung cancer cell line, the inhibition of cell viability by the bispecific antibody was tested. Briefly, H1975 lung cancer cells were cultured in RPMI 1640 supplemented with 1% penicillin / streptomycin and 10% FBS. The cells were trypsinized and seeded into round-bottom 96-well 3D culture plates (3,000 cells / 120 μl / well) and incubated for 3 - 4 hours until they aggregated to form 3D spheroids. A stock master plate of the EGFRxNRP1 bispecific antibody serially diluted 6-fold from 5 μM to 0.6 pM was prepared. The H1975 cells were treated with 30 μl of the EGFRxNRP1 bispecific antibody and incubated for 72 hours (duplicate). Then, CellTiter-Glo 3D reagent and 25 μl of serum-free medium were added to each well, wrapped with foil, and incubated on a rocker for 7 minutes. The plates were read on a Varioskan Lux Multimode Plate Reader and analyzed with Prism9 software.

[0234] Table 5 shows the IC50 of the EGFRxNRP1 antibody. The EGFRxNRP1 bispecific antibody construct (SEQ ID NOs: 1 and 12) with an NRP1 binding domain at the C-terminus without being fused to the scFv has a high IC50 value (1.96 μM) as shown in Figure 12A compared to other EGFRxNRP1 antibody constructs (see Figure 12B). The IC50 value data suggests that the single-chain variable fragment (scFv) containing the NRP1 binding domain of the EGFRxNRP1 antibody is important for the function of the EGFRxNRP1 antibody in inhibiting tumor cell growth.

[0235]

Table 5

[0236] Example 5. Tumor growth inhibitory effect in H1975 xenograft mouse model cell line The tumor growth inhibitory ability of the EGFRxNRP1 antibody was tested in a xenograft mouse model of the H1975 cell line. All cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were maintained at 37°C in a 5% CO2 incubator in RPMI 1640 (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (HyClone, Logan, UT, USA) and 2 mmol / L glutamine (HyClone, Logan, UT, USA).

[0237] Xenograft model Experiments and procedures using mice were conducted in accordance with the guidelines of the US Department of Agriculture, the Department of Health and Human Services, and the NIH for the humane care and use of laboratory animals. Six- to eight-week-old female athymic mice (nu / nu) from Charles River Laboratories (Wilmington, MA, USA) were housed under pathogen-free conditions and allowed free access to experimental food and water. Prior to transplantation of the tumor cell line, all cell lines were screened for infectious agents (such as mycoplasma). Xenografts were established by subcutaneous injection of 100 μl per mouse of a 1:1 mixture of Matrigel (Corning, Corning, NY, USA) containing (NCI-H1975) cells into the right flank. In the efficacy study, randomization was performed and the tumors were allowed to reach 150 - 300 mm 7 prior to treatment initiation (7 - 10 mice per group). 3

[0238] Treatment and tumor measurement The antibody was diluted with sterile phosphate-buffered saline (Corning, Corning, NY, USA) and administered by intraperitoneal injection. The total dose administered per mouse was 100 μl, administered at the dose indicated on the day of randomization and then twice weekly thereafter.

[0239] Digital calipers were used to measure the size of the tumors, and the tumor volume was calculated as V=(W 2 ​Calculated using the formula (W × L) / 2 (where W is the width of the tumor and L is the length of the tumor).

[0240] Statistical analysis GraphPad Prism 7 software (La Jolla, CA, USA) was used for statistical analysis. Results were shown as mean values. Data of the control group and the experimental group at each time point or endpoint were compared using Student's t-test. For growth curve analysis including longitudinal data by repeated measurement, type II ANOVA was used. p < 0.05 was considered as significant difference. TGI% was calculated according to the formula TGI(%) = (V C1 - V t1 ) / (V C0 - V t0 ) × 100. Here, V c1 and V t1 are the mean tumor volumes of the control group and the treatment group at the end point of the test, and V C0 and V t0 are the mean tumor volumes of the control group and the treatment group at the start point of the experiment, respectively. Table 6 shows the tumor growth inhibition by EGFRxNRP1 antibody in the Xenograph mouse model and the tumor volume percentage of the treatment group vs. the control group. The curves in FIGS. 13A - 13F show the mean tumor volume over the number of days after transplantation of the mice treated with EGFRxNRP1 antibody. The EGFRxNRP1 bispecific antibody construct 11 having the scFv NRP1 binding domain has the highest tumor growth inhibition effect. For example, treatment with EGFRxNRP1 bispecific antibody construct 11 (SEQ ID NOs: 11 and 12) resulted in a 100% tumor growth inhibition effect.

[0241]

Table 6

[0242] Example 6. EGFR by construct 11 in RH1975 cells T790 / L858R Degradation EGFR tyrosine kinase inhibitors (TKIs) have been widely used in the treatment of cancers with constitutively active EGFR mutations by binding to the catalytic site of EGFR and inhibiting its kinase activity. However, acquired resistance can be induced. Gefitinib, a first-generation EGFR TKI, induces the T790M mutation in most patients after treatment. Afatinib, a second-generation EGFR TKI, binds irreversibly to EGFR but lacks selectivity for EGFR WT and EGFR T790M and causes side effects. Osimertinib, a third-generation EGFR TKI, is effective against tumors with EGFR T790M mutations, but there are also patients who show acquired resistance such as the C797S mutation. Furthermore, NSCLC with EGFR WT does not respond to TKIs. Therefore, targeting EGFR by inducing degradation can be a more effective approach to suppressing EGFR in cancer treatment, regardless of the EGFR mutation status.

[0243] Anti-EGFR antibodies inhibit the activation of EGFR signaling via ligands and induce the endocytosis of receptors, but the EGFR receptors are mostly recycled. The ability of the EGFRxNRP1 bispecific antibody construct 11 to degrade double-mutant EGFR T790 / L858R was evaluated in cancer cells, and the relationship between the anti-cancer activity of construct 11 and the EGFR protein level was verified. EGFR T700M / L858RH1975 cells with mutations were grown in tissue culture plates using RPMI 1640 medium supplemented with 10% fetal bovine serum. They were incubated for 16 hours with construct 11 that was fixed (150 nM), or construct 11 serially diluted 6-fold from the highest concentration of 1 μM to the lowest concentration of 0.77 nM. The proteasome inhibitor MG132 (10 μM) or the lysosomal proteolysis inhibitor bafilomycin A1 (Baf A1, 200 nM) was used in combination with the EGFRxNRP1 bispecific antibody therapy to clarify the mechanism of action of the EGFRxNRP1 antibody. Cell lysates were prepared by collecting cells with a cell scraper and incubating them on ice for 20 minutes using NP40 lysis buffer containing protease / phosphate inhibitors. After centrifugation at 13,000 rpm for 10 minutes, the supernatant was collected into a new tube, protein quantification was performed using the BCA protein assay, and after mixing with NuPAGE LDS sample buffer and LDS sample reducing buffer, it was denatured at 70 °C for 10 minutes. 20 μg of cell lysate was added to a 4-12% bis-tris gel together with 4 μl of PageRuler Plus prestained ladder and reacted at 200 V for 45 minutes. The gel was washed with distilled water and transferred to a membrane using the IBlot2 Drying blotting system. Anti-EGFR, anti-NRP1, and anti-β-actin were incubated overnight at 4 °C in 5% milk in 1xTBST. The next day, the membrane was washed 3 times with TBST for 10 minutes each and incubated with the secondary antibody in 5% milk at room temperature for 1 hour. The membrane was washed 3 times with 1xTBST for 10 minutes each. Then, the membrane was incubated with SuperSignal Femto chemiluminescent substrate for 5 minutes and imaged using the BioRad ChemiDoc Touch Imaging system.

[0244] Human NSCLC H1975 cells (EGFR T790 / L858R ) were treated with an anti-NRP1 antibody (anti-NRP1 mAb), panitumumab (Pnm, an anti-EGFR antibody), anti-NRP1 + panitumumab, and the EGFRxNRP1 bispecific antibody construct 11 (SEQ ID NOs: 11 and 12) (150 nM) for 16 hours. The protein levels of EGFR, NRP1, and actin were measured by Western blot.

[0245] In the cells treated with construct 11, the levels of both total EGFR (tEGFR) and NRP1 were very low. In contrast, the levels of EGFR in the cells treated with anti-NRP1 mAb, panitumumab (Pnm), or anti-NRP1 mAb and panitumumab were similar to those in the untreated cells (Figure 14). This result suggests that the degradation of EGFR and NRP1 is achieved only by the EGFRxNRP1 antibody construct 11 that binds to EGFR T790 / L858R and NRP1 simultaneously.

[0246] Degradation of EGFR and NRP1 by the EGFRxNRP1 bispecific antibody in H1975 cells T790 / L858R was restored by co-treatment with the lysosomal proteolysis inhibitor bafilomycin A1 (Baf A1, 200 nM), but not by co-treatment with the proteasome inhibitor MG132 (10 μM) (Figure 15). Similar experiments were performed using Baf A1 inhibitor and MG132 inhibitor to examine the degradation of various EGFR forms (WT or mutants) by construct 11 in other NSCLC cell lines. The EGFR of PC9 cells del19 , the EGFR of H1299 (NRAS Q61K ) cells WT , the EGFR of HCC44 (KRAS G12C ) cells WT , the EGFR del19 / NRP1 in HCC827 cells, and the EGFR G12C / NRP1 in H358 (KRAS WT ) cells were examined for degradation (data not shown). For all EGFR types and cell lines examined, it was confirmed that receptor degradation by construct 11 was restored by co-treatment with the lysosomal proteolysis inhibitor Baf A1, but not by co-treatment with the proteasome inhibitor MG132.

[0247] Integrating these data, the EGFRxNRP1 antibody construct 11 induces the degradation of EGFR and NRP1 via the lysosomal proteolysis pathway. The drawings show that the EGFRxNRP1 bispecific antibody binds to EGFR and NRP1 simultaneously, resulting in the degradation of EGFR through the lysosomal degradation pathway and indicating that the binding to NRP1 is essential for the degradation of EGFR (Figure 16). The effect of the EGFR antibodies panitumumab, amivantamab, and construct-11 on the degradation of EGFR T790 / L858R in H1975 cells was evaluated. No clear degradation of tEGFR was observed in either panitumumab-treated cells or amivantamab-treated cells, whereas administration of construct 11 significantly reduced the EGFR T790 / L858R (tEGFR) protein level even at a low concentration of 0.77 nM (Figure 16). Construct 11 can be used for the treatment of tumors with EGFR mutations as a novel and unique antibody-based EGFR receptor degrader. Furthermore, the EGFRxNRP1 bispecific antibody construct 11 degraded EGFR with mutations including single mutation (Del19), double mutation (T790M / L858R), triple mutation (T790M / C797S / L858R), and insertion (InsEx20) (data not shown).

[0248] Thus, in summary, the EGFRxNRP1 bispecific antibody construct 11 was shown to degrade both wild-type and mutant EGFR by a lysosome-mediated degradation pathway in a wide panel of NSCLC cell lines.

[0249] Example 7. Effect of EGFRxNRP1 bispecific antibody on antitumor activity in osimertinib-resistant xenograft model mice Osimertinib is a third-generation irreversible EGFR TKI that targets activating mutations (L858R and deletion of chromosome 19) and the T790M mutation. Resistance to osimertinib has become a major obstacle in the treatment of EGFR-mutant NSCLC. In recent studies, the combination of lazertinib and amivantamab in osimertinib-resistant patients showed an overall response rate (ORR) of 36%. In preclinical trials using the H1975-HGF xenograft mouse model, the combination of lazertinib and amivantamab was found to be significantly effective.

[0250] To investigate the effect of the EGFRxNRP1 bispecific antibody construct 11 on osimertinib-resistant tumors, xenograft mice with osimertinib-sensitive, osimertinib-resistant, or osimertinib-refractory tumors were treated with osimertinib (1 mg / kg, intraperitoneal (i.p.), QD), panitumumab (5 mg / kg, i.p., BIW), construct-11 (6.87 μg / kg, i.p., BIW), amivantamab (5.05 mg / kg, i.p., BIW), and IgG1 isotype (5 mg / kg, i.p., BIW) as a control. Osimertinib was formulated for intraperitoneal administration by dissolving the dry powder in a small amount (10% of the final volume) of dimethyl sulfoxide. Osimertinib and the antibodies were diluted in sterile phosphate-buffered saline and administered by injecting a total volume of 100 μl per mouse per dose from the day of randomization as described above. V = (W 2To calculate the tumor volume using the formula (W×L) / 2 (where W is the width of the tumor and L is the length of the tumor), mb measurements were taken using a digital caliper. GraphPad Prism 7 software (La Jolla, CA, USA) was used for statistical analysis. The results were shown as mean ± SEM. Data at each time point or at the end point between the control group and the experimental group were compared using Student's t-test. In xenograft mice (H1975) sensitive to osimertinib, construct 11 was highly effective in suppressing tumor growth in mice compared to mice treated with amivantamab. Administration of construct 11 continued to suppress tumor growth until 42 days after transplantation, but the tumor did not respond effectively to osimertinib, panitumumab, or amivantamab (Figure 18A). The osimertinib-resistant H1975-OR cell line was developed by culturing H1975 cells in a medium with the osimertinib concentration increased up to 4 μM. In xenograft mice (H1975-OR) resistant to osimertinib, the construct-11 administration group also showed stronger tumor growth suppression compared to the amivamb treatment group (Figure 18B). The osimertinib-insensitive H1975-HGF cell line was developed by overexpressing hepatocyte growth factor (HGF) in H1975 cells and activating the cMET signal to reduce the dependence of H1975 cell line growth on the EGFR signal. In xenograft mice (H1975-HGF) insensitive to osimertinib, treatment with the EGFRxNRP1 bispecific antibody construct 11 showed a tumor growth suppression effect similar to that of amivamb treatment (Figure 18C). These results demonstrate that the EGFRxNRP1 bispecific antibody construct 11 induced strong degradation of EGFR and had antitumor activity in osimertinib-sensitive H1975. The efficacy of the EGFRxNRP1 bispecific antibody construct 11 in various EGFR T790 / L858R induced strong degradation and had antitumor activity in osimertinib-sensitive H1975. Various EGFR T790M / L858R The efficacy of the EGFRxNRP1 bispecific antibody construct 11 in xenograft mouse models is summarized in Table 7 below. The EGFRxNRP1 bispecific antibody construct 11 is also applicable to other receptor tyrosine kinase inhibitor (RTK)-resistant tumors.

[0251]

Table 7

[0252] The inventors of the present invention have demonstrated that the EGFRxNRP1 bispecific antibody inhibits the growth of tumor cells and reduces the cell viability. Further, it was verified in Example 6 using a lysosomal proteolysis inhibitor or a proteasome inhibitor that when the EGFRxNRP1 bispecific antibody is administered, EGFR is degraded in the lysosomal degradation pathway (see Fig. 15). Importantly, the degradation of EGFR was detected only when the EGFRxNRP1 bispecific antibody bound to both EGFR and NRP1. The presence of NRP1 is essential for the degradation of the target protein, i.e., the functionality of the bispecific binding molecule.

[0253] The present disclosure provides a novel antibody-based ApReptor platform, which has several advantages compared to other degraders, including (i) not requiring an E3 / ubiquitin-specific protein (USP), (ii) not requiring a linker and a catalytic enzyme, (iii) preclinical PoC validation, (iv) preclinical validation in a drug resistance model, and (v) broad application to disease-related extracellular receptors.

[0254] This antibody-based ApReptor platform is modular, selective, and provides a simple gene-encoding strategy, which induces lysosomal delivery of extracellular and surface target receptors that are widely or tissue-specifically distributed. The AbReptor platform holds great potential for personalized medicine that can be customized according to the patient's genetic background.

[0255] Example 8. Degradation of cMET receptor by cMETxNRP1 bispecific antibody construct In this example, a bispecific antibody (BsA) construct similar to construct 11 was prepared, which maintained the anti-NRP1 antibody binding arm of BsA but used an anti-cMET antibody as the other binding arm of BsA (instead of the anti-EGFR of construct 11). The K of the NRP1 binding of the cMETxNRP1 bispecific antibody constructD was 3.99x10 -10 M. The K D of cMET binding of the cMETxNRP1 bispecific antibody construct was <0.1 nM, which was equivalent to the K D of cMET binding of the anti-cMET mAb alone.

[0256] Degradation of NRP1 and cMET by the cMETxNRP1 BsA construct was examined in HCC827 cells, ACHN cells, and H1975 cells, and the results are shown in FIGS. 19A-19C, respectively. The cells were untreated (control), treated with the anti-cMET antibody alone, or treated with the cMETxNRP1 BsA construct. As a result, it was shown that only treatment with the BsA construct resulted in degradation of NRP1. Furthermore, treatment with the BsA construct led to more advanced degradation of cMET in all three cell lines examined than treatment with the anti-cMET alone. From these results, it was confirmed that the BsA construct containing anti-cMET has the ability to degrade cMET in cancer cells expressing cMET.

[0257] Example 9. Degradation of HER2 receptor by HER2xNRP1 bispecific antibody construct In this example, a bispecific antibody (BsA) construct similar to construct 11 was prepared, in which the anti-NRP1 antibody-binding arm of the BsA was maintained, but an anti-HER2 antibody was used as the other binding arm of the BsA (instead of the anti-EGFR of construct 11). The K D of NRP1 binding of the HER2xNRP1 bispecific antibody construct was 2.44x10 -10 M. The K D of HER2 binding of the HER2xNRP1 bispecific antibody construct was <0.1 nM, which was equivalent to the K D of HER2 binding of the anti-HER2 mAb alone.

[0258] Degradation of NRP1 and HER2 by the HER2xNRP1 BsA construct was examined in BT474 cells, and the results are shown in Figure 20. Cells were treated with non-treatment (control), anti-HER2 antibody alone, or the HER2xNRP1 BsA construct. As a result, it was shown that only treatment with the BsA construct (as expected) resulted in the degradation of NRP1. Furthermore, treatment with the BsA construct promoted the degradation of HER2 in the examined cell lines more than treatment with the anti-HER2 drug alone. From these results, it was confirmed that the BsA construct containing anti-HER2 has the ability to degrade HER2 in HER2-expressing cancer cells.

[0259] Example 10. Degradation of IGF1R receptor by IGF1RxNRP1 bispecific antibody construct In this example, a bispecific antibody (BsA) construct similar to construct 11 was prepared, in which the anti-NRP1 antibody-binding arm of the BsA was maintained, but an anti-IGF1R antibody was used as the other binding arm of the BsA (instead of the anti-EGFR of construct 11). The K of NRP1 binding of the IGF1RxNRP1 bispecific antibody construct D was <0.1 nM. The K of IGF1R binding of the IGF1RxNRP1 bispecific antibody construct D was <0.1 nM, which was equivalent to the K of IGF1R binding of the anti-IGF1R mAb alone. D

[0260] Degradation of NRP1 and IGF1R by the IGF1RxNRP1 BsA construct was examined in MCF-7 cells and ACHN cells, and the results are shown in Figures 21A-21B, respectively. Cells were treated with non-treatment (control), anti-IGF1R antibody alone, or the IGF1RxNRP1 BsA construct. As a result, it was shown that only treatment with the BsA construct (as expected) resulted in the degradation of NRP1. Furthermore, treatment with the BsA construct promoted the degradation of IGF1R in both examined cell lines more than treatment with the anti-IGF1R alone. From these results, it was confirmed that the BsA construct containing anti-IGF1R has the ability to degrade IGF1R in IGF1R-expressing cancer cells.

[0261] Array List Summary TIFF2025525096000024.tif211164 TIFF2025525096000025.tif212164 TIFF2025525096000026.tif212164 TIFF2025525096000027.tif216164 TIFF2025525096000028.tif215164 TIFF2025525096000029.tif216164 TIFF2025525096000030.tif216164 TIFF2025525096000031.tif216164 TIFF2025525096000032.tif216164 TIFF2025525096000033.tif216164 TIFF2025525096000034.tif214164 TIFF2025525096000035.tif212164 TIFF2025525096000036.tif213164 TIFF2025525096000037.tif216164 TIFF2025525096000038.tif216164 TIFF2025525096000039.tif215164 TIFF2025525096000040.tif217164 TIFF2025525096000041.tif213164 TIFF2025525096000042.tif216164 TIFF2025525096000043.tif216164 TIFF2025525096000044.tif30164

Claims

1. (a) A target protein binding domain that specifically binds to a receptor tyrosine kinase (RTK); and (b) An NRP1 binding domain that binds to NRP1, comprising an antibody or a neuropilin-1 (NRP1) binding fragment thereof, A bispecific binding molecule comprising: wherein the binding of the bispecific binding molecule to the target protein and NRP1 results in lysosomal degradation of the target protein in target cells. A bispecific binding molecule.

2. The bispecific binding molecule according to claim 1, wherein the RTK is not an epidermal growth factor receptor (EGFR).

3. The bispecific binding molecule according to claim 1, wherein the RTK is selected from the HER family of receptors, insulin growth factor receptor (IGFR), Met receptor tyrosine kinase (MET), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), and vascular endothelial growth factor (VEGFR).

4. The bispecific binding molecule according to claim 1, wherein the receptor tyrosine kinase is EGFR.

5. The bispecific binding molecule according to claim 1, wherein the receptor tyrosine kinase is cMET.

6. The bispecific binding molecule according to claim 1, wherein the receptor tyrosine kinase is HER2.

7. The bispecific binding molecule according to claim 1, wherein the receptor tyrosine kinase is IGF1R.

8. The bispecific binding molecule according to any one of claims 1 to 7, wherein the target cell is a cancer cell.

9. The bispecific binding molecule according to claim 8, wherein the cancer cell is selected from the group consisting of lung cancer, breast cancer, colorectal cancer, head and neck cancer, esophageal gastric cancer, liver cancer, glioblastoma, prostate cancer, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, and pancreatic cancer.

10. The bispecific binding molecule according to claim 8, wherein the cancer cell is a non-small cell lung cancer (NSCLC) cell.

11. The bispecific binding molecule according to any one of claims 1 to 7, wherein the target protein binding domain and the NRP1 binding domain are each independently selected from the group consisting of IgG, half antibody, single domain antibody, nanobody, Fab, monospecific Fab2, Fc, scFv, minibody, IgNAR, V-NAR, hcIgG, VHH domain, camel antibody, and peptibody.

12. The NRP1 binding domain is (i) An antibody heavy chain variable (VH) domain comprising CDR1, CDR2, and CDR3 regions (HCDR1, HCDR2, and HCDR3, respectively), wherein HCDR1 consists of the sequence shown in SEQ ID NO: 79, HCDR2 consists of the sequence shown in SEQ ID NO: 80, and HCDR3 consists of the sequence shown in any one of SEQ ID NOs: 81-84; and (ii) An antibody light chain variable (VL) domain comprising CDR1, CDR2, and CDR3 regions (LCDR1, LCDR2, and LCDR3, respectively), wherein LCDR1 consists of the sequence shown in any one of SEQ ID NOs: 85-87, LCDR2 consists of the sequence shown in SEQ ID NO: 88, and LCDR3 consists of the sequence shown in SEQ ID NO: 89 The bispecific binding molecule according to claim 1, comprising.

13. (i) HCDR1 consists of the sequence shown in SEQ ID NO: 79, HCDR2 consists of the sequence shown in SEQ ID NO: 80, and HCDR3 consists of the sequence shown in any one of SEQ ID NO: 84; and (ii) LCDR1 consists of the sequence shown in SEQ ID NO: 85, LCDR2 consists of the sequence shown in SEQ ID NO: 88, and LCDR3 consists of the sequence shown in SEQ ID NO: 89, The bispecific binding molecule according to claim 12.

14. The bispecific binding molecule according to any one of claims 1 to 7 and 12 to 13, which is part of an antibody-drug conjugate (ADC).

15. A nucleic acid encoding the bispecific binding molecule according to any one of claims 1 to 7 and 12 to 13.

16. An expression vector comprising the nucleic acid according to claim 15.

17. A cell capable of protein expression comprising the expression vector according to claim 16.

18. Use of the bispecific binding molecule according to any one of claims 1 to 7 and 12 to 13 in the manufacture of a medicament for treating cancer in a subject.

19. A method for inducing lysosomal degradation of a target protein in a cell, the method comprising contacting the cell with the bispecific binding molecule according to any one of claims 1 to 7 and 12 to 13 such that lysosomal degradation of the target protein is induced in the cell.

20. The method according to claim 19, wherein the cell contains one or more mutations in the target protein and / or overexpresses the target protein.

21. The method according to claim 19, wherein the target protein is EGFR.

22. The method according to claim 19, wherein the target protein is not EGFR.

23. The method according to claim 19, wherein the target protein is cMET.

24. The method according to claim 19, wherein the target protein is HER2.

25. The method according to claim 19, wherein the target protein is IGF1R.

26. The method according to claim 19, wherein the cell is resistant or unresponsive to the inhibitor of the target protein.

27. A method for inhibiting tumor growth in a subject having a tumor, comprising administering to the subject a bispecific binding molecule according to any one of claims 1 to 7 and 12 to 13 such that tumor growth in the subject is inhibited.

28. The method according to claim 27, wherein the tumor contains one or more mutations in the target protein and / or overexpresses the target protein.

29. The method according to claim 27, wherein the target protein is EGFR.

30. The method according to claim 27, wherein the target protein is not EGFR.

31. The method according to claim 27, wherein the target protein is cMET.

32. The method according to claim 27, wherein the target protein is HER2.

33. The method according to claim 27, wherein the target protein is IGF1R.

34. The method according to claim 27, wherein the tumor is resistant or unresponsive to the inhibitor of the target protein.

35. The method according to claim 27, wherein the bispecific binding molecule is administered intravenously, intraperitoneally, intrathecally, intracerebroventricularly, or into the brain parenchyma.

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