Compositions for targeted lysosomal degradation and methods of use thereof

Abstract

The present disclosure provides compositions based on a degradation agent of a bispecific binding molecule that degrades a target protein, such as a protein on a cancer cell, via a lysosomal pathway. The bispecific binding molecule specifically binds to a target protein of interest and to neuropilin-1 (NRP1). The present disclosure also provides methods of using the bispecific binding molecule-based degradation agents, such as enhancing the therapeutic efficacy of cancer treatments.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 369,948, filed on July 30, 2022, and U.S. Provisional Application No. 63 / 423,454, filed on November 7, 2022, both of which are hereby incorporated by reference in their entirety. Background Art

[0003] Protein degradation regulates many aspects of cellular homeostasis. Endogenous protein degradation machinery has been reprogrammed to eliminate various intracellular substrates through an approach called targeted protein degradation. Targeted protein degradation (TPD) has emerged as a promising therapeutic strategy superior to conventional inhibition. Unlike inhibitors, degraders achieve catalytic and persistent knockdown of protein levels. Proteolysis targeting chimeras (PROTACs) consist of two parts, which bind the target and the ubiquitin ligase (E3), separated by a flexible linker. However, due to their intracellular mechanism of action, PROTACs are limited to targeting intracellular proteins.

[0004] Lysosomal degradation platforms have been developed, such as cytokine receptor targeting chimeras (KineTac) and lysosomal targeting chimeras (LYTAC), which use E3 / USP to eliminate pathogenic proteins. The KineTacs platform is a genetically encoded bispecific antibody consisting of a cytokine arm that binds its cognate cytokine receptor and a target binding moiety for a protein of interest. However, KineTac is limited to cytokine receptors applied to T cells, and if the target protein is also present on T cells, the target protein can be degraded. The LYTAC platform composed of antibody-glycan conjugates degrades cell surface and extracellular proteins by shuttling the target protein to the lysosome for degradation. Therefore, there is a need in the art for additional methods for lysosomal degradation platforms. Summary of the invention

[0005] The present disclosure relates to novel and unique bispecific binding molecules, which are composed of fully recombinant bispecific binding domains that utilize 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. Therefore, the present disclosure provides a degrader based on a bispecific binding molecule that degrades a target protein, such as a target protein on a cancer cell. The bispecific binding molecules specifically bind to a target protein of interest and neuropilin-1 (NRP1). The present disclosure further relates to methods of using NRP1-dependent bispecific binding molecules that induce degradation of a target protein through a lysosomal degradation pathway. The present disclosure provides methods of inhibiting tumor growth using bispecific binding molecules, such as enhancing the therapeutic efficacy of cancer treatment.

[0006] The present disclosure relates to a bispecific binding molecule comprising a target protein binding domain and a neuropilin-1 (NRP1) binding domain, the binding domain being an antibody or its NRP-1 binding fragment. In some embodiments, the bispecific binding molecule comprises 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-associated. In some embodiments, the binding of the bispecific binding molecule to NRP1 results in internalization of the target protein bound to the bispecific binding molecule. In some embodiments, the binding of the bispecific binding molecule to NRP1 subsequently induces lysosomal degradation of the target protein.

[0007] In certain embodiments, the target protein binding domain of the bispecific molecule binds to a receptor tyrosine kinase (RTI). Thus, in one aspect, the present disclosure relates to a bispecific binding molecule comprising:

[0008] (a) a target protein binding domain that specifically binds to a receptor tyrosine kinase (RTK); and

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

[0010] The binding of the bispecific binding molecule to the target protein and to NRP1 results in lysosomal degradation of the target protein in the target cell.

[0011] In one embodiment, the RTK is EGFR. In another embodiment, the RTK is not epidermal growth factor receptor (EGFR) (ie, the target protein is a RTK, provided that the RTK is not EGFR).

[0012] In other embodiments, the RTK is selected from an EGFR family receptor, a HER family receptor, an insulin growth factor receptor (IGFR), a Met receptor tyrosine kinase (MET), a platelet-derived growth factor receptor (PDGFR), a fibroblast growth factor receptor (FGFR), and a vascular endothelial growth factor (VEGFR).

[0013] In one embodiment, the receptor tyrosine kinase is cMET.In one embodiment, the receptor tyrosine kinase is HER2.In one embodiment, the receptor tyrosine kinase is IGF1R.

[0014] In one embodiment, the target cell is a cancer cell. In an embodiment, the cancer cell is selected from lung cancer, breast cancer, colon and rectal cancer, head and neck cancer, esophageal and gastric cancer, liver cancer, glioblastoma, prostate cancer, cervical cancer, ovarian cancer, bladder cancer, kidney cancer and pancreatic cancer. In one embodiment, the cancer cell is a non-small cell lung cancer (NSCLC) cell.

[0015] In an embodiment, the target protein binding domain and the NRP1 binding domain are each independently selected from IgG, half antibodies, single domain antibodies, nanobodies, Fab, monospecific Fab2, Fc, scFv, minibodies, IgNAR, V-NAR, hcIgG, VHH domains, camelid antibodies, and peptibodies.

[0016] In one embodiment, the NRP1 binding domain

[0017] Include:

[0018] (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

[0019] (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.

[0020] In one embodiment,

[0021] (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

[0022] (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.

[0023] In other aspects, the disclosure relates to nucleic acids encoding the bispecific binding molecules of the disclosure, expression vectors comprising the nucleic acids of the disclosure, and cells capable of protein expression comprising the nucleic acids of the disclosure (eg, expression vectors).

[0024] In another aspect, the disclosure relates to use of a bispecific binding molecule of the disclosure in the manufacture of a medicament for treating cancer in a subject.

[0025] In another aspect, the disclosure relates to a method for inducing lysosomal degradation of a target protein in a cell, the method comprising contacting a cell with a bispecific binding molecule of the disclosure, such that lysosomal degradation of the target protein is induced in the cell. In embodiments, the cell comprises one or more mutations in the target protein and / or overexpresses the target protein. In embodiments, the cell is resistant or refractory to the responsiveness of an inhibitor of the target protein.

[0026] In one embodiment, the target protein is EGFR. In one embodiment, the target protein is 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.

[0027] 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 present disclosure, such that growth of the tumor in the subject is inhibited. In embodiments, the tumor comprises one or more mutations in the target protein and / or overexpresses the target protein. In embodiments, the tumor is resistant or refractory to the responsiveness of the inhibitor of the target protein.

[0028] In one embodiment, the target protein is EGFR. In one embodiment, the target protein is 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.

[0029] In an embodiment, the bispecific binding molecule is administered intravenously, intraperitoneally, intrathecally, intracerebroventricularly, or intraparenchymalally.

[0030] 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-associated. In some embodiments, the target protein is a cell surface receptor protein. In some embodiments, the target protein is a transmembrane protein.

[0031] 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).

[0032] In some embodiments, 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.

[0033] In some embodiments, the target protein includes EGFR. In some embodiments, EGFR is selected from EGFR / HER1 / Erb1, HER2 / ErbB2, HER3 / ErbB3 and HER4 / ErbB4. In some embodiments, the target protein is EGFR.

[0034] In some embodiments, the target protein comprises FGFR.In some embodiments, FGFR1 is selected from FGFR1, FGFR2, FGFR3 and FGFR4.

[0035] In some embodiments, the target protein comprises the receptor tyrosine kinase MET. In some embodiments, the receptor tyrosine kinase MET is MET or macrophage-stimulating protein receptor (MST1R / RON).

[0036] In some embodiments, the target protein comprises PDGFR.In some embodiments, the PDGFR is selected from PDGFR, PDGFRα, PDGFRβ, CSF-1R, Kit, and FLT-3.

[0037] In some embodiments, the target protein comprises VEGFR.In some embodiments, the VEGFR is selected from VEGFR1, VEGFR2 and VEGFR3.

[0038] In some embodiments, the receptor tyrosine kinase is selected from EGFR / HER1 / Erb1, HER2 / ErbB2, HER3 / ErbB3, HER4 / ErbB4 VEGFR1, VEGFR2, and VEGFR3.

[0039] In some embodiments, target proteins include receptor serine / threonine kinases (RSTKs), G protein-coupled receptors (GPCRs), immune checkpoint receptors, and ion channel receptors.

[0040] In some embodiments, the target protein includes 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.

[0041] In some embodiments, the target protein includes a G protein coupled receptor (GPCR). In some embodiments, the GPCR is selected from 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, chemokine receptor 1, CCR1, CX3CR1, ACKR3, CCK1 receptor, GPR3, GPR12, GPR17, GPR32 and GPR35.

[0042] In some embodiments, the target protein includes an immune checkpoint receptor. In some embodiments, the immune checkpoint receptor is selected from 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.

[0043] In some embodiments, the target protein includes an ion channel receptor. In some embodiments, the ion channel receptor is selected from 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.

[0044] In some embodiments, the target protein includes a membrane-associated target protein, wherein 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 a membrane-associated target protein. In some embodiments, the target cell includes a tumor cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the cancer cell is selected from lung cancer, breast cancer, colon and rectal cancer, head and neck cancer, esophageal and gastric cancer, liver cancer, glioblastoma, prostate cancer, cervical cancer, ovarian cancer, bladder cancer, kidney cancer and pancreatic cancer. In some embodiments, the target cell includes an immune cell.

[0045] In some embodiments, the target protein binding domain and the NRP1 binding domain are each independently selected from IgG, half antibodies, single domain antibodies, nanobodies, Fab, monospecific Fab2, Fc, scFv, minibodies, IgNAR, V-NAR, hcIgG, VHH domains, camelid antibodies, and peptibodies.

[0046] 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, IgG1 is human IgG1 or a variant thereof.

[0047] In some embodiments, the bispecific binding molecule comprises: two identical heavy chain polypeptides comprising a first heavy chain fused to a first single-chain variable fragment (scFv) by a peptide linker to produce a first heavy chain fusion polypeptide and a second heavy chain 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 two identical light chains comprising a first light chain and a second light chain, wherein each heavy chain fusion polypeptide sequence is any one of 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 shown in SEQ ID NO: 13, wherein n is each independently an integer between 1-20. In some embodiments, the disclosure further provides nucleotide sequences encoding each heavy chain fusion polypeptide shown in any one of SEQ ID NO: 15-24 or 40 and the light chain polypeptide shown in SEQ ID NO: 25.

[0048] In another embodiment, the bispecific binding molecule comprises: (a) two identical heavy chain polypeptides comprising a first heavy chain fused to a first single-chain variable fragment (scFv) via a peptide linker to produce a first heavy chain fusion polypeptide and a second heavy chain fused to a second scFv via a peptide linker to produce a second heavy chain fusion polypeptide, 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 located at the C-terminus of CH3; and (ii) the light chain polypeptide comprises 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 a target protein binding domain that binds to a target protein and an scFv comprising an NRP1 binding domain that binds to NRP1.

[0049] In some embodiments, the NRP1 binding domain of the bispecific binding molecule comprises:

[0050] (i) an antibody heavy chain variable (VH) domain containing 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

[0051] (ii) an antibody light chain variable (VL) domain containing 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.

[0052] In another embodiment,

[0053] (iii) HCDR1 consists of the sequence shown in SEQ ID NO:79, HCDR2 consists of the sequence shown in SEQ ID NO:80, and HCDR3 consists of the sequence shown in any one of SEQ ID NO:84; and

[0054] (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.

[0055] In some embodiments, the binding affinity (K) of the bispecific binding molecule for a target protein is D )<0.1 nM. In some embodiments, the binding affinity (K D ) is in the range of 0.1 nM-100 nM. In some embodiments, the binding affinity (K) of the binding antibody to the target protein is D ) is the binding affinity of the bispecific binding molecule to NRP1 (K D ), at least 2, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or more than 100 times larger than the

[0056] In some embodiments, the bispecific binding molecule (bispecific antibody) is part of an antibody-drug conjugate (ADC), wherein the bispecific binding molecule is conjugated to the drug via a linker. Non-limiting examples of suitable drugs for ADC compounds are provided herein.

[0057] 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 EGFR in vitro. T790M / L858In some embodiments, the degradation of the target protein results in inhibition of target cell proliferation. In some embodiments, the EGFRxNRP1 bispecific antibody reduces in vitro cell viability.

[0058] In some embodiments, the bispecific binding molecules reduce tumor volume in an osimertinib-sensitive xenograft mouse model. In some embodiments, the bispecific binding molecules reduce tumor volume in an osimertinib-resistant or osimertinib-refractory xenograft mouse model. In some embodiments, treatment with the bispecific binding molecules enhances anti-tumor efficacy.

[0059] In some embodiments, the nucleic acid encoding the bispecific binding molecule is as shown in Table 2. In some embodiments, the nucleic acid is operably linked to a promoter.

[0060] An expression vector comprising a nucleic acid of a bispecific binding molecule is provided. In some embodiments, the vector further comprises a promoter, wherein the promoter is operably linked to the nucleic acid.

[0061] The present disclosure provides cells capable of protein expression, comprising nucleic acids of bispecific binding molecules. In some embodiments, the engineered cells comprise cancer cells.

[0062] In another embodiment, the disclosure provides a method for preparing a bispecific binding molecule, wherein the method comprises (a) providing a cell capable of protein synthesis comprising a nucleic acid disclosed herein; and (b) inducing expression of the bispecific binding molecule.

[0063] 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 an engineered cell, and a pharmaceutically acceptable carrier.

[0064] The present disclosure also provides use of a bispecific binding molecule in the manufacture of a medicament for treating cancer in a subject.

[0065] The present disclosure provides methods for treating cancer in a subject. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a bispecific binding molecule, nucleic acid, vector, engineered cell, or pharmaceutical composition provided herein. In some embodiments, the bispecific binding molecule is administered intraperitoneally.

[0066] The present disclosure further provides methods of preventing cancer in a subject.In some embodiments, methods of inhibiting tumor cell growth are provided.

[0067] In some embodiments, a kit comprises one or more unit doses of a pharmaceutical composition and instructions for providing the one or more unit doses of the pharmaceutical composition to a subject in need thereof.

[0068] In some embodiments, the disease comprises a neoplastic disease, an inflammatory disease, a metabolic disease, or a neurological disease.

[0069] In certain embodiments, neoplastic diseases include lung cancer, breast cancer, colon and rectal cancer, head and neck cancer, esophageal and gastric cancer, liver cancer, glioblastoma, prostate cancer, cervical cancer, ovarian cancer, bladder cancer, kidney cancer and pancreatic cancer. In one embodiment, lung cancer is non-small cell lung cancer (NSCLC).

[0070] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 : Schematic representation of a bispecific binding molecule that specifically binds to a target protein and neuropilin-1 (NRP1).

[0072] Figure 2A -2K: Graph showing the binding kinetics of isolated bispecific EGFRxNRP1 bispecific antibodies to recombinant human EGFR (huEGFR). The EGFRxNRP1 antibodies tested herein for binding affinity determination are as follows: Each EGFRxNRP1 bispecific antibody consists of a heavy chain fusion polypeptide and a light chain polypeptide. They are construct 1 (SEQ ID NO: 1 and 12), construct 2 (SEQ ID NO: 2 and 12), construct 3 (SEQ ID NO: 3 and 12), construct 4 (SEQ ID NO: 4 and 12), construct 5 (SEQ ID NO: 5 and 12), construct 6 (SEQ ID NO: 6 and 12), construct 7 (SEQ ID NO: 7 and 12), construct 8 (SEQ ID NO: 8 and 12), construct 9 (SEQ ID NO: 9 and 12), construct 10 (SEQ ID NO: 10 and 12), construct 11 (SEQ ID NO: 11 and 12). The binding affinity to huEGFR was quantified by the Octet Red 96 system (Fortebio). Each row of sensorgrams on the graph represents the binding kinetics at a specific concentration (10, 5, 2.5 nM from left to right).

[0073] Figure 3A-3K: Graph showing the binding kinetics of isolated bispecific EGFRxNRP1 antibodies to recombinant human NRP1 (huNRP1). The EGFRxNRP1 bispecific antibody constructs tested for binding affinity determination herein are as follows: Each EGFRxNRP1 bispecific antibody consists of a heavy chain fusion polypeptide and a light chain polypeptide. They are construct 1 (SEQ ID NO: 1 and 12), construct 2 (SEQ ID NO: 2 and 12), construct 3 (SEQ ID NO: 3 and 12), construct 4 (SEQ ID NO: 4 and 12), construct 5 (SEQ ID NO: 5 and 12), construct 6 (SEQ ID NO: 6 and 12), construct 7 (SEQ ID NO: 7 and 12), construct 8 (SEQ ID NO: 8 and 12), construct 9 (SEQ ID NO: 9 and 12), construct 10 (SEQ ID NO: 10 and 12), construct 11 (SEQ ID NO: 11 and 12). Binding affinity to huNRP1 was quantified by the Octet Red 96 system (Fortebio). Each row of sensorgrams on the graph represents the binding kinetics at a specific concentration (10, 5, 2.5 nM from left to right).

[0074] Figure 4 : Effect of EGFRxNRP1 bispecific antibody construct 1 on VEGF-mediated VEGFR2 phosphorylation. The expression levels of total VEGFR and p-VEGFR2 were determined by EGFRxNRP1 bispecific antibody construct 1 (SEQ ID NO: 1 and 12). HUVEC cells were pre-incubated for 30 min in the presence or absence of construct 1 (1 μM) and treated with VEGF165 (2.2 ng / ml) for another 10 min to induce VEGFR2 activation. VEGFR2 signaling activation was detected by western blotting of whole cell lysates using anti-phosphorylated VEGFR2 (Y1175). Relative p-VEGFR2 expression was quantified by normalization with total VEGFR2 using Image J software densitometry analysis. The numbers shown below the pVEGFR2 blot are the normalized numbers of pVEGFR2 relative to total VEGFR2.

[0075] Figure 5A-5B : Effect of EGFRxNRP1 bispecific antibody construct 2 on VEGF-mediated VEGFR2 phosphorylation. The expression levels of total VEGFR and p-VEGFR2 by EGFRxNRP1 bispecific antibody construct 2 (SEQ ID NO: 2 and 12) were determined by Western blotting using anti-phosphorylated VEGFR2 (Y1175). Figure 5A). HUVEC cells were pre-incubated with construct 2 (from right to left, 6-fold serial dilution concentrations starting from 1 μM) for 30 min and treated with VEGF165 (2.2 ng / ml) for another 10 min to induce VEGFR2 activation. ( Figure 5A The inhibition of VEGFR2 phosphorylation by construct 2 was calculated as % P-VEGFR2 ( Figure 5B ). (n=2)

[0076] Figure 6A-6B : Effect of EGFRxNRP1 bispecific antibody construct 3 on VEGF-mediated VEGFR2 phosphorylation. Expression levels of total VEGFR and p-VEGFR2 ( Fig. 6A The inhibition of VEGFR2 phosphorylation by construct 3 was calculated as % P-VEGFR2 relative to the control using Prism9 ( Figure 6B ). (n=2) Experimental methods and materials Figure 5A and 5B Same as described in .

[0077] Figure 7A-7B : Effect of EGFRxNRP1 bispecific antibody construct 4 on VEGF-mediated VEGFR2 phosphorylation. Expression levels of total VEGFR and p-VEGFR2 ( Fig. 7A The inhibition of VEGFR2 phosphorylation by EGFRxNRP1 bispecific antibody construct 4 was calculated as % P-VEGFR2 ( Figure 7B ). (n=2)

[0078] Figures 8A-8B : Effect of EGFRxNRP1 bispecific antibody construct 5 on VEGF-mediated VEGFR2 phosphorylation. Expression levels of total VEGFR and p-VEGFR2 ( Fig. 8A The inhibition of VEGFR2 phosphorylation by EGFRxNRP1 bispecific antibody construct 5 was calculated as % P-VEGFR2 ( Figure 8B ).

[0079] (n=2)

[0080] Figures 9A-9B: Effect of EGFRxNRP1 bispecific antibody construct 6 on VEGF-mediated VEGFR2 phosphorylation. Expression levels of total VEGFR and p-VEGFR2 ( Fig.9A The inhibition of VEGFR2 phosphorylation by EGFRxNRP1 bispecific antibody construct 6 was calculated as % P-VEGFR2 ( Fig. 9B ). (n=2)

[0081] Figures 10A-10B : Effect of EGFRxNRP1 bispecific antibody construct 7 on VEGF-mediated VEGFR2 phosphorylation. Expression levels of total VEGFR and p-VEGFR2 ( Fig. 10A The inhibition of VEGFR2 phosphorylation by EGFRxNRP1 bispecific antibody construct 7 was calculated as % P-VEGFR2 ( Fig. 10B ). (n=2)

[0082] Figures 11A-11B : Effect of EGFRxNRP1 bispecific antibody construct 11 on VEGF-mediated VEGFR2 phosphorylation. Expression levels of total VEGFR and p-VEGFR2 ( Fig.11A The inhibition of VEGFR2 phosphorylation by EGFRxNRP1 bispecific antibody construct 11 was calculated as % P-VEGFR2 ( Fig. 11B ). (n=2).

[0083] Figures 12A-12B : Effect of EGFRxNRP1 bispecific antibody on the growth rate of H1975 cells. Cells were incubated with various concentrations of EGFRxNRP1 bispecific antibody for 72 hours, and cell growth was assessed and expressed as a growth rate relative to the control (%). Construct 1 (SEQ ID NO: 1 and 12) ( Fig. 12A ); and construct 3 (SEQ ID NOs: 3 and 12), construct 7 (SEQ ID NOs: 7 and 12), construct 9 (SEQ ID NOs: 9 and 12), and construct 11 (SEQ ID NOs: 11 and 12) ( Fig. 12B ).

[0084] Figures 13A-13F: Antitumor activity of EGFRxNRP1 bispecific antibodies in the H1975 xenograft mouse model. Mice were treated with various EGFRxNRP1 bispecific antibody constructs, panitumumab or vehicle (control) at various doses. Construct 1 (SEQ ID NO: 1 and 12) was administered at 10 mg / kg, ip, BIW ( Fig.13A ); Construct 1 (SEQ ID NO: 1 and 12), at 5 mg / kg ( Fig. 13B ); SEQ ID NO: 2 (Construct 2), at 6.825 mg / kg ( Fig. 13C ); construct 3 (SEQ ID NO: 3 and 12), at 5 mg / kg ( Fig.13D ); Construct 7 (SEQ ID: 7 and 12), at 6.85 mg / kg ( Fig.13E ); and construct 7, construct 9 (SEQ ID NOs: 9 and 11) and construct 11 (SEQ ID NOs: 11 and 12), respectively, at 6.87 mg / kg ( Fig.13F ) or PBS as a negative control was administered to mice via the intraperitoneal route (ip).

[0085] EGFRxNRP1 bispecific antibody constructs 3, 7, 9, and 12 inhibited tumor growth more effectively than construct 1, which has the NRP1 binding domain in CH3 but no scFv.

[0086] Fig.14 :EGFRxNR:1 bispecific antibody construct 11 induces EGFR T790M / L858R and strong degradation of NRP1. H1975 cells were incubated with panitumumab, anti-NRP1 mAb, anti-NRP1 mAb plus panitumumab (Pnm), or construct 11, and Western blots of separated whole cell lysates were probed to detect EGFR and NRP1 using monoclonal anti-EGFR and anti-NRP1 antibodies, respectively. Nonspecific protein actin was used as a loading control.

[0087] Fig.15 : The EGFRxNRP1 bispecific antibody construct 11 induced the degradation of EGFR and NRP1 through the lysosomal degradation pathway. The degradation of EGFR and NRP1 by construct 11 was restored by co-treatment with the lysosomal protein degradation inhibitor bafilomycin A1 but not with the proteasome inhibitor MG132.

[0088] Fig.16 : Schematic representation of the proposed EGFRxNRP1 bispecific antibody-mediated EGFR degradation mechanism.

[0089] Fig.17: EGFRxNR:1 bispecific antibody construct 11 (SEQ ID NO: 11 and 12) mediated EGFR in H1975 cells T790M / L858R H1975 cells were treated with construct 11, panitumumab, or ervantumab at concentrations between 0.77 nM and 1 μM (from right to left, 6-fold serial dilutions of 1 μM) for 16 hours. Whole cell lysates were used for Western blotting to detect EGFR and actin. Actin was used as a loading control. In H1975 cells, construct 11 treatment induced EGFR degradation even at a concentration of 0.77 nM. T790M / L858R strong degradation.

[0090] Figures 18A-18C : Antitumor activity of EGFRxNRP1 bispecific antibody construct 11 (SEQ ID NO: 11 and 12) in various osimertinib xenograft mouse models. Construct 1 was evaluated in osimertinib-sensitive H1975 ( Fig.18A ), osimertinib-resistant H1975-OR ( Fig.18B ) and osimertinib-refractory H1975-HGF( Fig. 18C ) Inhibitory effect on tumor growth in a xenograft mouse model. Xenograft mice were treated with bs construct 11, osimertinib, panitumumab, ervantumab, or IgG1 isotype control, and tumor volume (mm) was quantified at the indicated days after implantation. 3 ).

[0091] Figures 19A-19C Graph demonstrating the degradation of cMET and NRP1 following treatment with the cMETxNRP1 bispecific antibody construct (PTX-414) compared to an anti-cMET monoclonal antibody (PTX-413). Fig.19A Results for HCC827 cells are shown. Fig.19B Results for ACHN cells are shown. Fig.19C Results for H1975 cells are shown.

[0092] Fig. 20 Graph demonstrating degradation of HER2 and NRP1 following treatment of BT474 cells with the HER2xNRP1 bispecific antibody construct (PTX-402) compared to an anti-HER2 monoclonal antibody (PTX-401).

[0093] Figures 21A-21B Graph demonstrating degradation of IGF1R and NRP1 following treatment with an IGF1RxNRP1 bispecific antibody construct (PTX-418) compared to an anti-IGF1R monoclonal antibody (PTX-417). Fig.21A Results for MCF-7 cells are shown. Fig. 21BResults for ACHN cells are shown. DETAILED DESCRIPTION

[0094] definition

[0095] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Therefore, the following terms are intended to have the following meanings:

[0096] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0097] As used herein, the term "human EGFR" or "EGFR" refers to human epidermal growth factor receptor protein (UniProKB / Swiss-Pro No. P00533) and includes any variants, isoforms and species homologs of EGFR that are naturally expressed by cells (including tumor cells) or expressed on cells transfected with an EGFR gene or cDNA.

[0098] As used herein, the term "human NRP1" or "NRP1" refers to human neuropilin-1 protein (UniProKB / Swiss-Pro No. 014786) and includes any 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.

[0099] As used herein, the term "receptor tyrosine kinase" or "Receptor Tyrosine Kinase" or "RTK" refers to a protein that acts as a receptor (ie, binds a ligand) and phosphorylates tyrosine residues.

[0100] As used herein, the term "non-receptors tyrosine kinase" or "Non-Receptor Tyrosine Kinase" or "non-RTK" refers to a protein that is not a RTK, ie, a protein that is not a receptor and / or does not phosphorylate tyrosine residues.

[0101] As used herein, the terms "administering" or "administration" of a disclosed bispecific binding molecule or polypeptide thereof include delivering a polypeptide or composition of the invention as described herein, or a prodrug or other pharmaceutically acceptable derivative thereof, to a subject using any suitable formulation or route of administration, e.g., as described herein.

[0102] The term "and / or", when used in a list of two or more items, means that any one of the listed items may be employed alone or any combination of two or more of the listed items may be employed.

[0103] As used herein, "treatment," "treat," or "treating" are used in reference to a disease or disorder, meaning that at least an improvement in the symptoms associated with the disorder afflicting a subject is achieved, wherein improvement is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, such as a symptom associated with the disorder being treated. The term "treatment" or "treating" refers to a method for obtaining a therapeutic benefit. A therapeutic benefit is determined by whether a tumor shrinks, remains the same size, or progression-free survival time is increased compared to a placebo. Thus, treatment includes prevention (i.e., reducing the risk of clinical symptoms developing, including causing clinical symptoms not to develop, such as preventing disease progression) and inhibition (i.e., preventing the development or further development of clinical symptoms, such as alleviating or completely inhibiting active disease).

[0104] As used herein, "subject" may refer to any animal suffering from cancer, such as a mammal, such as an experimental animal, a farm animal, a pet, etc. In some embodiments, the animal is a primate, preferably a human. As used herein, the terms "subject" and "subject" are used interchangeably. The terms "subject" and "subject" refer to animals (e.g., birds, such as chickens, quail or turkeys, or mammals), particularly "mammals", including non-primates (e.g., cattle, pigs, horses, sheep, rabbits, guinea pigs, rats, cats, dogs and mice) and primates (e.g., monkeys, chimpanzees and humans), and more specifically humans. In one embodiment, the subject is a non-human animal, such as a farm animal (e.g., horse, cattle, pigs or sheep) or a pet (e.g., dog, cat, guinea pig or rabbit). In a preferred embodiment, the subject is a "human".

[0105] As used herein, the term "fusion" refers to unifying two molecules with the same or different functions or structures, and the method of fusion may include any physical, chemical or biological method capable of combining a peptide with a protein, a small molecule drug, a nanoparticle or a liposome. Preferably, the fusion may be mediated by a linker peptide, and for example, the linker peptide may be fused to the C-terminus of an antibody light chain variable region (Fc) fragment. Alternatively, two molecules may be fused by integrating multiple domains within a polypeptide sequence.

[0106] As used herein, the term "joint" or "flexible joint" is a molecule or peptide that connects two polypeptide subunits together. The joint peptide sequence may include the amino acid sequence subunit (GGGGS)n, where n defines the number of subunit repeats. The number of subunit repeats determines the flexibility of the joint peptide. A flexible peptide joint allows greater flexibility between two binding domains.

[0107] As used herein, "effective amount" refers to an amount sufficient to cause a desired anticancer response. In the present invention, the desired biological response is to inhibit cell proliferation. The exact amount of the bispecific binding molecule administered to the subject will depend on the mode of administration, the type and severity of the cancer, and the characteristics of the subject, such as general health, age, sex, weight, and tolerance to the drug. A skilled technician will be able to determine the appropriate dose depending on these and other factors. When administered together with other anticancer agents, such as when administered together with chemotherapy, the "effective amount" of the second drug will depend on the type of drug used. For approved drugs, suitable doses are known and can be adjusted by a skilled technician according to the condition of the subject, the type of cancer being treated, and the amount of the polypeptide administered herein. In the case where the amount is not explicitly noted, an effective amount should be assumed. For example, the bispecific antibodies disclosed herein can be administered to subjects at weekly or biweekly intervals in a dosage range of about 0.01-100 mg / kg body weight / day.

[0108] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

[0109] As used herein, the term "variant" describes a sequence that has changes from the wild-type, conventional or primary sequence. These changes may take the form of deletions, substitutions or insertions of amino acids or nucleotides. A variant may contain one or more, including a combination of sequence changes.

[0110] The term "reduce" or other forms of the word, such as "reducing" or "reduction", generally refers to a reduction in an event or characteristic (e.g., one or more symptoms, or the binding of one protein to another). It should be understood that this is generally related to some standard or expected value, in other words, it is relative, but does not always require reference to a standard or relative value.

[0111] As used in the context of "binding affinity", the term "affinity" refers to the reduced affinity of one molecule for another molecule. For example, in some embodiments, a protein, domain or motif can specifically bind to a particular target, such as a peptide, polypeptide, protein, carbohydrate, saccharide, polysaccharide, glycosaminoglycan or any epitope thereof, with a given affinity. The term "affinity" refers to the sum strength of non-covalent interactions between a single binding domain of a molecule and its binding target or partner (e.g., an antigen). The affinity of a molecule for its target can be measured using the dissociation constant (K D ) indicates that K D are the dissociation and association rate constants (k off and k on The strength or affinity of a binding interaction can be measured using the dissociation constant (K D ) is used to represent the smaller K D =K indicates greater affinity. The binding properties (affinity) of a selected polypeptide can be quantified using methods well known in the art. One such method entails measuring the rates of formation and dissociation of the antigen binding domain / antigen complex, where these rates depend on the concentration of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates equally in both directions. Thus, the "binding rate constant" (K on ) and the “dissociation rate constant” (K off ) Both can be determined by calculating the concentration and the actual association and dissociation rates, (see Nature 361: 186-87 (1993)).

[0112] K off / K on The ratio of 2 / 3 eliminates all parameters not related to affinity and is equal to the dissociation constant K. D (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). In some embodiments, when the equilibrium binding constant (K D ) is ≤1 μM, the recombinant polypeptide of the present invention can specifically bind to the epitope. In some embodiments, when the equilibrium binding constant (K D ) is ≤100 nM, the recombinant polypeptide of the present invention can specifically bind to the epitope. In some embodiments, when the equilibrium binding constant (K D ) is ≤10 nM, the recombinant polypeptide of the present invention can specifically bind to the epitope. In some embodiments, when the equilibrium binding constant (K D) is ≤100 pM to about 1 pM, the recombinant polypeptide of the present invention can specifically bind to the epitope, as measured by an assay such as surface plasmon resonance (SPR), Octet assay, or similar assays known to those skilled in the art. In some embodiments, K D Can be 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).

[0113] Thus, equivalent affinities may include different rate constants as long as the ratio of the rate constants remains the same. Thus, in some embodiments, "reduced binding" refers to a decrease in the affinity of the corresponding interaction. Conversely, "increased binding" refers to an increase in the binding affinity of the corresponding interaction.

[0114] As used herein, the term "isotype" refers to the immunoglobulin class (eg, IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by the heavy chain constant region genes.

[0115] In the context of the present invention, the term "bispecific binding molecule" refers to an antibody that has two different antigen-binding regions defined by different antibody sequences.

[0116] "EGFRxNRP1 bispecific antibody" or "anti-EGFRxNRP1 bispecific antibody" is a bispecific binding molecule comprising two different antigen binding domains, one of which specifically binds to the antigen EGFR1 and one of which specifically binds to NRP1. Similar nomenclature is used throughout for other bispecific binding molecules that bind targets other than EGFR, such as "HER2xNRP1 bispecific antibody" or "cMETxNRP1 bispecific antibody" to describe bispecific binding molecules comprising one antigen binding domain that specifically binds to HER2 or cMET, respectively, and another antigen binding domain that specifically binds to NRP1.

[0117] As used herein, the term "heavy chain" may be interpreted as including a full-length heavy chain comprising a heavy chain variable region domain (VH), which includes an amino acid sequence having a variable region sequence sufficient to confer antigen specificity, and three heavy chain constant region domains CH1, CH2 and CH3, or fragments thereof. In addition, as used herein, the term "light chain" may be interpreted as including a full-length light chain comprising a light chain variable region domain (VL), which includes an amino acid sequence having a variable region sequence sufficient to confer antigen specificity and a light chain constant region domain (CL), fragments thereof. As used herein, the term "Fab" refers to an antigen-binding region. Fab consists of a variable heavy chain and light chain and a constant heavy chain and light chain.

[0118] As used herein, the term "percent identity" between two sequences (e.g., amino acid or nucleotide sequences) refers to the percentage of positions (out of a possible 100%) that are identical (with appropriate insertions or deletions for optimal alignment) when optimally aligned and compared. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / number of total positions x 100), taking into account the number of gaps and the length of each gap, which need to be introduced to achieve optimal alignment of the two sequences. Comparison of sequences and determination of the percent identity between two sequences can be achieved using a mathematical algorithm, as described in the following non-limiting examples. Methods and algorithms for determining the % homology between two protein sequences are well established in the art.

[0119] For example, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol. (48): 444-453) algorithm, which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. In addition, a protein amino acid sequence can be used as a "query sequence" to search public databases, for example to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215: 403-10. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0120] As used herein, the term "monoclonal antibody" refers to an antibody molecule prepared with a single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to antibodies displaying a single binding specificity, which antibodies have variable and constant regions derived from human germline immunoglobulin sequences.

[0121] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which an expression vector, e.g., an expression vector encoding an antibody of the invention, has been introduced. Recombinant host cells include, e.g., transfectomas such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6 or NSO cells, and lymphocytes.

[0122] The term "treatment" refers to the administration of an effective amount of a therapeutically active bispecific binding molecule of the invention with the goal of alleviating, ameliorating, preventing or eradicating (curing) symptoms or a disease state.

[0123] The term "effective amount" or "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result at the desired dosage and time period. The therapeutically effective amount of the bispecific binding molecule may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the bispecific binding molecule to elicit a desired response in the subject. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.

[0124] As used herein, the terms "combination" or "co-administration" are used interchangeably to refer to the use of more than one therapy (e.g., one or more prophylactic and / or therapeutic agents). The use of these terms does not limit the order in which therapies (e.g., prophylactic and / or therapeutic agents) are administered to a subject.

[0125] As used herein, the term "synergistic" refers to a combination of a polypeptide of the invention and another therapy (eg, a prophylactic or therapeutic agent) that is more effective than the additive effects of the therapies.

[0126] The term "pharmaceutically acceptable salts" is meant to include salts of the active bispecific antibodies that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the bispecific antibodies described herein.

[0127] The term "parenteral" as used herein includes, but is not limited to, subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0128] The term "carrier" refers to a vehicle used to formulate a composition, and may be composed of a variety of excipients.

[0129] As used herein, the term "excipient" refers to any pharmacologically inactive, natural or synthetic ingredients or substances prepared together with (e.g., concomitantly) or afterwards with the active ingredient of the present invention. In some embodiments, the excipient may be any additive, adjuvant, adhesive, filler (bulking agent), carrier, coating, diluent, disintegrant, filler (filler), glidant, lubricant, preservative, vehicle or combination thereof that can be given together with the recombinant polypeptide of the present invention, and or useful in preparing the composition of the present invention. Excipients include any such materials that are non-toxic and do not interact with other ingredients of the composition known in the art. In some embodiments, when preparing the composition for the purpose of filling the composition, the excipient may be prepared together with the recombinant polypeptide (therefore often referred to as a bulking agent (bulking agents), filler (filler) or diluent). In other embodiments, excipients can be used to give active ingredient enhancement in the final dosage form, such as promoting absorption and / or solubility. In still other embodiments, excipients 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., the composition is in the physical form of a dry granule, or a dry flowable powder). Reference to an excipient includes both one and more than one such excipient. Suitable pharmaceutical excipients are described by EW Martin in Remington's Pharmaceutical Sciences, the disclosure of which is incorporated herein by reference in its entirety.

[0130] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to mean the inclusion of stated steps, elements or integers, or groups of steps, elements or integers, but not the exclusion of any other steps, elements or integers, or groups of elements, steps or integers.

[0131] All patent applications, patents, and printed publications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Furthermore, all patent applications, patents, and printed publications cited herein are incorporated by reference in their entirety, except for any definitions, subject matter disclaimers, or disclaimers, and unless the incorporated material is inconsistent with the explicit disclosure herein, in which case the language of the disclosure controls.

[0132] The scope of the present invention is not limited by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will become apparent to those skilled in the art from the above description and the accompanying drawings. Such modifications are expected to fall within the scope of the appended claims. It should be further understood that all values ​​are approximate and are provided for description only.

[0133] Bispecific antibodies with EGFR binding domain and NRP1 binding domain (EGFRxNRP Ab)

[0134] As described above, the present invention relates to bispecific binding molecules that specifically bind to a target protein and neuropilin-1 (NRP1).

[0135] Bispecific antibodies have many advantages over monoclonal antibodies. First, the manufacture of bispecific binding molecules is more efficient because it is a molecule with two effective targets. For example, bispecific binding molecules can target a variety of immune cell receptors (CD3, CD16 or CD47) or immune checkpoint proteins (PD1, LAG-3 or 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 1NRP1. Bispecific antibodies for anticancer therapy are designed to improve anticancer efficacy.

[0136] EGFR binding domain

[0137] Epidermal growth factor receptor (EGFR) is one of the most frequently altered oncogenes in solid tumors. Increased EGFR signaling can drive proliferation and cell survival in many cancer types, including breast cancer, prostate cancer, non-small cell lung cancer (NSCLC), esophageal and gastric cancer, liver, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, pancreatic cancer, colon cancer and rectal cancer. Increased EGFR signaling can occur from EGFR overexpression, EGFR mutations or mutations of transduction factors that lead to constitutive activation within the EGFR signaling pathway, and / or increased levels of EGFR cognate ligands such as EGF, tumor necrosis factor-α (TGF-α), amphiregulin (AREG), epigen, β-cells, heparin-binding EGF (HB-EGF) and epidermal regulatory proteins. EGFR signaling activates downstream signaling cascades, including RAS-RAF-MEK-ERK and PI3K-Akt-mTOR axes, which lead to proliferation and cancer cell survival. Unfortunately, anti-EGFR drugs, including tyrosine kinase inhibitors, monoclonal antibodies, and radiation therapy, are effective only in a few cancer types, such as metastatic colorectal cancer, non-small cell lung cancer (NSCLC), and advanced head and neck cancer. In addition, treatment improves survival in only some subjects, and initial responses often end in drug resistance. Therefore, new rational designs are needed to improve the efficacy of treatment.

[0138] Embodiments of the present disclosure further provide methods of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a bispecific binding molecule. In some embodiments, the present disclosure provides for use of the disclosed bispecific binding molecules in the manufacture of a medicament for treating cancer.

[0139] In certain embodiments of the bispecific binding molecule, the disclosure provides a method for treating EGFR overexpression cancer in a subject in need, including administering a therapeutically effective amount of the bispecific binding molecule and / or a pharmaceutically acceptable carrier or diluent provided herein. In certain embodiments, the cancer overexpressed by EGFR is non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, colorectal cancer, head and neck squamous cell carcinoma, gastric junction adenocarcinoma, gastroesophageal junction adenocarcinoma, liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, renal cancer and pancreatic cancer or any other solid tumor tissue overexpressing EGFR or EGFR kinase domain mutations. In some embodiments of the method of treatment, the method further includes administering to the subject a drug that increases cell EGFR expression.

[0140] In certain embodiments of the method of treatment, the cancer in which EGFR is overexpressed is resistant to treatment with an anti-EGFR monoclonal antibody. In certain embodiments of the method of treatment, the anti-EGFR monoclonal antibody is amivantamab, cetuximab, depatuxizumab, depatuxizumab mafodotin, duligotuzumab, futuximab, GC1118, Imagatuzumab, matuzumab, necitumumab, nimotuzumab, panitumumab, zalutumumab or HumMR1. In some embodiments of the method of treatment, the subject is human. In some embodiments, EGFR-overexpression is resistant or refractory to EGFR tyrosine kinase inhibitors. In some embodiments, the EGFR-overexpressing cancer is refractory to osimertinib.

[0141] Antibodies are proteins that bind to antigens with high specificity and high affinity and can neutralize antigen activity. Anti-EGFR antibodies block ligand-activated EGFR signaling and induce receptor endocytosis, leading to EGFR degradation in the proteasome. When mutations occur in the EGFR binding domain of the antibody, the subject may acquire anti-EGFR antibody resistance during the course of treatment. Chimeric mouse / human monoclonal cetuximab and fully humanized monoclonal antibody panitumumab have different binding domains for EGFR, and therefore panitumumab is still effective after resistance to cetuximab is generated, and vice versa. Therefore, another anti-EGFR antibody targeting a different EGFR binding domain can be given to a subject who has acquired resistance to an anti-EGFR antibody.

[0142] Panitumumab is an IgG2 anti-EGFR humanized monoclonal antibody that binds to EGFR with approximately 8-fold higher affinity than the IgG1 anti-EGFR chimeric human / mouse monoclonal antibody cetuximab (Garcia-Foncillas et al. 2019). Antibody binding can induce immune responses prior to induction of endocytosis. Panitumumab binding induces antibody-dependent cell-mediated cytotoxicity (ADCC) and initiates antibody-dependent cellular phagocytosis (ADCP) by activating 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 to induce ADCC. Activated NK cells secrete perforins and granzymes, which lead to cancer cell lysis and release of immunostimulatory molecules such as interferon-γ (IFN-γ), TNF-α, chemokines, and granulocyte macrophage colony-stimulating factor (GM-CSF). Cytokine secretion by NK cells stimulates dendritic cell maturation, NK cell cross-talk, and co-expression of CD137. CD137 expression recruits anti-EGFR CD8 + T cells, leading to increased killing of cancer cells expressing EGFR. Mature dendritic cells further activate NK cells and present tumor antigens to cytotoxic CD8 + T cells. The IgG1 Fc region also binds to Fc receptors on macrophages or plasmacytoid dendritic effector cells or the first subunit of the C1 complement complex (C1q) to initiate antibody-dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC) by the cells, respectively. Like cetuximab, the anti-EGFR antibodies cetuximab, nemotuzumab, and nimotuzumab also have an IgG1 Fc region for ADCC induction.

[0143] NRP1 binding domain

[0144] Cancer cells overexpress pro-angiogenic factors that promote rapid growth of new blood vessels. Blood vessels around tumors are abnormal, with capillary constriction that reduces total blood flow to the tumor. Reduced blood flow rate increases tumor interstitial fluid pressure and reduces drug flow from blood vessels to the tumor (Milosevic et al., 1999). Unlike normal tissue, the lack of lymphatic vessels in tumor tissue also contributes to abnormal angiogenesis and high tumor interstitial pressure. Inhibiting angiogenesis by targeting vascular endothelial growth factor-A (VEGF165) can normalize blood vessel formation, 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 can have adverse side effects and are only effective within a narrow exposure range (Kamba and McDonald, 2007).

[0145] Neuropilin-1 (NRP1) and neuropilin-2 (NRP2) receptors are multifunctional single-pass transmembrane glycoproteins that play important roles in angiogenesis and lymphangiogenesis, respectively. The C-terminal region of each VEGF ligand family and Sema3 ligand that binds to NRP1 and NRP2 binds to the arginine binding pocket in the b1 domain of NRP1 and NRP2 (Parker et al., 2012). Binding to the arginine binding pocket occurs through a motif of R / KxxR / K (R = arginine, K = lysine, and x = any amino acid), which is usually present in the C-terminal region of NRP binding ligands and is referred to as the "C-terminal rule" (CendR) (Teesalu et al. 2009). Proteins or peptides containing the C-terminal rule sequence are able to bind to NRPs through the C-terminal arginine (Arg) or lysine (Lys) residue (Zanuy et al., 2013).

[0146] Selective targeting of NRP1 is crucial for anti-EGFR cancer therapy, as silencing NRP2 significantly increases EGFR expression in lung and gastric cancer cells (Rizzolio et al., 2017). In addition, NRP1 is overexpressed in many cancer cells, including colon cancer, melanoma, astrocytoma, lung cancer, prostate cancer, and pancreatic ductal adenocarcinoma, and plays a crucial role in cancer progression (Graziani and Lacal, 2015). In addition to NRP1 overexpression in a variety of cancers, NRP1 is also overexpressed in tumor-associated endothelial cells. NRP1 acts as a coreceptor for ligands involved in angiogenesis, such as vascular endothelial growth factor (VEGF). 165 ), 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 thereby aids in VEGFR signaling, leading to increased angiogenesis. NRP1 also binds to secreted class 3 semaphorin ligands (Sema3A, Sema3B, Sema3C, Sema3D, Sema3E, Sema3F, Sema3G) to act as a coreceptor for plexin family receptors, which also regulate angiogenesis.

[0147] Blocking the binding of NRP1 ligands to the anti-NPR1 specific binding domain reduced the expression of the endothelial adhesion molecule VE-cadherin, resulting in increased extravasation of anti-EGFR antibodies from the blood vessels. Increased extravasation also reduced tumor interstitial pressure, allowing the drug to flow more freely from the blood vessels to the tumor environment.

[0148] The epithelial barrier around solid tumors is composed of intercellular spaces densely filled with interstitial epithelial cells connected by intercellular adhesion factors, and prevents therapeutic agents from penetrating tumors. Overexpression of E-cadherin helps intercellular adhesion. Since substances that reduce E-cadherin have been found in viruses (adenovirus-3), this is a reported case in which only a part (JO-1) of the protein constituting the virus that has the activity of reducing the E-cadherin of cells in tight binding is co-administered with antibodies, thereby increasing the anti-cancer effect of antibodies (Beyer et al. 2011). Targeting NRP1 reduces the expression of epithelial barrier adhesion molecules E-cadherin and integrin β1 subunits, which are overexpressed in many solid tumors and increase extracellular matrix connections, resulting in reduced drug penetration. In addition, integrin β1 subunits are involved in activating growth factor receptor-mediated cell proliferation.

[0149] In conclusion, targeting NRP1 reduced VEGF165 binding, as well as the expression of VE-cadherin, E-cadherin, and integrin β1, which together served to reduce angiogenesis, tumor interstitial pressure, and increase the extravasation and tumor penetration of anti-EGFR antibodies.

[0150] Cancer cells produce immune checkpoint molecules that inhibit immune responses. NRP1 is an immune checkpoint molecule, and its expression is increased in tumor-associated endothelial cells and cancer cells. NRP1 expression increases Treg activity and decreases tumor-specific CD8 + T cell response to induce immunosuppression (Chuckran et al. 2020). Therefore, in addition to increasing drug extravasation and penetration, the NRP1 binding domain also acts as an immune checkpoint inhibitor. In still another aspect, the present disclosure provides a bispecific binding molecule that targets NRP1 to reduce cancer-mediated checkpoint molecule immunosuppression, allowing natural immunity at the tumor site, and increasing the likelihood of tumor response.

[0151] The anti-NRP1 antibody MNRP1685A, also known as vesencumab, competitively binds to VEGF165 on NRP1 and acts to inhibit VEGF signaling through VEGFR2, thereby affecting angiogenesis, cell survival, migration, adhesion and invasion (Pan Q et al. 2007). However, subjects treated with vesencumab in a Phase I trial of advanced solid tumors suffered intolerable side effects, including gastrointestinal bleeding, fungemia, duodenal obstruction, thrombocytopenia, proteinuria, alopecia, dysphonia, fatigue and nausea, which led to further abortion of the trial (Weekes et al. 2014, Patnaik et al. 2014).

[0152] NRP1 is active as a homodimer or heterodimer, while monomeric peptides, such as the cleaved penetrating peptide iRGD, have only a weak ability to modulate NRP1 bioactivity (Sugahara et al. 2010). Therefore, peptides that selectively bind to NRP1 as a homodimer to modulate bioactivity are preferred. Unfortunately, although the Fc heavy chain-fused NRP-binding peptide A22p is presented as a dual peptide (homodimer), A22p binds to both NRP1 and NRP2 (Shin et al. 2014). Therefore, it is important to identify effective homodimer and non-toxic NRP1 selective targeting molecules.

[0153] The NRP1 binding domain comprises an antibody or an NRP1 binding fragment thereof. For example, all or a portion of an NRP1 antibody known in the art or an anti-NRP1 antibody provided herein may be used. Non-limiting examples of anti-NRP1 mAb heavy chain polypeptide sequences are set forth in SEQ ID NOs: 41-47, which include an N-terminal NPR1 binding domain. In some embodiments, the monoclonal antibody heavy chain polypeptide comprises an N-terminal NRP1 binding domain comprising a variable heavy chain (VH) and a constant heavy chain 1 (CH1), and an Fc domain comprising a constant heavy chain 2 (CH2) and a constant heavy chain 3 (CH3). In some embodiments, the monoclonal antibody comprises a light chain polypeptide sequence as shown in any one of SEQ ID NOs: 48-54. Thus, the heavy chains of SEQ ID NOs: 41-47 can be paired with the light chains of SEQ ID NOs: 48-54, respectively. The polynucleotide sequences encoding the heavy and light chain polypeptides are shown in SEQ ID NOs: 55-61 and 62-68, respectively.

[0154] Bispecific antibodies with affinity for EGFR and NRP1

[0155] The present disclosure provides a bispecific antibody comprising a heavy chain fusion polypeptide, wherein the N-terminal EGFR binding domain is located at the N-terminus of the heavy chain polypeptide and the NRP1 binding domain is located at the C-terminus. The heavy chain fusion polypeptide of the bispecific binding molecule comprises an EGFR binding region at the N-terminus, which includes a variable heavy chain (VH) and a constant heavy chain 1 (CH1); an Fc domain comprising a constant heavy chain 2 (CH2), 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 consists of a variable heavy chain (VH) and a variable light chain (VL) fused by a flexible peptide linker.

[0156] Schematic diagram of representative bispecific antibody structures of the present disclosure is shown in Figure 1 .

[0157] Exemplary bsAb heavy chain polypeptide sequences are shown 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 shown in SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 40, respectively. In some aspects, the bispecific antibody further comprises a light chain polypeptide sequence comprising a variable light chain and a constant light chain, which is paired with the heavy chain polypeptide and is shown in SEQ ID NO: 12. The polynucleotide sequence encoding the light chain polypeptide of SEQ ID NO: 12 is shown in SEQ ID NO: 25. In some aspects, the present disclosure provides polynucleotide sequences for encoding the GGGGS subunit, and these sequences are shown in SEQ ID NOs: 26-38. Table 1 lists the polypeptide sequences of the bispecific antibodies, and Table 2 lists the polynucleotide sequences encoding the polypeptides. Example 1 describes a method for the generation of bispecific antibodies.

[0158] In some aspects, the bispecific antibody comprises an anti-EGFR binding arm comprising (or consisting of) a VH amino acid sequence as shown in SEQ ID NO: 69 and a VL amino acid sequence as shown in SEQ ID NO: 70. In some aspects, the bispecific antibody comprises an anti-EGFR binding arm comprising a heavy chain CDR1, CDR2, and CDR3 region comprising (or consisting of) the amino acid sequences shown in SEQ ID NO: 71, 72, and 73, respectively. In some aspects, the bispecific antibody comprises an anti-EGFR binding arm comprising a heavy chain CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NO: 73. In some aspects, the bispecific antibody comprises an anti-EGFR binding arm comprising a light chain CDR1, CDR2, and CDR3 region comprising (or consisting of) the amino acid sequences shown in SEQ ID NO: 74, 75, and 76, respectively. In some aspects, the bispecific antibody comprises an anti-EGFR binding arm comprising a light chain CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NO: 76. In some embodiments, the anti-EGFR binding arm comprises (or consists of) one or more sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.8% identical to any of the above VH, VL, HCDR or LCDR sequences.

[0159] In some aspects, the bispecific antibody comprises an anti-NRP1 binding arm comprising (or consisting of) a VH amino acid sequence as shown in SEQ ID NO: 77 and a VL amino acid sequence as shown in SEQ ID NO: 78. In some aspects, the bispecific antibody comprises an anti-NRP1 binding arm comprising a heavy chain CDR1, CDR2, and CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NO: 79, 80, and 81, respectively. In some aspects, the bispecific antibody comprises an anti-NRP1 binding arm comprising a heavy chain CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NO: 81. In some aspects, the bispecific antibody comprises an anti-NRP1 binding arm comprising a light chain CDR1, CDR2, and CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NO: 82, 83, and 84, respectively. In some aspects, the bispecific antibody comprises an anti-NRP1 binding arm comprising a light chain CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NO: 84. In some embodiments, the anti-NRP1 binding arm comprises (or consists of) one or more sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.8% identical to any of the above VH, VL, HCDR or LCDR sequences.

[0160] Thus, in an embodiment, the invention relates to a composition comprising a bispecific binding molecule comprising:

[0161] (a) two identical heavy chain polypeptides and the first heavy chain polypeptide is fused to a first scFv via a peptide linker to produce a first heavy fusion polypeptide, and the second heavy chain is fused to a second scFv via a peptide linker to produce a second heavy fusion polypeptide, wherein the first and second scFvs are identical and bind to NRP1; and

[0162] (b) two identical light chains comprising a first light chain and a second light chain,

[0163] The VL and VH of the heavy and light chain polypeptides comprise a target protein binding domain that binds to the target protein and a scFv comprising an NRP1 binding domain that binds to NRP1.

[0164] In some embodiments of the bispecific binding molecule, the heavy chain fusion polypeptide is paired with a light chain polypeptide. In some embodiments, the heavy chain polypeptide comprises (a) two identical heavy chain polypeptides and the first heavy chain polypeptide is fused to the first scFv via a peptide linker to produce a first heavy fusion polypeptide, and the second heavy chain is fused to the second scFv via a peptide linker to produce a second heavy 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 fusion and light chain polypeptides comprise a target protein binding domain that binds to a target protein and an scFv containing an NRP1 binding domain that binds to NRP1.

[0165] Bispecific antibodies having different binding affinities for EGFR and NRP1 are provided. In one embodiment, it is preferred that the binding affinity for NRP1 is lower than that for EGFR. In some embodiments,

[0166] Accordingly, the present invention relates to bispecific binding molecules comprising:

[0167] (a) two identical heavy chain polypeptides and the first heavy chain polypeptide is fused to a first scFv via a peptide linker to produce a first heavy fusion polypeptide, and the second heavy chain is fused to a second scFv via a peptide linker to produce a second heavy fusion polypeptide, wherein the first and second scFvs are identical and bind to NRP1; and

[0168] (b) two identical light chains comprising a first light chain and a second light chain,

[0169] The VL and VH of the heavy and light chain polypeptides comprise a target protein binding domain that binds to the target protein and a scFv comprising an NRP1 binding domain that binds to NRP1.

[0170] In some embodiments of the bispecific binding molecule, the heavy chain fusion polypeptide is paired with a light chain polypeptide. In some embodiments, the heavy chain polypeptide comprises (a) two identical heavy chain polypeptides and the first heavy chain polypeptide is fused to the first scFv via a peptide linker to produce a first heavy fusion polypeptide, and the second heavy chain is fused to the second scFv via a peptide linker to produce a second heavy 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 fusion and light chain polypeptides comprise a target protein binding domain that binds to a target protein and an scFv containing an NRP1 binding domain that binds to NRP1.

[0171] In some embodiments, the disclosure provides polynucleotide sequences encoding GGGGS subunits, and these sequences are shown in SEQ ID NOs: 26-38. Table 1 lists the polypeptide sequences of the bispecific binding molecules, and Table 2 lists the polynucleotide sequences encoding the polypeptides.

[0172] Example 1 describes methods for generating bispecific binding molecules as described in Example 1.

[0173] In some embodiments, the EGFR binding domain of the bispecific binding molecule has an affinity (K) for EGFR in the subnanomolar range (<0.1 nM). D In some embodiments, the NRP1 binding domain of the bispecific binding molecule has an NRP1 affinity (K) ranging from 70 nM for a bispecific binding molecule construct having the polypeptide sequence of SEQ ID NO: 1 to the subnanomolar range for bispecific antibodies having the polypeptide sequences shown in SEQ ID NOs: 2, 3, 5, 7, 10, and 11. D ). Thus, in one aspect, the present disclosure provides a bispecific antibody having an EGFR binding domain with an affinity for EGFR in the subnanomolar range and an NRP1 binding domain with an affinity for NRP1 in the subnanomolar range, as shown in Table 2 of Example 2. Figure 2A -2K shows the binding affinity curve of the bispecific binding molecule to immobilized huEGFR. In some embodiments, the binding affinity of the bispecific binding molecule to EGFR is <0.1 nM. In some embodiments, the binding affinity of the bispecific binding molecule to EGFR is <0.01 nM. Figure 3A -3K shows the binding affinity curve of the bispecific binding molecule to immobilized huNRP1. In some embodiments, the binding affinity of the bispecific binding molecule to NRP1 is in the range of 0.01 nM-1000 nM. In some embodiments, the binding affinity of the bispecific binding molecule to NRP1 is in the range of 0.1 nM-100 nM.

[0174] One aspect of the present disclosure provides bispecific antibodies with asymmetric binding affinity for EGFR and NRP1, see Table 2. In some embodiments, the K of the EGFR binding domain to EGFR is D K of NRP1 binding domain to NRP1 D The asymmetric affinity provides better homing of cancer cells expressing EGFR while reducing the cytotoxicity of targeting NRP1.

[0175] Blocking VEGF binding to NRP1 with the NRP1 binding domain of the bispecific binding molecule inhibits VEGFR2 activation as measured by VEGFR2 phosphorylation. D 69.7 nM, but did not inhibit VEGFR phosphorylation ( Figure 4 In some embodiments, the bispecific binding molecules inhibit VEGFR2 phosphorylation to prevent angiogenesis, see Table 3 and Figure 5A-5B as well as Figures 11A-11B In some embodiments, the inventors have found that the dissociation constant (K D ) does not reflect the ability of the bispecific binding molecule to inhibit VEGFR2 phosphorylation. For example, the bispecific binding molecule having heavy chain SEQ ID NO: 6 inhibited VEGFR2 phosphorylation by 50% at 10,130 nM (IC 50 ), but NRP1 K D The bispecific binding molecule with heavy chain SEQ ID NO: 4 inhibited 50% VEGFR2 phosphorylation (IC 50 ), but NRP1 K D The value of 5-nitro-1-nitro-2-nitro-4 ...

[0176] In some embodiments, the bispecific antibodies inhibit cancer cell growth. In some embodiments, the bispecific binding molecule Fc domain comprising constant heavy chains 2 and 3 (CH2 and CH3) belongs to the IgG1 or IgG2 subclass. In some embodiments, the present disclosure provides a bispecific binding molecule having an EGFR binding domain and an NRP1 binding domain of a scFv, which robustly inhibits cell proliferation in the cancer cell line H1975, Tables 4 and Fig. 12A and 12B This result indicates that the scFv is essential for the function of the bispecific binding molecule as a degrader. NRP1 Example 4 describes the use of the bispecific binding molecule IC for measuring cell growth inhibition in the H1975 cancer cell line. 50 method.

[0177] Additional bispecific antibodies with binding affinity for NRP1 and receptor tyrosine kinases (RTKs)

[0178] In addition to the EGFRxNRP1 bispecific antibody constructs described above, in other embodiments, the present disclosure provides additional bispecific antibody constructs having binding affinity for NRP1 and a receptor tyrosine kinase (RTK) other than EGFR. Non-limiting examples include the following representative constructs.

[0179] cMETxNRP1 bispecific construct

[0180] In one embodiment, the RTK is a cMET family member. In one embodiment, the RTK is cMET. Receptor degradation mediated by the cMETxNRP1 bispecific antibody construct is described in detail in Example 8.

[0181] In an embodiment, the cMETxNRP1 bispecific antibody comprises an anti-NRP1 binding arm comprising a heavy chain CDR1, CDR2, and CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NOs: 79, 80, and 81, respectively. In one embodiment, the bispecific antibody comprises an anti-NRP1 binding arm comprising a heavy chain CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NO: 81. In an embodiment, the bispecific antibody comprises an anti-NRP1 binding arm comprising a light chain CDR1, CDR2, and CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NOs: 82, 83, and 84, respectively. In one embodiment, the bispecific antibody comprises an anti-NRP1 binding arm comprising a light chain CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NO: 84.

[0182] In an embodiment, the cMET binding arm comprises a sequence of an anti-cMET monoclonal antibody (mAb) known and available in the art, such as the heavy and light chain CDRs (1-3) of the mAb or the VH / VL polypeptides of the mAb. Many anti-cMET mAbs have been described in the art, including, for example, in U.S. Patents US 7,476,724, US 8,673,302, and US 9,068,011, in US Patent Publications US2013 / 0216527, US2019 / 0248907, and US2021 / 0087278, and in PCT Publication WO 2010 / 064089, the entire contents of each of which (including 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 in the art as LY2875358) (see, e.g., Rosen et al. (2017) Clin. Cancer Res. 23:1910-1919; Yan et al. (2018) Invest. New Drug 36:536-544); onartuzumab (see, 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 (see, e.g., Camidge et al. (2018) Annals Oncol. 29:496-497; Camidge et al. (2022) JTO Clin. Res. Rep. 3:100262), the entire contents of each of which are expressly incorporated herein by reference.

[0183] HER2xNRP1 Bispecific Constructs

[0184] In one embodiment, the RTK is a HER family member. In one embodiment, the RTK is HER2. Receptor degradation mediated by HER2xNRP1 bispecific antibody constructs is described in detail in Example 9.

[0185] In an embodiment, the HER2xNRP1 bispecific antibody comprises an anti-NRP1 binding arm comprising a heavy chain CDR1, CDR2, and CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NOs: 79, 80, and 81, respectively. In one embodiment, the bispecific antibody comprises an anti-NRP1 binding arm comprising a heavy chain CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NO: 81. In an embodiment, the bispecific antibody comprises an anti-NRP1 binding arm comprising a light chain CDR1, CDR2, and CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NOs: 82, 83, and 84, respectively. In one embodiment, the bispecific antibody comprises an anti-NRP1 binding arm comprising a light chain CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NO: 84.

[0186] In embodiments, the HER2 binding arm comprises the sequence of anti-HER2 monoclonal antibodies (mAbs) known and available in the art, such as the heavy and light chain CDRs (1-3) of mAbs or the VH / VL polypeptides of mAbs.Many anti-HER2 mAbs have been described in the art, including, for example, in U.S. Patent No. 10,377,825, in U.S. Patent Publication US2009 / 0226466, US 2010 / 0047230, US 2011 / 0313137, US 2012 / 0309942, US 2015 / 0322162, US 2017 / 0066829 and US2018 / 0201692 and in PCT Publication WO 2013 / 075382, the entire contents of each of which (including anti-cHER2 antibody sequences) are expressly incorporated herein by reference. Non-limiting examples of anti-HER2 mAbs known in the art include trastuzumab (see, e.g., Romond et al. (2005) New Engl. J. Med. 353: 1673-1684; Hudis (2007) New Engl. J. Med. 357: 39-51) and pertuzumab (see, 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 each 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.

[0187] IGF1RxNRP1 bispecific constructs

[0188] In one embodiment, the RTK is a member of the IGFR family. In one embodiment, the RTK is IGF1R. Receptor degradation mediated by the IGF1RxNRP1 bispecific antibody construct is described in detail in Example 10.

[0189] In an embodiment, the IGF1RxNRP1 bispecific antibody comprises an anti-NRP1 binding arm comprising a heavy chain CDR1, CDR2, and CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NOs: 79, 80, and 81, respectively. In one embodiment, the bispecific antibody comprises an anti-NRP1 binding arm comprising a heavy chain CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NO: 81. In an embodiment, the bispecific antibody comprises an anti-NRP1 binding arm comprising a light chain CDR1, CDR2, and CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NOs: 82, 83, and 84, respectively. In one embodiment, the bispecific antibody comprises an anti-NRP1 binding arm comprising a light chain CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NO: 84.

[0190] In an embodiment, the IGF1R binding arm comprises a sequence of an anti-IGF1R monoclonal antibody (mAb) known and available in the art, such as the heavy and light chain CDRs (1-3) of the mAb or the VH / VL polypeptides of the mAb. Many anti-IGF1R mAbs have been described in the art, including, for example, in U.S. Pat. Nos. 10,106,614, 10,112,998, and 10,519,245, in U.S. Pat. Nos. 2010 / 0143340 and 2014 / 0079665, and in PCT Publication No. WO 2011 / 057064, each of which is expressly incorporated herein by reference in its entirety (including anti-IGF1R antibody sequences). Non-limiting examples of anti-IGF1R mAbs known in the art include ganitumab (also known in the art as AMG 479) (see, e.g., Moody et al. (2004) J. Endocrinol. 221: 145-155; Tap et al. (2012) J. Clin. Oncol. 30: 1849-1856), figitumumab (see, e.g., Molife et al. (2010) Brit. J. Cancer 103:332-339; Langer et al. (2014) J. Clin. Oncol. 32:2059-2066), cixutumumab (see, e.g., Rathkopf et al. (2011) J. Clin. Oncol. 29:e15081; Schwartz et al. (2013) Lancet 14:371-382), and dalotuzumab (see, e.g., Scartozzi et al. (2010) Curr. Opin. Mol. Therap. 12:361-371; Atzori et al. (2011) Clin. Cancer Res. 17:6304-6312), the entire contents of each of which are expressly incorporated herein by reference.

[0191] Bispecific Binding Molecules Tumor Growth Inhibition in Xenograft Mouse Models

[0192] In some aspects of the present disclosure, treatment with a bispecific antibody reduces tumor growth. In some embodiments, a bispecific binding molecule having a heavy chain of SEQ ID NO: 11 and a light chain of SEQ ID NO: 12 significantly reduces tumor growth rate. In some aspects, the addition of a scFv comprising an NRP1 binding domain significantly improves the efficacy of tumor growth inhibition. Example 5 describes methods and results of treating an H1975 xenograft mouse model with a bispecific antibody for tumor growth inhibition. Table 5 shows the percentage of tumor growth and inhibition. Figures 13A-13FTumor growth within days after implantation is shown.

[0193] Biological effects of bispecific binding molecules

[0194] In summary, the present disclosure provides bispecific antibodies that combine EGFR binding domains with NRP1 binding domains to promote homing of high EGFR expressing tumors by increasing EGFR affinity. The bispecific binding molecules will have a variety of biological effects that are combined to reduce cancer cell proliferation and survival, including reducing abnormal angiogenesis, increasing extravasation and penetration by reducing VE-cadherin, E-cadherin and integrin β1 expression. NRP1 targeting further reduces EGFR surface aggregation to enhance EGFR downregulation and inhibit NRP1 checkpoint immunosuppression. The combination of these features provides a bispecific binding molecule with a powerful ability to reduce EGFR-mediated cancer cell proliferation and survival.

[0195] FcRn binding for the transcytosis cycle

[0196] ~150k D The size of the antibody ensures a long serum half-life, thereby obtaining a lasting therapeutic effect. In addition, the antibody IgG Fc portion composed of 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 and recycled back to the serum through transcytosis, which further prolongs the antibody serum half-life. Cancers with low FcRn or FcγR expression are associated with poor prognosis (Pyzik et al., 2019), and therefore, FcRn- or FcγR-mediated circulation plays an important role in maintaining antibody serum concentrations. IgG1 antibodies have more efficient FcRn and FcγR circulation processes than IgG2 antibodies. Therefore, having an IgG1 Fc domain in a therapeutic antibody helps maintain drug treatment levels and reduce the frequency of administration, while shortening the half-life will be ideal for diagnostic testing or toxicity control.

[0197] In some embodiments, a polypeptide of the invention comprises an immunoglobulin domain, including a polypeptide comprising an immunoglobulin domain comprising an Fc domain selected from the IgG1 subclass.

[0198] Expression constructs

[0199] As used herein, the term "vector" or "expression vector" refers to a tool for expressing a target gene in a host cell. For example, a vector may include a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector such as an adenoviral vector, a retroviral vector, and an adeno-associated viral vector. Recombinant vectors can be produced by operating plasmids commonly used in related fields (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), phages (e.g., Agt4AB, A-Charon, AAz1, and M13, etc.), or viruses (e.g., CMV, SV40, etc.). In some embodiments, the expression vector comprises nucleic acids encoding the heavy chain fusion polypeptide and the light chain polypeptide of the bispecific binding molecule, wherein the nucleic acid sequence encoding the heavy chain fusion polypeptide is any one of SEQ ID NOs: 15-24 and the nucleic acid sequence encoding the light chain polypeptide is SEQ ID NO: 25.

[0200] In another aspect, the present disclosure provides isolated polynucleotides encoding heavy and light chain amino acid sequences of bispecific binding molecules. The polynucleotides encoded in the recombinant vector can be operably connected to a promoter. As used herein, the term "operably connected" refers to the functional connection between a nucleotide expression control sequence (such as a promoter sequence) and a second nucleotide sequence. Therefore, the regulatory sequence can control the transcription and / or translation of the second nucleotide sequence. Recombinant vectors can generally be constructed as vectors for cloning or for expression. As vectors for expression, vectors commonly used in the relevant art for expressing foreign proteins from plants, animals or microorganisms can be used. Recombinant vectors can be constructed by various methods known in the relevant art. Recombinant vectors can be constructed using eukaryotic cells as hosts, with fl replication origins, SV40 replication origins, pMB1 replication origins, glandular replication origins, AAV replication origins, CMV replication origins, and BBV replication origins, but are not limited thereto.

[0201] In addition, a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian cell virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a cytomegalovirus (CMV) promoter, or a thymidine kinase (TK) promoter of a 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 where the antibody has a peptide fused thereto, 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. For the latter, the two vectors can be introduced into a host cell by co-transformation and targeted transformation. Therefore, in another aspect, the present disclosure includes a vector containing a nucleic acid disclosed in SEQ ID NO: 14-38. Example 1 describes the characteristics of a vector that can be used to produce a bispecific antibody.

[0202] Another aspect of the present disclosure provides host cells transformed with recombinant vectors. Any type of host cell known in the relevant art can be used as a host cell. Examples of prokaryotic cells include strains such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, or strains belonging to the genus Bacillus (such as Bacillus subtilis and Bacillus thuringiensis), Salmonella typhimurium, Serratia marcescens and intestinal flora and strains such as various Pseudomonas species (Pseudomonas Spp.) and the like. Prokaryotic cell transformation can be used to clone plasmids on a large scale. Prokaryotic host cells may lack post-translational modifications required for antibody assembly and structure, and therefore, a preferred embodiment is vector transformation in eukaryotic host cells, such as yeast (Saccharomyces cerevisiae), insect cells, plant cells, mammalian cells, for example 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, etc. In some embodiments, the host cell comprises an expression vector, a nucleic acid encoding a heavy chain fusion polypeptide and a light chain polypeptide of the bispecific binding molecule, wherein the nucleic acid sequence encoding the heavy chain fusion polypeptide is any one of SEQ ID NOs: 15-24, and the nucleic acid sequence encoding the light chain polypeptide is SEQ ID NO: 25.

[0203] Another aspect of the present disclosure provides a method for preparing a peptide that specifically binds to NRP1, comprising culturing the above-mentioned host cell. Polynucleotides and recombinant vectors comprising polynucleotides can be inserted into host cells using insertion methods well known in the relevant art. For example, when the host cell is a prokaryotic cell, it can be transferred according to the CaCl2 method or electroporation method, and when the host cell is a eukaryotic cell, the vector can be transferred to the host cell by various methods, including microscopic injection, calcium phosphate precipitation, electroporation, liposome-mediated transformation and gene bombardment, etc., but the transfer method is not limited thereto. The method for selecting the transformed host cell can be easily performed using the phenotype expressed by the selected marker according to the well-known method in the relevant art. For example, when the selected marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a culture medium containing an antibiotic, an acceptable salt thereof and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients, or carriers appropriately selected according to the intended form of administration and consistent with conventional pharmaceutical practice.

[0204] Antibody-drug conjugates

[0205] In another aspect, the present disclosure provides an antibody-drug conjugate (ADC), comprising a bispecific antibody (protein degrader) of the present disclosure conjugated to a drug or other functional compound (also referred to as a payload). ADC technology is established in the art (e.g., reviewed in Strohl and Strohl (eds.) "Ch. 15: Antibody-Drug Conjugates" in Therapeutic Antibody Engineering (2012); Tumey (2020) Methods Mol. Biol. 2078: 1-22), and many ADC therapies are approved by the FDA.

[0206] In an ADC, the payload is conjugated to the bispecific antibody, such as via a linker and / or by using site-specific conjugation chemistries established in the art.

[0207] The use of linkers in ADCs is reviewed, for example, in Nareshkumar et al. (2015) Pharm. Res. 32: 3526-3540. In one embodiment, the linker is a cleavable linker. In another embodiment, the linker is a non-cleavable linker. Suitable linkers based on chemical motifs are available in the art, including disulfides, hydrazones, peptides, and thioethers.

[0208] The use of various site-specific conjugation chemistries in ADCs is reviewed, for example, in Zhou et al. (2017) Biomedicines 5: 64. These methods couple payloads to specifically defined sites in the antibody portion of the ADC (i.e., the bispecific antibodies of the present disclosure), including cysteine, glutamine, unnatural amino acids, short peptide tags, and glycans.

[0209] In one embodiment, the payload used in the ADC is toxic to cells (i.e., exhibits cytotoxicity). In another embodiment, the payload used in the ADC is non-toxic to cells (i.e., does not exhibit cytotoxicity).

[0210] In embodiments, the useful load used in ADC acts as a cytotoxic agent or a cytolytic agent. In embodiments, the useful load used in ADC acts to enhance one or more pharmacokinetic properties of the molecule, such as serum half-life. In embodiments, the useful load used in ADC acts to target the molecule to a microenvironment of interest (e.g., a target cell type or tissue). In embodiments, the useful load used in ADC imparts one or more immunomodulatory properties to the molecule. In embodiments, the useful load used in ADC imparts one or more enzyme properties to the molecule.

[0211] In an embodiment, the payload used in the ADC is a cytotoxic or cytolytic agent, non-limiting examples of which include small molecule drugs (eg, chemotherapeutic agents), protein toxins, bacterial toxins, cytolytic proteins / peptides, and radionuclides.

[0212] In an embodiment, the payload conjugated to the bispecific antibody acts by a mechanism selected from the group consisting of targeting folate receptors, inhibiting microtubules, cleaving DNA, and inhibiting TOP1.

[0213] In an embodiment, the payload conjugated to the bispecific antibody belongs to a class of payloads selected from maytansinoids (eg, maytansinoid DM4), MMAE / auristatins, and camptothecins.

[0214] In an embodiment, the payload conjugated to the bispecific antibody is selected from vedotin, emtansine, govitecan, talirine, ozogamicin, pasudotox, deruxtecan, mafodotin, soravtansine, and tesinine.

[0215] In an embodiment, the payload conjugated to the bispecific molecule is a radionuclide, non-limiting examples of which include 90 Y and 111 In.

[0216] In an embodiment, the payload conjugated to the bispecific molecule is a protein toxin, such as Pseudomonas endotoxin or diphtheria toxin.

[0217] In an embodiment, the payload conjugated to the bispecific molecule is an immunomodulatory peptide, such as Fas ligand (FasL).

[0218] In an embodiment, the payload conjugated to the bispecific molecule is a bioactive peptide, such as one that prolongs pharmacological half-life (e.g., GLP1) or targets specific cells. For example, calcitonin has been used to target ADCs to osteoclasts (Newa et al. (2011) Pharm. Res. 28: 1131-1143).

[0219] In an embodiment, the payload conjugated to the bispecific molecule is an enzyme.

[0220] In an embodiment, the payload conjugated to the bispecific molecule is an oligonucleotide.

[0221] Pharmaceutical compositions

[0222] Aspects of the present disclosure further include compositions. According to some embodiments, the compositions of the present disclosure include bispecific binding molecules of the present disclosure. For example, the bispecific binding molecules can be any bispecific binding molecules described in the bispecific antibody section above, and for the sake of brevity, these descriptions are included herein but not repeated.

[0223] The polypeptides described herein can be formulated into a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. In one embodiment, the present disclosure provides a pharmaceutical composition comprising the disclosed polypeptide and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. In one embodiment, the present invention is a pharmaceutical composition comprising an effective amount of the disclosed bispecific antibody or pharmaceutically.

[0224] Pharmaceutically acceptable carriers or excipients may contain inert ingredients that do not excessively inhibit the biological activity of the polypeptide. Pharmaceutically acceptable carriers should be biocompatible, for example, non-toxic, non-inflammatory, non-immunogenic, or have no other undesirable reactions or side effects after administration to a subject. Standard pharmaceutical preparation techniques may be used.

[0225] As used herein, pharmaceutically acceptable carriers, adjuvants or vehicles include any and all solvents, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., such as suitable for the desired specific dosage form. Remington's Pharmaceutical Sciences, 16th edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers for preparing pharmaceutically acceptable compositions and known techniques for their preparation. Unless any conventional carrier medium is incompatible with the polypeptide described herein, such as by producing any undesirable biological effect or otherwise interacting with any other component of the pharmaceutically acceptable composition in a harmful manner, its use is considered within the scope of the present invention. As used herein, the phrase "side effect" includes unwanted and adverse effects of therapy.

[0226] Materials that can serve as pharmaceutically acceptable carriers for antibodies can increase conformational stability, reduce protein dynamics, inhibit aggregation, and protect proteins from adsorption to liquid-air interfaces, and include, but are not limited to, cyclodextrin hydrogels, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as Tween 80, phosphates, glycine, sorbic acid or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride or zinc salts), colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, methylcellulose, hydroxypropyl methylcellulose, lanolin, sugars such as lactose, glucose and sucrose Sugars; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol and phosphate buffered solutions, and other nontoxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the composition, according to the judgment of the formulator.

[0227] In some embodiments, the compositions of the present invention include pharmaceutically acceptable salts. When the polypeptides of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such polypeptides with a sufficient amount of the desired base, either pure or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts or similar salts. When the polypeptides of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such polypeptides with a sufficient amount of the desired acid, either pure or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid or phosphorous acid, and 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, and the like. Also included are salts of amino acids, such as arginine salts, and salts of organic acids, such as glucuronic acid or galacturonic acid, and the like. (See, e.g., Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific polypeptides disclosed herein contain both basic and acidic functionalities that allow the polypeptides to be converted into base or acid addition salts.

[0228] Therefore, the disclosed polypeptides may exist as salts, such as salts with pharmaceutically acceptable acids. The present invention includes such salts. Non-limiting examples of such salts include hydrochlorides, hydrobromides, phosphates, sulfates, mesylates, nitrates, maleates, acetates, citrates, fumarates, propionates, tartrates (e.g., (+)-tartrates, (-)-tartrates, or mixtures thereof, including racemic mixtures), succinates, benzoates, and salts with amino acids such as glutamic acid, and quaternary ammonium salts (e.g., methyl iodide, ethyl iodide, etc.). These salts can be prepared by methods known to those skilled in the art.

[0229] The neutral forms of the polypeptide are preferably regenerated by contacting the salt with a base or acid and isolating the parent polypeptide in the conventional manner.The parent form of the polypeptide may differ from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0230] Certain polypeptides of the present invention may exist in unsolvated forms as well as solvated forms (including hydrated forms). Typically, solvated forms are equivalent to unsolvated forms and are included within the scope of the present invention. Certain polypeptides of the present invention may exist in a variety of crystalline or amorphous forms. Typically, all physical forms are equivalent for the purposes contemplated by the present invention and are contemplated to be within the scope of the present invention.

[0231] In some embodiments, subcutaneous formulations may contain recombinant human PH20 hyaluronidase (rHuPH20) to facilitate dispersion of the antibody from the injection site.

[0232] In some embodiments of the bispecific binding molecule, the pharmaceutical composition comprises 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 comprises a therapeutically effective amount of the bispecific binding molecule and one or more therapeutic agents for chemotherapy.

[0233] In some embodiments, the kit comprises one or more unit doses of a pharmaceutical composition comprising a bispecific binding molecule herein and a pharmaceutically effective carrier, and instructions for administering the one or more unit doses of the pharmaceutical composition to a subject in need thereof.

[0234] Giving Methods

[0235] The compositions of the invention may be administered to a subject in need of cancer treatment. The term "administration" or "administering" refers to the act of providing a composition of the invention, such as a polypeptide or a pharmaceutically acceptable salt thereof, to a subject in need of cancer treatment.

[0236] As used herein, "intermittent administration" includes administering a drug over a period of time (which may be considered a "first administration period"), followed by a period during which the composition is not taken or is taken at a lower maintenance dose (which may be considered a "resting period"), followed by a period during which the composition is administered again (which may be considered a "second administration period"). Typically, during the second phase of administration, the dosage level of the drug will match that administered during the first administration period, but may be increased or decreased as medically necessary.

[0237] In some embodiments, the compositions of the present invention can be administered orally, as suppositories, topical contact, intravenously, parenterally, intraperitoneally, intramuscularly, intralesionally, intrathecally, intranasally or subcutaneously, or a sustained-release device such as a miniature osmotic pump can be implanted to give the subject. Therefore, administration can be by any route, including parenterally and through mucosa (e.g., oral, sublingual, palate, gums, nose, vagina, rectum or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, etc. Other delivery methods include, but are not limited to, using liposome preparations, intravenous infusion or via implantation of reservoirs, etc. Specifically, the compositions are administered orally, intraperitoneally or intravenously.

[0238] In some embodiments, the bispecific binding molecule composition is administered by systemic intravenous (IV) or oral route for intestinal cancer, such as gastric cancer or intestinal cancer (Tashima et al., 2021). The formulation for delivery can be optimized by conventional, 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, and elixirs. In addition to the active polypeptide, the liquid dosage form may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0239] In some embodiments, the bispecific binding molecule composition is administered by subcutaneous injection. The injectable bispecific binding molecule preparation can be sterilized, for example, by filtering through a bacteria-retaining filter, or by incorporating a sterilant dispersed in sterile water or other sterile injection medium before use. In order to prolong the effect of the polypeptide described herein, it is often desirable to slow down the absorption of the polypeptide from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous material with poor water solubility. The absorption rate of the polypeptide then depends on its dissolution rate, which in turn can depend on crystal size and crystalline form. Alternatively, delayed absorption of the polypeptide form administered parenterally is achieved by dissolving or suspending the polypeptide in an oil vehicle. An injectable storage depot form is made by forming a microcapsule matrix of the polypeptide in a biodegradable polymer such as polylactide-polyglycolide. Depending on the ratio of the polypeptide to the polymer and the properties of the specific polymer used, the rate of polypeptide release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the polypeptide in liposomes or microemulsions that are compatible with body tissues.

[0240] The sterile injectable form of the composition described herein may be an aqueous or oily suspension. These suspensions may be prepared using suitable dispersing or wetting agents and suspending agents according to techniques known in the art. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example as solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, non-volatile oils are conventionally used as solvents or suspension media. For this purpose, any mild non-volatile oil may be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, may be used to prepare injections as natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially polyoxyethylated versions thereof. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethyl cellulose or similar dispersants, which are commonly used to prepare pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers, which are commonly used in the manufacture of pharmaceutically acceptable dosage forms may also be used for the purposes of formulation.

[0241] In some embodiments, the formulation includes agents such as excipients, buffers, isotonic agents, preservatives, surfactants, and preferably zinc. The formulation may also include excipients or agents for polypeptide stabilization, such as buffers, reducing agents, bulk proteins or carbohydrates. Bulk proteins that can be used to formulate at least one polypeptide composition include albumin, protamine, and the like. Typical carbohydrates that can be used to formulate at least one polypeptide include sucrose, mannitol, lactose, trehalose, glucose, and the like. The bispecific binding molecule formulation may also include a surfactant that reduces or prevents surface-induced aggregation of at least one polypeptide caused by atomization of the solution when forming an aerosol. Various conventional surfactants may be used, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. The amount will generally be in the range of about 0.001%-4% by weight of the formulation. Particularly preferred surfactants for the purposes of the present invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, and the like. Additional agents known in the art for formulating polypeptides, such as antibody proteins, may also be included in the formulation.

[0242] Target protein of interest

[0243] Genomic studies have revealed various types of alterations in genes encoding receptor tyrosine kinases (RTKs) such as EGFR, HER2 / Erb2, and MET, as well as many others known to be associated with a variety of human diseases, especially in cancer. For example, EGFR mutations in non-small cell lung cancer (NSCLC), MET exon 14 skipping mutations in NSCLC, RET mutations in medullary thyroid carcinoma, ALK translocations and ROS1 translocations in NSCLC, and HER2 amplification or overexpression in breast cancer have been reported.

[0244] The present invention provides a bispecific binding molecule that binds to EGFR and then degrades EGFR through a lysosomal degradation pathway. 20 receptor tyrosine kinases including the EGFR family, FGFR family, PDGFR family, MET family, and VEGFR family are known to be co-receptors of NRP1 (see Critchley et al., Cells (2018) 7 (3): 22).

[0245] In some embodiments, the target proteins of the bispecific binding molecules herein include, but are not limited to, oncogenic receptors, such as receptor tyrosine kinases (RTKs) and receptor serine / threonine kinases (RSTKs), the fibroblast growth factor receptor (FGFR) family, the platelet-derived growth factor receptor (PDGFR) family, the tyrosine kinase MET family, and the vascular endothelial growth factor (VEGFR) family.

[0246] In some embodiments, the target protein is a RTK, with the proviso that the target protein is not EGFR.

[0247] In some embodiments, receptor tyrosine kinase receptors include HER1 / ErbBl, HER2 / ErbB2, HER3 / ErbB3 and HER4 / ErbB4, FGFR1, FGFR2, FGFR3 and FGFR4, Met and Ron, PDGFR family PDGFRα, PDGFRβ, CSF-1R, Kit and FLT-3, and VEGFR1, VEGFR2 and VEGFR3.

[0248] In some embodiments, the target proteins of the bispecific binding molecules herein include, but are not limited to, receptor serine / threonine kinases, G-protein coupled receptors, immune checkpoint receptors, and ion channel receptors.

[0249] In some embodiments, the receptor serine / threonine kinase is 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 receptor (GPCR) is 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.

[0250] In some embodiments, the G-protein coupled receptor is selected from the group consisting of a GPCR, a CCR, and a CXCR.

[0251] In some embodiments, the immune checkpoint receptor is selected from CTLA4, PD-L1, PD-1, and integrin.

[0252] In some embodiments, the ion channel receptor is a voltage-gated receptor.

[0253] In some embodiments of the bispecific binding molecules, target proteins of interest herein include, but are not limited to, disease-associated proteins.

[0254] Reagent test kit

[0255] Aspects of the present disclosure further include kits. In certain embodiments, these kits can be used to practice the methods of the present disclosure, such as methods including administering the pharmaceutical composition of the present disclosure to a subject to enhance anti-tumor immunity in the subject, administering the pharmaceutical composition of the present disclosure to a subject to enhance or suppress an immune response in the subject, etc.

[0256] Thus, in certain embodiments, the kits of the present disclosure comprise one or more unit doses of the pharmaceutical composition of the present disclosure, and instructions for administering the pharmaceutical composition to a subject in need thereof. The pharmaceutical composition included in the kit may include any bispecific antibody of the present disclosure, such as any bispecific binding molecule described above, which will not be repeated herein for the sake of brevity.

[0257] The kit of the present disclosure may include a certain amount of composition, present in unit dose, such as ampoule or multiple dose form.Therefore, in certain embodiments, these kits may include one or more (such as two or more) unit doses (such as ampoule) of the composition comprising the bispecific binding molecules of the present disclosure.As used herein, the term "unit dose" refers to a physical discrete unit suitable as a unit dose for human and animal subjects, each unit containing a predetermined number of compositions calculated in an amount sufficient to produce a desired effect.The amount of a unit dose depends on various factors, such as the specific bispecific binding molecules used, the effect to be achieved, and the pharmacodynamics associated with the bispecific binding molecules in an individual.In still other embodiments, these kits may include a single multiple dose of the amount of the composition.In certain embodiments, the kit of the present disclosure includes instructions for administering one or more unit doses of a subject's pharmaceutical composition that needs to enhance anti-tumor immunity.According to some embodiments, the kit of the present disclosure includes instructions for administering one or more unit doses of a subject's pharmaceutical composition that needs to enhance or suppress immune response.

[0258] Combination therapy

[0259] In some embodiments, the polypeptide or its pharmaceutically acceptable salt or solvate (e.g., hydrate) alone or in combination with another suitable chemotherapeutic agent (e.g., oxaliplatin, irinotecan or FOLFOX) can be used to achieve an effective amount of a bispecific antibody in the method or pharmaceutical composition of the present invention. When "combination therapy" is used, a first amount of a polypeptide or its pharmaceutically acceptable salt or solvate (e.g., hydrate) and a second amount of another suitable chemotherapeutic agent can be used to achieve an effective amount.

[0260] Co-administration includes administering the first and second amounts of the polypeptide and the chemotherapeutic agent in a substantially simultaneous manner, such as in a single pharmaceutical composition, e.g., a capsule having a fixed ratio of the first and second amounts, or each in a plurality of separate capsules. In addition, such co-administration also includes using each polypeptide in a sequential manner in either order.

[0261] When co-administration involves separate administration of a first amount of a polypeptide and a second amount of an additional therapeutic agent, the administration of the polypeptide is close enough in time to have the desired therapeutic effect. For example, the time period between each administration that can produce the desired therapeutic effect can range from a few minutes to a few hours, and can be determined by considering the characteristics of each drug, such as efficacy, solubility, bioavailability, plasma half-life, and kinetic characteristics. For example, the polypeptide and the second therapeutic agent can be administered in any 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.

[0262] More specifically, the bispecific binding molecules of the invention can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of a second anticancer agent to a subject.

[0263] It will be appreciated that the method of co-administering a first amount of a bispecific binding molecule and a second amount of an additional therapeutic agent may result in an enhanced or synergistic therapeutic effect, wherein the combined effect is greater than the additive effect that would result from separate administration of the first amount of the polypeptide and the second amount of the additional therapeutic agent.

[0264] The synergistic effect of a combination of therapies (e.g., a combination of preventive or therapeutic agents) can allow the use of lower doses of one or more therapies and / or less frequent administration of the therapies to a subject. The ability to utilize lower doses of therapies (e.g., preventive or therapeutic agents) and / or to administer the therapies less frequently can reduce the toxicity associated with administering the therapies to a subject without reducing the efficacy of the therapies in treating cancer. In addition, the synergistic effect can result in increased efficacy of the drug in preventing, managing, or treating disorders. Finally, the synergistic effect of a combination of therapies (e.g., a combination of preventive or therapeutic agents) can avoid or reduce adverse or unwanted side effects associated with the use of either therapy alone.

[0265] When combination therapy using a bispecific binding molecule of the invention is combined with another anticancer agent, both therapeutic agents can be administered so that the period of time between each administration can be longer (eg, days, weeks, or months).

[0266] The presence of synergy can be determined using a suitable method for evaluating drug interactions. Suitable methods include, for example, the Sigmoid-Emax equation (Holford, NHG and Scheiner, LB, Clin.Pharmacokinet.6:429-453 (1981)), the Loewe additivity equation (Loewe, S. and Muischnek, H., Arch.Exp.PatholPharmacol.114:313-326 (1926)) and the median-effect equation (Chou, TC and Talalay, P., Adv.Enzyme Regul.22:27-55 (1984)). Each of the above-mentioned equations can be applied together with experimental data to generate corresponding graphs to help evaluate the effect of drug combinations. The corresponding graphs associated with the above-mentioned equations are concentration-effect curves, isobologram curves and combination index curves, respectively.

[0267] method

[0268] Aspects of the present disclosure include methods of using the bispecific antibodies of the present disclosure. These methods are useful in a variety of contexts, including in vitro and / or in vivo research and / or clinical applications.

[0269] In certain aspects, methods of enhancing anti-tumor immunity in a subject in need thereof are provided. Such methods include administering to an individual an effective amount of a pharmaceutical composition of the disclosure, such as a pharmaceutical composition comprising a bispecific binding molecule of the disclosure comprising a target protein binding domain and an NRP1 binding domain. In certain embodiments, these methods are used to enhance antibody-dependent lysosomal degradation of a target protein and / or cytotoxicity to cancer cells in an individual.

[0270] In certain aspects, methods of inhibiting or suppressing tumor cell growth in a subject in need thereof are provided. Such methods comprise administering to a subject an effective amount of a pharmaceutical composition of the disclosure, such as a pharmaceutical composition comprising a bispecific binding molecule of the disclosure comprising a target protein binding domain and an NRP1 binding domain.

[0271] Subjects in need thereof may suffer from a cell proliferative disorder. "Cell proliferative disorder" means that one or more cell subpopulations in a multicellular organism undergo unwanted cell proliferation, resulting in damage, such as pain or shortened life expectancy to the organism. Cell proliferative disorders include, but are not limited to, cancer, precancerous, benign tumors, vascular proliferative disorders (e.g., arthritis, restenosis, etc.), fibrotic disorders (e.g., cirrhosis, atherosclerosis, etc.), psoriasis, epidermal and dermoid cysts, lipomas, adenomas, capillary and cutaneous hemangiomas, lymphangiomas, nevus lesions, teratomas, nephromas, myofibromatosis, bone-forming tumors, dysplastic masses, mesangial cell proliferative disorders, etc.

[0272] In some embodiments, the subject suffers from cancer. The subject method can be used to treat various cancers. As used herein, "tumor" refers to the growth and proliferation of all tumor cells, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe the physiological conditions of mammals, which are generally characterized by unregulated cell growth / proliferation. Examples of cancers that can be treated using the subject method 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, bile duct cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, various types of head and neck cancer, etc. In certain embodiments, the subject suffers from a cancer selected from: solid tumors, recurrent glioblastoma multiforme (GBM), non-small cell lung cancer, metastatic melanoma, melanoma, peritoneal cancer (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 non-small cell lung cancer, rhabdomyosarcoma, metastatic renal cell carcinoma, basal cell carcinoma (basal cell epithelioma) and gliosarcoma. In some aspects, the subject suffers from a cancer selected from melanoma, Hodgkin's lymphoma, renal cell carcinoma (RCC), bladder cancer, non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC).

[0273] The bispecific antibodies of the present disclosure can be administered via an administration route selected from oral (e.g., in tablet form, capsule form, liquid form, etc.), parenteral (e.g., by intravenous, intraarterial, subcutaneous, intramuscular or epidural injection), topical, intranasal or intratumoral administration.

[0274] The bispecific antibodies of the present disclosure can be administered in a therapeutically effective amount of a pharmaceutical composition. "Therapeutically effective amount" means a dosage sufficient to produce a desired result, such as an amount sufficient to achieve a beneficial or desired therapeutic (including preventive) result compared to a control, such as alleviating the symptoms of cancer and / or immune disorders. With regard to cancer, in some embodiments, a therapeutically effective amount is sufficient to slow the growth of a tumor, reduce the size of a tumor, and / or the like. An effective amount can be administered in one or more administrations.

[0275] Aspects of the present disclosure include methods for treating individual cancer and / or immune disorders. Treatment means at least improving one or more symptoms associated with individual cancer and / or immune disorders, wherein improvement is broadly used to refer to at least reducing the size of a parameter, such as a symptom associated with the treated cancer and / or immune disorder. Therefore, treatment also includes a situation in which cancer and / or immune disorders or at least one or more symptoms associated therewith are completely suppressed, such as preventing occurrence or stopping, such as terminating, so that the subject no longer suffers from cancer and / or immune disorders or at least is characterized by a symptom of cancer and / or immune disorders.

[0276] The present invention is further illustrated by the following examples, which should not be construed as further limiting.The contents of the figures and all references, patents and co-published patent applications cited throughout this application are expressly incorporated herein by reference.

[0277] Example

[0278] The examples in this specification are not intended to, and should not be used to, limit the invention; they are provided merely to illustrate the invention.

[0279] Example 1. Generation of bispecific antibodies

[0280] The designed proteins were produced by codon-optimized gene synthesis and inserted into pcDNA3.4 as an expression vector using Not I and Hind III restriction enzymes. Table 1 shows the sequence of the heavy chain polypeptide shown in SEQ ID NO: 1 and the heavy chain fusion polypeptides of the bispecific binding molecules in SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 39 used herein, which are combined with the light chain polypeptide sequence shown in SEQ ID NO: 12. Table 2 shows the polynucleotide sequence encoding the heavy chain polypeptide shown in SEQ ID NO: 14, and the polynucleotide sequence encoding the heavy chain polypeptide of the bispecific antibody shown in SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 40. The polynucleotide sequence encoding the light chain polypeptide combined with the heavy chain polypeptide of the bispecific antibody is set forth in SEQ ID NO: 25. The sequences shown in SEQ ID NO: 26-38 disclose the polynucleotide sequences of the GGGGS subunits used to encode the peptide linker. The constructed expression vector includes a signal peptide for optimizing transcription, and a Kozak sequence may be included in the 5' untranslated region.

[0281] To obtain the amount of plasmid construct for transfection, transform the plasmid construct into One Shot TM Top10 E. coli competent cells were then cultured overnight. The construct plasmid was obtained using the PureLinkTM HiPure Expi Plasmid Megaprep Kit.

[0282] Fusion proteins were transiently expressed in a CHO-S system (Thermo Fisher Scientific Inc.). Proteins were expressed individually according to the manufacturer's instructions. Briefly, OPTIPRO TM SFM and ExpiFectamine TM Prepare a total of 0.8 μg of plasmid DNA with a 1:1 ratio of light chain to heavy chain per mL of CHO-S culture. 6 CHO-S cells were added at a viable cell density of 10 cells / mL and greater than 98% viability. The cells were shaken in a Nalgene at 125 RPM and 19 mm orbital. TM The cell cultures were incubated overnight in disposable PETG Erlenmeyer flasks at 37°C, 80% humidity, 8% CO2. The next day, the cultures were enhanced (ExpiCHO TM Enhancer; Thermo Fisher Scientific Inc.) and feeding (ExpiCHO TMFeed; Thermo Fisher Scientific Inc.) and transferred to 32°C, 80% humidity, 5% CO2, with 125RPM and 19mm orbital shaking. A second feeding was performed on the 5th day, and the culture was returned to 32°C. Until the 12th day harvest. Harvesting was achieved by centrifugation at 4000xg 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.

[0283] exist (GE Healthcare Life Sciences) using MabSelect prismA TM Resin (GE Healthcare Life Sciences) purifies the sterilized supernatant of all antibodies. Use 50mM sodium phosphate, 150mM NaCl, pH 7.0 buffer to balance the resin. Then the antibody supernatant is loaded into the column. Use 50mM sodium phosphate, 150mM NaCl, pH 7.0 buffer to wash the resin until the chromatographic baseline returns to the column equilibrium level. Then use 100mM sodium acetate, 20% glycerol (pH 3.0) to elute, and collect fractions. Immediately neutralize the fractions with 1M Tris (pH 9). The fractions containing obvious absorbance at a wavelength of 280nm are merged into an Amicon 10-kDa ultrafiltration device for buffer exchange. Use storage buffer (phosphate buffered saline) to remove the elution buffer by centrifugation, and half dilute 7 times in an Amicon concentrator. Submit the material for SEC and then store at 4°C.

[0284] Cation exchange chromatography was used to purify the antibody. The cation exchange chromatography column (Capto SImpAct) was disinfected with 1M NaOH and rinsed with MQ. Equilibration was completed with 50mM NaAc pH 5.5 (starting buffer) and 50mM NaAc pH5.5, 1M NaCl (elution buffer). Protein A purified antibody was loaded at a concentration of 1-2g antibody / mL resin. The column was then washed with 50mM NaAc (pH5.5). The antibody product was then eluted using a gradient of 5%-60% elution buffer in 25 column volumes. Each peak in the CEX purification was collected separately and used Ultra-15 centrifugal filter devices were used for concentration via centrifugation at 4000 xg and the buffer was then exchanged into PBS.

[0285] Size exclusion chromatography (SEC) analysis was performed on an Agilent Infinity 1260II Quaternary Pump High Performance Liquid Chromatography (HPLC) system with a diode array UV detector WR. Twenty (20) μg of antibody material was injected onto an XBridge Protein BEH SEC column. 2.5 μm, 4.6 mm x 150 mm column. The mobile phase was 100 mM phosphate, 300 mM sodium chloride (pH 7.0 at 50°C) and the flow rate was 0.3 mL / min. The antibody material was detected at 220, 280 and 330 nm wavelengths at 1 Hz sampling rate during a 10 minute acquisition period.

[0286] Table 1. Amino acid sequences of bispecific binding molecules

[0287]

[0288]

[0289]

[0290]

[0291]

[0292] Table 2. Nucleotide sequences encoding bispecific binding molecule polypeptides

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307] Example 2. Binding affinity of bispecific binding molecules to EGFR and NRP1

[0308] Octet red 96 system was used for BLI binding studies to evaluate the binding of bispecific antibodies to recombinant huEGFR and huNRP1. In brief, commercially derived biotinylated huEGFR and huNRP1 were fixed on streptavidin (Sa) biosensors and queried with the generated constructs for binding and characterization. Using BLI technology, both kinetics and binding affinity (equilibrium binding constant, KD) were evaluated for binding to the constructs and evaluated in a bivalent form. These studies were conducted to determine whether antibodies targeting tumor-related essential receptors (TAER-TAB) bind cancer targets (EGFR and NRP1) and evaluate their affinity for targets. Constructs and KD determinations for binding huEGFR and huNRP1 were performed using the Octet Red 96 system.

[0309] huEGFR and huNRP1 (in their respective immobilization columns) were immobilized to the Sa biosensor at a concentration of 0.2 μg / ml in 2X kinetic buffer (loading signal range of 0.2-0.4 nanometers), with a loading time of 180 seconds for each. Loading was performed for 60 seconds after the baseline. The association of the construct was started at 100nM (10nM for high affinity binding), followed by 2 1:1 serial dilutions and then a fourth well (blank) containing only buffer. The construct dissociated after the baseline well. Reference sensors were generated by applying the constructs to a blank AMC biosensor or streptavidin biosensor surface. The association and dissociation steps were 600 seconds each. The data were analyzed using Octet analysis software, applying 1:1 and 2:1 model fits, and the dissociation constants K are reported. D (M).

[0310] All EGFRxNRP1 bispecific antibody constructs, including Construct 1 (SEQ ID NOs: 1 and 12) and Construct 11 (SEQ ID NOs: 11 and 12), had double-digit picomolar binding affinities to EGFR. See Table 3. For NRP1 binding, EGFRxNRP1 bispecific antibody constructs 2, 3, 5, 7, 9, 10, and 11 exhibited subnanomolar affinities, while Construct 1 bound to NRP1 at double-digit nanomolar levels. Figure 2A -2K shows the binding affinity curve of the bispecific antibody to immobilized huEGFR. Figure 3A-3K shows the binding affinity curve of EGFRxNRP1 antibody to immobilized huNRP1.

[0311] Table 3. Binding affinity of EGFRxNRP1 antibodies to EGFR and NRP1

[0312]

[0313]

[0314] Example 3. Inhibition of VEGFR2 signaling in HUVEC cells

[0315] The inhibition of VEGFR2 signaling by EGFRxNRP1 bispecific antibodies in HUVEC cells was tested by probing electrophoretic cell lysates with anti-phosphorylated VEGFR2 based on Western blot. HUVEC cells were grown overnight in EBM-2 medium in 6-well plates supplemented with EGM-2 SingleQuots. The next day, the cells were incubated for 4 hours with 2ml F-12K medium with 0.1mg / ml heparin, endothelial cell growth supplements and 10% FBS. In order to observe the dose-dependent response to EGFRxNRP1 antibodies, cells were treated with EGFRxNRP1 bispecific antibodies diluted 6 times from the highest concentration of 1μM to the lowest concentration of 0.005μM in serum-free medium for 30 minutes, followed by 2.2ng / ml of VEGF165 or control for 10 minutes. Cell lysates were prepared by collecting cells with a cell scraper and incubating on ice for 20 minutes with 100μl of NP-40 lysis buffer and protease / phosphatase inhibitors. The supernatant lysate was collected into a new tube after centrifugation at 13,000 rpm for 10 minutes, protein quantification was measured using BCA protein assay, and denatured at 70°C for 10 minutes after mixing with NuPAGE LDS sample buffer and LDS sample reducing buffer.

[0316] Cell lysate equal to 10 μg was loaded into a 4-12% Bis-Tris gel with 4 μl of PageRuler Plus pre-stained protein ladder and run at 200V for 45 minutes. The gel was washed with distilled water and transferred to the membrane using the IBlot2Drying blotting system. The membrane was blocked with 5% milk in 1x TBST for 1 hour at room temperature and incubated overnight with anti-phosphorylated VEGFR2 (Y1175) in 5% milk in 1xTBST at 4°C. The next day, the membrane was washed 3 times with TBST for 10 minutes and 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. Then, the membrane was incubated with SuperSignal Femto chemiluminescent substrate for 1-2 minutes and imaged with Amersham ImageQuant 800. The blots were then stripped with Restore Plus Western Blot Stripping Buffer for 15 minutes at room temperature on a shaker, followed by 3 washes with 1x TBST and the procedure was repeated starting with milk blocking followed by incubation of primary anti-VEGFR2 antibody.

[0317] The intensity of the western blot bands of phosphorylated VEGFR2 (Y1175) and VEGFR2 was analyzed using ImageJ software. The obtained band density of phosphorylated VEGFR2 (Y1175) was normalized by VEGFR2, and the inhibition of VEGFR2 phosphorylation by EGFRxNRP1 bispecific antibody construct 11 was calculated by the ratio relative to VEGF165 control cell lysate (positive control). The IC50 of EGFRxNRP1 bispecific antibody for VEGFR2 phosphorylation 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 molecules of EGFRxNRP1 bispecific antibody construct 1 (SEQ ID NOs: 1 and 12) does not inhibit VEGF165-mediated VEGFR phosphorylation. Figures 5-11 show Western blots and IC50 graphs for inhibition of VEGFR2 phosphorylation after incubation with bispecific antibodies. The IC50s of the bispecific antibodies are shown in Table 4.

[0318] Table 4. Inhibition of VEGFR signaling by EGFRxNRP1 antibodies

[0319] EGFRxNRP1 BsA construct SEQ ID NO (heavy chain and light chain) picture VEGF inhibition (nM) Construct 1 SEQ ID NO:1,12 3 n / a (less than 5% inhibition at 1 μM) Construct 2 SEQ ID NO:2,12 4 12.9 Construct 3 SEQ ID NO:3,12 5 3.3 Construct 4 SEQ ID NO:4,12 6 410 Construct 5 SEQ ID NO:5,12 7 61 Construct 6 SEQ ID NO:6,12 8 10130 Construct 7 SEQ ID NO:7,12 9 1.74 Construct 11 SEQ ID NO:11,12 10 20

[0320] Example 4: Effect of EGFRxNRP1 bispecific antibody on cell viability

[0321] The inhibition of cell viability by bispecific antibodies was tested in the H1975 lung cancer cell line. In brief, H1975 lung cancer cells were grown in RPMI1640 supplemented with 1% penicillin / streptomycin and 10% FBS. The cells were trypsinized and seeded into a round-bottomed 96-well 3D culture plate (3,000 cells / 120 μl / well) and incubated for 3-4 hours until they aggregated to form 3D spheroids. A 6-fold serial dilution of 5 μM-0.6 pM EGFRxNRP1 bispecific antibody reserve master plate was prepared. H1975 cells were treated with 30 μl of EGFRxNRP1 bispecific antibody and incubated for 72 hours (in duplicate). Subsequently, CellTiter-Glo 3D reagent and 25 μl serum-free culture medium were added to each well, wrapped with foil, and incubated on a shaker for 7 minutes. The plate was read with a Varioskan Lux multi-mode plate reader and analyzed with Prism9 software.

[0322] Table 5 shows the IC50 of EGFRxNRP1 antibodies. Compared with other EGFRxNRP1 antibody constructs, the EGFRxNRP1 bispecific antibody constructs (SEQ ID NOs: 1 and 12) with the NRP1 binding domain at the C-terminus without fusion with scFv had a high IC50 value (1.96 μM), as shown in Table 5. Fig. 12A See Fig. 12B The IC50 value data indicated that the short chain variable fragment (scFv) comprising the NRP1 binding domain of the EGFRxNRP1 antibody was essential for the function of the EGFRxNRP1 antibody in inhibiting tumor cell growth.

[0323] Table 5. Cell growth inhibition by EGFRxNRP1 antibodies

[0324]

[0325] Example 5: Tumor Growth Inhibition in the H1975 Xenograft Mouse Model Cell Line

[0326] The ability of EGFRxNRP1 antibodies to inhibit tumor growth was tested in a H1975 cell line xenograft mouse model. All cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained 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) at 37°C in a 5% CO2 incubator.

[0327] Xenograft Models

[0328] Experiments and procedures involving mice were performed in accordance with the policies of the U.S. Department of Agriculture, the Department of Health and Human Services, and the NIH on the humane care and use of laboratory animals. Female athymic (nu / nu) mice aged 6-8 weeks from Charles River Laboratories (Wilmington, MA, USA) were housed in pathogen-free conditions with laboratory food and water ad libitum. Prior to implantation of tumor cell lines, all cell lines were screened for infectious agents (mycoplasma, etc.). The cells were incubated with 1x 10 50 μL of PBS mixed 1:1 in Matrigel (Corning, Corning, NY, USA). 7 Xenografts were established by subcutaneous injection of 100 μl of (NCI-H1975) cells / mouse into the right flank. For efficacy studies, tumors were allowed to reach 150-300 mm before randomization and treatment initiation. 3 (7-10 mice per group).

[0329] Treatment and tumor measurements

[0330] Antibodies were diluted in sterile phosphate-buffered saline (Corning, Corning, NY, USA) and given via ip injection at the indicated dose levels on the day of randomization in a total volume of 100 μl per mouse per treatment and then twice weekly.

[0331] Tumor size was measured using a digital caliper and the formula V = (W 2 × L) / 2, where W is the tumor width and L is the tumor length.

[0332] Statistical analysis

[0333] Statistical analysis was performed using GraphPad Prism 7 software (La Jolla, CA, USA). Results are shown as mean values. Data at a single time point or endpoint were compared between the control and experimental groups using Student's t-test. For growth curve analysis involving longitudinal data with repeated measures, type II ANOVA was used. Statistical differences were considered significant when p < 0.05. The formula TGI (%) = (V C1 -V t1 ) / (V C0 -V t0 )X 100 to calculate TGI%, where V C1 and V t1 are the mean tumor volumes of the control and treatment groups at the end of the study, and V C0 and V t0The mean tumor volumes of the control and treatment groups at the beginning of the experiment, respectively. Table 6 shows the inhibition of tumor growth and treatment-control percentage tumor volume by EGFRxNRP1 antibodies in a xenograft mouse model. The curves in Figures 13-13F show the mean tumor volumes of mice treated with EGFRxNRP1 antibodies within a few days after implantation. The EGFRxNRP1 bispecific antibody construct 11 with a scFv NRP1 binding domain had the highest tumor growth inhibition, for example, treatment with the EGFRxNRP1 bispecific antibody construct 11 resulted in 100% tumor growth inhibition.

[0334] Table 6. Tumor Growth Inhibition and Treatment-Control Percent Tumor Volume

[0335] Bispecific binding peptides TGI % Average T / C% SEQ ID NO:1,12 39.94 74.58％ SEQ ID NO:2,12 23.94 81.91％ SEQ ID NO:3,12 71.13 37.15％ SEQ ID NO:7,12 81.09％ 28.08％ SEQ ID NO:9,12 94.96％ 18.86％ SEQ ID NO:11,12 102.56％ 12.54％

[0336] Example 6. EGFR in H1975 cells by construct 11 T790 / L858R Degradation

[0337] EGFR tyrosine kinase inhibitors (TKIs) have been widely used to treat cancers with constitutively active EGFR mutations by binding to the catalytic site of EGFR and blocking its kinase activity. However, they can also induce acquired resistance and. The first-generation EGFR TKI gefitinib induces T790M mutations in most patients after treatment. The second-generation EGFR TKI afatinib binds irreversibly to EGFR, but its effects on EGFR WT and EGFR T790M The third-generation EGFR TKI osimertinib is not selective and causes adverse reactions. T790M Although tumors with EGFR mutations are effective, some patients may develop acquired resistance, such as C797S mutations. In addition, NSCLC with EGFR WT is unresponsive to TKIs. Therefore, targeting EGFR by inducing degradation may be a more effective approach to inhibit EGFR in cancer treatment, regardless of EGFR mutation status.

[0338] Anti-EGFR antibodies can block ligand-mediated activation of EGFR signaling and induce receptor endocytosis, but EGFR receptors are mostly recycled. The EGFRxNRP1 bispecific antibody construct was evaluated in cancer cells to degrade double-mutant EGFR T790 / L858R to verify the association between the anticancer activity of construct 11 and EGFR protein levels. T700M / L858RMutated H1975 cells were grown in RPMI1640 medium with 10% fetal bovine serum in tissue culture plates. Cells were incubated for 16 hours with fixed (150 nM) or 6-fold serial dilutions from the highest concentration of 1 μM to the lowest concentration of 0.77 nM construct 11. Proteasome inhibitor MG132 (10 μM) or lysosomal protein degradation inhibitor bafilomycin A1 (Baf A1, 200 nM) were used together with EGFRxNRP1 bispecific antibody treatment to determine the mechanism of action of EGFRxNRP1 antibodies. Cell lysates were prepared by collecting cells with a cell scraper and incubating on ice for 20 minutes with NP40 lysis buffer with protease / phosphate inhibitors. The supernatant lysate was collected into a new tube after centrifugation at 13,000 rpm for 10 minutes, protein quantification was measured using the BCA protein assay, and denatured at 70°C for 10 minutes after mixing with NuPAGE LDS sample buffer and LDS sample reduction buffer. Cell lysate equal to 20 μg was loaded into a 4-12% Bis-Tris gel along with 4 μl of PageRuler Plus prestained ladder and run 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 1x TBST for 1 hour at room temperature and incubated overnight at 4°C with anti-EGFR, anti-NRP1, and anti-β-actin in 5% milk in 1xTBST. The next day, the membrane was washed 3 times for 10 minutes with TBST and incubated with secondary antibodies in 5% milk for 1 hour at room temperature. The membrane was washed 3 times for 10 minutes with 1xTBST. The membrane was then incubated with SuperSignal Femto chemiluminescent substrate for 5 minutes and imaged with the BioRad ChemiDoc Touch imaging system.

[0339] Human NSCLC H1975 cells (EGFR T790 / L858R ) for 16 hours. EGFR, NRP1 and actin levels were measured by Western blotting.

[0340] Levels of both total EGFR (tEGFR) and NRP1 were very low in cells treated with construct 11. In contrast, levels of EGFR in cells treated with anti-NRP1 mAb, panitumumab (Pnm), or anti-NRP1 mAb plus panitumumab were similar to those in untreated cells ( Fig.14). This result suggests that the degradation of EGFR and NRP1 is only through simultaneous interaction with EGFR T790 / L858R The EGFRxNRP1 antibody construct 11 that binds to NRP1 was realized.

[0341] Co-treatment with the lysosomal protein degradation inhibitor bafilomycin A1 (Baf A1, 200 nM) restored the EGFRxNRP1 bispecific antibody's ability to inhibit EGFR in H1975 cells. T790 / L858R and NRP1 degradation, but co-treatment with the proteasome inhibitor MG132 (10 μM) did not ( Fig.15 ). Similar experiments were performed using Baf A1 and MG132 inhibitors to examine the degradation of various EGFR forms (WT or mutant) by construct 11 in other NSCLC cell lines. Degradation was examined for: EGFR in PC9 cells del19 、H1299(NRAS Q61K )EGFR in cells WT 、HCC44(KRAS G12C )EGFR in cells WT , EGFR in HCC827 cells del19 / NRP1 and H358 (KRAS G12C )EGFR in cells WT / NRP1 (data not shown). For all EGFR forms and cell lines examined, the results demonstrated that receptor degradation of construct 11 was restored by co-treatment with the Baf A1 lysosomal protein degradation inhibitor, but not by co-treatment with the MG132 proteosomal inhibitor.

[0342] Taking this data together, EGFRxNRP1 antibody construct 11 induces degradation of EGFR and NRP1 via the lysosomal protein degradation pathway. The figure depicts that simultaneous binding of the EGFRxNRP1 bispecific antibody to EGFR and NRP1 leads to degradation of EGFR via the lysosomal degradation pathway, and its binding to NRP1 is essential for the degradation of EGFR ( Fig.16 The effects of EGFR antibodies panitumumab, ervantumab, and construct-11 on EGFR in H1975 cells were evaluated. T790 / L858R Even at a low concentration of 0.77 nM, treatment with construct 11 significantly reduced EGFR T790 / L858R The levels of tEGFR (tEGFR) protein were significantly decreased in panitumumab-treated cells and ivantuzumab-treated cells, while no tEGFR degradation was evident in either panitumumab-treated or ivantuzumab-treated cells ( Fig.16). Construct 11 is a novel and unique antibody-based EGFR receptor degrader that can be used to treat tumors with EGFR mutations. In addition, EGFRxNRP1 bispecific antibody construct 11 degrades EGFR with mutations, including single mutation (Del 19), double mutation (T790M / L858R), triple mutation (T790M / C797S / L858R), and insertion (InsEx20) (data not shown).

[0343] Thus, in summary, the EGFRxNRP1 bispecific antibody construct 11 has been shown to degrade both wild-type and mutant forms of EGFR via a lysosome-mediated degradation pathway in a wide variety of NSCLC cell lines.

[0344] Example 7. Anti-tumor activity of EGFRxNRP1 bispecific antibody in osimertinib-resistant xenograft mouse model Sexual influence

[0345] Osimertinib is a third-generation irreversible EGFR TKI that targets activating mutations (L858R and chromosome 19 deletion) and T790M mutations. Resistance to osimertinib has become a major obstacle to the treatment of EGFR-mutant NSCLC. Recent studies have shown that the overall response rate (ORR) of osimertinib-resistant patients treated with lazertinib plus ervantumab was 36%. Preclinical studies in the H1975-HGF xenograft mouse model found that lazertinib plus ervantumab was significantly effective.

[0346] To determine the effect of EGFRxNRP1 bispecific antibody construct 11 on tumors resistant to osimertinib, osimertinib-sensitive, osimertinib-resistant or osimertinib-refractory xenograft mice were treated with osimertinib (1 mg / kg, ip, QD), panitumumab (5 mg / kg, ip, BIW), construct-11 (6.87 mg / kg, ip BIW), ervantumab (5.05 mg / kg, ip, BIW) and IgG1 isotype (5 mg / kg, ip, BIW) as a control. Osimertinib was formulated for ip administration by dissolving the dry powder in a small amount (10% of the final volume) of dimethyl sulfoxide. Osimertinib and antibodies were diluted in sterile phosphate-buffered saline and administered as indicated above, starting on the day of randomization, by injection in a total volume of 100 μl per mouse per treatment. Values ​​were measured using a digital caliper and the formula V = (W 2Tumor volume was calculated by RT-PCR (x L) / 2, where W is tumor width and L is tumor length. Statistical analysis was performed using GraphPad Prism 7 software (La Jolla, CA, USA). Results are shown as mean ± SEM. Student's t-test was used to compare data at a single time point or endpoint between the control and experimental groups. In osimertinib-sensitive xenograft mice (H1975), construct 11 was highly effective in inhibiting mouse tumor growth compared to mice treated with ivantazumab. Treatment with construct 11 continued to inhibit tumor growth for up to 42 days after implantation, while tumors did not respond effectively to osimertinib, panitumumab, and ivantazumab ( Fig.18A ). The osimertinib-resistant H1975-OR cell line was developed by growing H1975 cells in medium with increasing osimertinib concentrations up to 4 μM. In osimertinib-resistant xenograft mice (H1975-OR), construct-11-treated group also showed stronger tumor growth inhibition compared with ervantumab-treated group ( Fig.18B ). The osimertinib-refractory H1975-HGF cell line was developed to reduce the dependence of the H1975 cell line on EGFR signaling for its growth by overexpressing hepatocyte growth factor (HGF) in H1975 cells to activate cMET signaling. In osimertinib-refractory xenograft mice (H1975-HGF), treatment with the EGFRxNRP1 bispecific antibody construct 11 showed similar inhibitory efficacy on tumor growth as treatment with ervantumab ( Fig. 18C These results suggest that EGFRxNRP1 bispecific antibody construct 11 induces EGFR T790 / L858R EGFRxNRP1 bispecific antibody construct 11 showed strong degradation of EGFR and had antitumor activity in osimertinib-sensitive H1975. T790M / L858R The efficacy in the xenograft mouse model is summarized below in Table 7. EGFRxNRP1 bispecific antibody construct 11 may be applicable to other receptor tyrosine kinase inhibitor (RTK)-resistant tumors.

[0347] Table 7. EGFR T790 / L858 Preclinical proof of concept in various xenograft mouse models

[0348]

[0349] O: Great efficacy at low doses in sensitization mode

[0350] Δ: Ineffective at low doses and good efficacy at high doses

[0351] X: Ineffective even at high doses

[0352] We demonstrated that EGFRxNRP1 bispecific antibody inhibited tumor cell growth and reduced cell viability. In addition, EGFRxNRP1 bispecific antibody treatment degraded EGFR via the lysosomal degradation pathway, which was verified by experiments using lysosomal protein degradation inhibitors or proteasome inhibitors6. Fig.15 Importantly, 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.

[0353] The present disclosure provides a novel antibody-based receptor degrader (ApReptor) platform that has several advantages over other degraders: (i) no need for E3 / ubiquitin specific protein (USP), (ii) no need for linker and catalytic enzyme, (iii) preclinical PoC validation, (iv) preclinical validation in drug resistance models, and (v) application to a wide range of disease-related extracellular receptors.

[0354] This antibody-based receptor degrader (ApReptor) platform offers a modular, selective, and simple genetically encoded strategy for inducing lysosomal delivery of extracellular and surface target receptors with broad or tissue-specific distribution. The AbReptor platform holds great promise for personalized medicine and can be customized based on the patient's genetic background.

[0355] Example 8. Degradation of cMET receptor by cMETxNRP1 bispecific antibody construct

[0356] In this example, a bispecific antibody (BsA) construct similar to construct 11 was prepared, in which the anti-NRP1 antibody binding arm of BsA was maintained, but an anti-cMET antibody was used as the other binding arm of BsA (rather than anti-EGFR as in construct 11). K values ​​of NRP1 binding of cMETxNRP1 bispecific antibody constructs D is 3.99 x 10 -10 M. K of cMET binding of cMETxNRP1 bispecific antibody constructs D The K of cMET binding of anti-cMET mAb alone was <0.1 nM. D quite.

[0357] The 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 Figures 19A-19C. Cells were either untreated (control), treated with anti-cMET antibody alone, or treated with the cMETxNRP1 BsA construct. The results showed that (as expected), treatment with the BsA construct alone resulted in NRP1 degradation. In addition, in the three cell lines examined, treatment with the BsA construct resulted in greater cMET degradation than treatment with anti-cMET alone. These results confirm the ability of the anti-cMET-containing BsA construct to degrade cMET in cancer cells expressing cMET.

[0358] Example 9. Degradation of HER2 receptor by HER2xNRP1 bispecific antibody construct

[0359] In this example, a bispecific antibody (BsA) construct similar to construct 11 was prepared, in which the anti-NRP1 antibody binding arm of BsA was maintained, but an anti-HER2 antibody was used as the other binding arm of BsA (rather than anti-EGFR as in construct 11). K of NRP1 binding of HER2xNRP1 bispecific antibody constructs D 2.44×10 -10 M. HER2 binding K of HER2xNRP1 bispecific antibody constructs D The K of HER2 binding of anti-HER2 mAb alone was <0.1 nM. D quite.

[0360] The degradation of NRP1 and HER2 by the HER2xNRP1 BsA construct was examined in BT474 cells and the results are shown in Fig. 20 Cells were either untreated (control), treated with anti-HER2 antibody alone, or treated with the HER2xNRP1 BsA construct. The results showed that (as expected), treatment with the BsA construct alone resulted in NRP1 degradation. Furthermore, in the cell lines examined, treatment with the BsA construct resulted in greater HER2 degradation than treatment with anti-HER2 alone. These results demonstrate the ability of the anti-HER2-containing BsA construct to degrade HER2 in HER2-expressing cancer cells.

[0361] Example 10. Degradation of the IGF1R receptor by IGF1RxNRP1 bispecific antibody constructs

[0362] In this example, a bispecific antibody (BsA) construct similar to construct 11 was prepared, in which the anti-NRP1 antibody binding arm of BsA was maintained, but an anti-IGF1R antibody was used as the other binding arm of BsA (rather than anti-EGFR as in construct 11). K values ​​of NRP1 binding of IGF1RxNRP1 bispecific antibody constructs D The K of IGF1R binding of the IGF1RxNRP1 bispecific antibody construct was <0.1 nM. DThe K of IGF1R binding of anti-IGF1R mAb alone was <0.1 nM. D quite.

[0363] The 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 . Cells were either untreated (control), treated with anti-IGF1R antibody alone, or treated with the IGF1RxNRP1 BsA construct. The results show that (as expected), treatment with the BsA construct alone resulted in NRP1 degradation. Furthermore, in both cell lines examined, treatment with the BsA construct resulted in greater IGF1R degradation than treatment with anti-IGF1R alone. These results demonstrate the ability of the anti-IGF1R-containing BsA construct to degrade IGF1R in cancer cells expressing IGF1R.

[0364] Sequence Listing Overview

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

Claims

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

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

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

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

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

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

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

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

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

11. The bispecific binding molecule of any one of claims 1-7, wherein the target protein binding domain and the NRP1 binding domain are each independently selected from IgG, half antibodies, single domain antibodies, nanobodies, Fab, monospecific Fab2, Fc, scFv, minibodies, IgNAR, V-NAR, hcIgG, VHH domains, camelid antibodies, and peptibodies.

12. The bispecific binding molecule of claim 1, wherein the NRP1 binding domain Include: (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.

13. The bispecific binding molecule of claim 12, wherein: (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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

35. The method of claim 27, wherein the bispecific binding molecule is administered intravenously, intraperitoneally, intrathecally, intracerebroventricularly, or intraparenchymal.

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