Bispecific antibodies comprising an NRP1-binding domain and methods of use thereof

Abstract

The present disclosure provides a bispecific antibody, and a composition and method for treating cancer incorporating the bispecific antibody.The bispecific antibody comprises a polypeptide comprising an EGFR binding domain and a C-terminal neuropilin (NRP1) specific binding domain.A monoclonal antibody that specifically binds to NRP1 is also provided.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 325,312, filed March 30, 2022, and U.S. Provisional Application No. 63 / 325,317, filed March 30, 2022, the entire contents of both of which are incorporated by reference herein. [Background technology]

[0002] background Solid tumors and blood cancers, such as leukemia and lymphoma, are treated with therapeutic antibodies. Antibody treatments for solid tumors and blood cancers show different response rates. For example, the response rate of antibody treatments for blood cancers reaches 30-51%, whereas the response rate for solid tumors is only 8-15%. This is because cancer cells in the blood are accessible for antibody targeting, whereas efficacy for solid tumors depends on extravasation and tumor penetration.

[0003] Antibodies have reduced tumor penetration for several reasons, including 1) inability to penetrate the endothelial barrier due to the inherent property of antibody size of approximately 150 kDa, 2) high tumor interstitial fluid pressure, and 3) abnormal microstructural / physiological properties of tumor tissue, such as reduced perfusion, abnormal angiogenesis, reduced lymphangiogenesis, high cellular density, and high extracellular density (Jain and Stylianopoulos, 2010). For these reasons, the amount of antibody that accumulates in tumor tissue is very low (0.01–0.0001% of the injected dose per gram of tumor tissue), resulting in low response rates (Thurber et al. 2008). Therefore, development of antibody technologies that allow antibodies to selectively accumulate in tumor tissue and have high penetration ability in tumor tissue would reduce toxicity and improve therapeutic efficacy.

[0004] Efforts are being made to improve tumor penetration by making antibodies smaller, modulating antigen-binding specificity, and combining molecules that facilitate antibody administration and tumor tissue penetration. For example, antibody fragments such as antigen-binding fragments (Fab) (50 kDa), single-chain variable fragments (scFv) (30 kDa), and heavy chain variable domains (VH) (14 kDa) have been reduced in size to overcome the endothelial barrier. However, because antibody fragments lack Fc fragments and are small in size, they are quickly filtered from serum by the kidney after administration in the body, reducing their circulating half-life and therapeutic efficacy (Behr et al. 1998). Antibodies may be equipped with functions that improve tumor penetration. Summary of the Invention

[0005] overview The present disclosure provides bispecific antibodies comprising a neuropilin-1 receptor (NRP1) binding domain and a second binding domain that binds to a second target, such as a receptor tyrosine kinase (RTK) binding domain, e.g., an epidermal growth factor receptor (EGFR) binding domain. Thus, in one embodiment, the present disclosure provides a bispecific antibody comprising a first binding domain that binds to human neuropilin-1 receptor (NRP1) and a second binding domain that binds to human epidermal growth factor receptor (EGFR).

[0006] The present disclosure provides an NRP1 binding domain from an anti-NRP1 monoclonal antibody that forms a bispecific antibody with beneficial properties. For example, the NRP1xEGFR bispecific antibody of the present disclosure has enhanced ability to both internalize and degrade NRP1 and EGFR receptors compared to anti-EGFRmAb or anti-NRP1mAb alone or in combination. The present disclosure provides an EGFRxNRP1 bispecific antibody with high affinity to EGFR to provide high homing ability to tumors expressing EGFR. The bispecific antibody has asymmetric binding affinity to EGFR and NRP1, and the affinity to EGFR is at least 10 times higher than the affinity to NRP1. The NRP1 targeting portion of the antibody includes an NRP1 binding domain that shortens the occupancy time of NRP1 to reduce toxicity in healthy tissues. The combination of these features in a bispecific antibody significantly increases the possibility of exerting high therapeutic efficacy for cancer treatment while reducing toxic side effects.

[0007] In one aspect, the disclosure relates to a bispecific antibody comprising a first binding domain that binds to the human Neuropilin-1 receptor (NRP1) and a second binding domain that binds to a target other than NRP1, wherein the first binding domain comprises: i. an antibody heavy chain variable (VH) domain comprising CDR1, CDR2 and CDR3 regions (HCDR1, HCDR2 and HCDR3, respectively), wherein HCDR1 consists of the sequence set forth in SEQ ID NO: 79, HCDR2 consists of the sequence set forth in SEQ ID NO: 80, and HCDR3 consists of the sequence set forth in any one of SEQ ID NOs: 81-84; and ii. An antibody light chain variable (VL) domain comprising CDR1, CDR2 and CDR3 regions (LCDR1, LCDR2 and LCDR3, respectively), wherein LCDR1 consists of the sequence set forth in any one of SEQ ID NOs: 85-87, LCDR2 consists of the sequence set forth in SEQ ID NO: 88 and LCDR3 consists of the sequence set forth in SEQ ID NO: 89.

[0008] In certain embodiments, (i) HCDR1 consists of the sequence set forth in SEQ ID NO: 79, HCDR2 consists of the sequence set forth in SEQ ID NO: 80, and HCDR3 consists of the sequence set forth in any one of SEQ ID NOs: 84; and (ii) LCDR1 consists of the sequence set forth in SEQ ID NO: 85, LCDR2 consists of the sequence set forth in SEQ ID NO: 88, and LCDR3 consists of the sequence set forth in SEQ ID NO: 89.

[0009] In certain embodiments, the first binding domain comprises: (i) an antibody heavy chain variable (VH) domain comprising CDR1, CDR2 and CDR3 regions (HCDR1, HCDR2 and HCDR3, respectively), where HCDR1 comprises the sequence set forth in SEQ ID NO: 79, HCDR2 comprises the sequence set forth in SEQ ID NO: 80, and HCDR3 comprises the sequence set forth in any one of SEQ ID NOs: 81-84; and (ii) an antibody light chain variable (VL) domain comprising CDR1, CDR2 and CDR3 regions (LCDR1, LCDR2 and LCDR3, respectively), where LCDR1 comprises the sequence set forth in any one of SEQ ID NOs: 85-87, LCDR2 comprises the sequence set forth in SEQ ID NO: 88, and LCDR3 comprises the sequence set forth in SEQ ID NO: 89.

[0010] In certain embodiments, the second binding domain targets EGFR. For example, in one embodiment, the second binding domain is composed of an N-terminal EGFR-binding heavy chain variable domain (VH) and a heavy chain constant 1 domain (CH1), and the first binding domain is composed of a C-terminal NRP1-binding single chain variable fragment (scFv), where the N-terminal variable heavy chain (VH) and the C-terminal scFv binding domain are at opposite ends of a contiguous sequence. In one embodiment, the second binding domain further comprises a heavy chain constant 2 domain (CH2) and a heavy chain constant 3 domain (CH3). In one embodiment, the C-terminal NRP1-binding scFv comprises a light chain variable domain (VL) connected to the heavy chain variable domain (VH) by a flexible linker peptide. In one embodiment, the bispecific antibody further comprises a corresponding EGFR-binding light chain variable domain (VL) and a light chain constant domain (CL), where the bispecific antibody is composed of two polypeptides.

[0011] In certain embodiments, the second binding domain targeting EGFR comprises: (i) an antibody heavy chain variable (VH) domain comprising CDR1, CDR2 and CDR3 regions (HCDR1, HCDR2 and HCDR3, respectively), where HCDR1 consists of the sequence set forth in SEQ ID NO: 71, HCDR2 consists of the sequence set forth in SEQ ID NO: 72 and HCDR3 consists of the sequence set forth in SEQ ID NO: 73; and (ii) an antibody light chain variable (VL) domain comprising CDR1, CDR2 and CDR3 regions (LCDR1, LCDR2 and LCDR3, respectively), where LCDR1 consists of the sequence set forth in SEQ ID NO: 74, LCDR2 consists of the sequence set forth in SEQ ID NO: 75 and LCDR3 consists of the sequence set forth in SEQ ID NO: 76.

[0012] In certain embodiments, the bispecific antibody comprises a first binding domain (i.e., an NRP1-binding domain) comprising a VH domain comprising the sequence set forth in SEQ ID NO: 77 and a VL domain comprising the sequence set forth in SEQ ID NO: 78.

[0013] In certain embodiments, the bispecific antibody comprises a first binding domain (i.e., an NRP1-binding domain) comprising a VH domain comprising the sequence set forth in SEQ ID NO: 77 and a VL domain comprising the sequence set forth in SEQ ID NO: 78, and a second binding domain (i.e., an EGFR-binding domain) comprising a VH domain comprising the sequence set forth in SEQ ID NO: 69 and a VL domain comprising the sequence set forth in SEQ ID NO: 70.

[0014] In certain embodiments of the bispecific antibody, the first binding domain and the second binding domain comprise a bispecific antibody heavy chain comprising an amino acid sequence selected from SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 39. In certain embodiments, the bispecific antibody heavy chain is paired with an antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the bispecific antibody heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 11 or 39 and the bispecific antibody light chain comprises the amino acid sequence set forth in SEQ ID NO: 12.

[0015] In certain embodiments of the bispecific antibody, the binding affinity (K D In certain embodiments, the binding affinity (K D ) ranges from 0.1 nM to 100 nM. In certain embodiments, the EGFR-binding domain has a K D a K for EGFR that is at least 2-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 100-fold, or more than 100-fold D Includes.

[0016] In another aspect, the present disclosure relates to a pharmaceutical composition comprising a bispecific antibody of the present disclosure.

[0017] In another aspect, the present disclosure relates to a polynucleotide encoding a bispecific antibody of the present disclosure. In one embodiment, the bispecific antibody comprises a heavy chain polypeptide encoded by a polynucleotide sequence set forth in any one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 40. In one embodiment, the polynucleotide sequence is inserted into a vector for protein expression.

[0018] In another aspect, the disclosure relates to a method of internalizing a neuropilin-1 (NRP1) receptor by a cell, the method comprising contacting the cell with a bispecific antibody of the disclosure to cause NRP1 to be internalized by the cell. In another embodiment, the disclosure provides a method of internalizing an epidermal growth factor receptor (EGFR) by a cell, the method comprising contacting the cell with an NRP1 x EGFR bispecific antibody of the disclosure to cause EGFR to be internalized by the cell.

[0019] In another aspect, the present disclosure relates to a method for treating cancer associated with abnormal NRP1 expression in a subject, comprising administering to the subject a bispecific antibody of the present disclosure or a pharmaceutical composition thereof. In another embodiment, the present disclosure provides a method for treating cancer associated with abnormal EGFR expression in a subject, comprising administering to the subject a NRP1 x EGFR bispecific antibody of the present disclosure or a pharmaceutical composition thereof. In one embodiment, the cancer is non-small cell lung cancer. In one embodiment, the method further comprises administering to the subject at least one additional chemotherapeutic agent for combination therapy.

[0020] In another aspect, the invention relates to the use of a bispecific antibody of the invention in the manufacture of a medicament for use in a treatment, such as for example the treatment of cancer, as described herein. In one embodiment, the invention relates to the use of an NRP1xEGFR bispecific antibody of the invention in the manufacture of a medicament for the treatment of a cancer associated with aberrant EGFR expression. [Brief description of the drawings]

[0021] The following figures are provided as examples and are not intended to limit the scope of the present invention.

[0022] [Figure 1-1]FIG. 1A Binding of bsAb with heavy chain polypeptide SEQ ID NO: 1 and recombinant human EGFR measured by Octet Red 96 sensorgrams shows dose-dependent binding of tumor-associated receptor-targeting antibody. Each line on the graph represents binding kinetics at a particular bsAb concentration (10, 5, 2.5 nM from left to right). FIG. 1B Binding of bsAb with heavy chain polypeptide SEQ ID NO: 2 and recombinant human EGFR measured by Octet Red 96 sensorgrams shows dose-dependent binding of tumor-associated receptor-targeting antibody. Each line on the graph represents binding kinetics at a particular bsAb concentration (10, 5, 2.5 nM from left to right). FIG. 1C Binding of bsAb with heavy chain polypeptide SEQ ID NO: 3 and recombinant human EGFR measured by Octet Red 96 sensorgrams shows dose-dependent binding of tumor-associated receptor-targeting antibody. Each line on the graph represents binding kinetics at a particular bsAb concentration (10, 5, and 2.5 nM from left to right). FIG. 1D Binding of bsAb with heavy chain polypeptide SEQ ID NO: 4 and recombinant human EGFR measured by Octet Red 96 sensorgrams shows dose-dependent binding of tumor-associated receptor-targeting antibodies. Each line on the graph represents binding kinetics at a particular bsAb concentration (10, 5, and 2.5 nM from left to right). FIG. 1E Binding of bsAb with heavy chain polypeptide SEQ ID NO: 5 and recombinant human EGFR measured by Octet Red 96 sensorgrams shows dose-dependent binding of tumor-associated receptor-targeting antibodies. Each line on the graph represents binding kinetics at a particular bsAb concentration (10, 5, and 2.5 nM from left to right). FIG. 1F Binding of bsAb with heavy chain polypeptide SEQ ID NO: 6 and recombinant human EGFR measured by Octet Red 96 sensorgrams shows dose-dependent binding of tumor-associated receptor-targeting antibodies. Each line on the graph represents binding kinetics at a particular bsAb concentration (10, 5, and 2.5 nM from left to right).

[0023] [Figure 1-2]FIG. 1G Binding of bsAb with heavy chain polypeptide SEQ ID NO: 7 and recombinant human EGFR measured by Octet Red 96 sensorgrams shows dose-dependent binding of tumor-associated receptor-targeting antibodies. Each line on the graph represents the binding kinetics at a particular bsAb concentration (10, 5, and 2.5 nM from left to right). FIG. 1H Binding of bsAb with heavy chain polypeptide SEQ ID NO: 8 and recombinant human EGFR measured by Octet Red 96 sensorgrams shows dose-dependent binding of tumor-associated receptor-targeting antibodies. Each line on the graph represents the binding kinetics at a particular bsAb concentration (10, 5, and 2.5 nM from left to right). FIG. 1I Binding of bsAb with heavy chain polypeptide SEQ ID NO: 9 and recombinant human EGFR measured by Octet Red 96 sensorgrams shows dose-dependent binding of tumor-associated receptor-targeting antibodies. Each line on the graph represents the binding kinetics at a particular bsAb concentration (10, 5, and 2.5 nM from left to right). Figure 1J Binding of bsAb with heavy chain polypeptide SEQ ID NO: 10 and recombinant human EGFR measured by Octet Red 96 sensorgrams represents dose-dependent binding of tumor-associated receptor-targeting antibodies. Each line on the graph represents binding kinetics at a particular bsAb concentration (10, 5, and 2.5 nM from left to right). Figure 1K Binding of bsAb with heavy chain polypeptide SEQ ID NO: 11 and recombinant human EGFR measured by Octet Red 96 sensorgrams represents dose-dependent binding of tumor-associated receptor-targeting antibodies. Each line on the graph represents binding kinetics at a particular bsAb concentration (10, 5, and 2.5 nM from left to right).

[0024] [Figure 2-1]FIG. 2A Binding of bsAb with heavy polypeptide SEQ ID NO: 1 to recombinant NRP1 measured by Octet Red 96 sensorgrams, showing dose-dependent binding of tumor-associated receptor-targeting antibodies. Each line on the graph represents the binding kinetics at a particular bsAb concentration (10, 5, and 2.5 nM from left to right). FIG. 2B Binding of bsAb with heavy polypeptide SEQ ID NO: 2 to recombinant NRP1 measured by Octet Red 96 sensorgrams, showing dose-dependent binding of tumor-associated receptor-targeting antibodies. Each line on the graph represents the binding kinetics at a particular bsAb concentration (10, 5, and 2.5 nM from left to right). FIG. 2C Binding of bsAb with heavy polypeptide SEQ ID NO: 3 to recombinant NRP1 measured by Octet Red 96 sensorgrams, showing dose-dependent binding of tumor-associated receptor-targeting antibodies. Each line on the graph represents the binding kinetics at a particular bsAb concentration (10, 5, and 2.5 nM from left to right). FIG. 2D Binding of bsAb with heavy polypeptide SEQ ID NO: 4 to recombinant NRP1 measured by Octet Red 96 sensorgrams, showing dose-dependent binding of tumor-associated receptor-targeting antibodies. Each line on the graph represents the binding kinetics at a particular bsAb concentration (10, 5, 2.5 nM from left to right). FIG. 2E Binding of bsAb with heavy polypeptide SEQ ID NO: 5 to recombinant NRP1 measured by Octet Red 96 sensorgrams, showing dose-dependent binding of tumor-associated receptor-targeting antibodies. Each line on the graph represents the binding kinetics at a particular bsAb concentration (10, 5, 2.5 nM from left to right). FIG. 2F Binding of bsAb with heavy polypeptide SEQ ID NO: 6 to recombinant NRP1 measured by Octet Red 96 sensorgrams, showing dose-dependent binding of tumor-associated receptor-targeting antibodies. Each line on the graph represents the binding kinetics at a particular bsAb concentration (10, 5, 2.5 nM from left to right).

[0025] [Figure 2-2]FIG. 2G Binding of bsAb with heavy polypeptide SEQ ID NO: 7 to recombinant NRP1 measured by Octet Red 96 sensorgrams, showing dose-dependent binding of tumor-associated receptor-targeting antibody. Each line on the graph represents the binding kinetics at a particular bsAb concentration (10, 5, 2.5 nM from left to right). FIG. 2H Binding of bsAb with heavy polypeptide SEQ ID NO: 8 to recombinant NRP1 measured by Octet Red 96 sensorgrams, showing dose-dependent binding of tumor-associated receptor-targeting antibody. Each line on the graph represents the binding kinetics at a particular bsAb concentration (10, 5, 2.5 nM from left to right). FIG. 2I Binding of bsAb with heavy polypeptide SEQ ID NO: 9 to recombinant NRP1 measured by Octet Red 96 sensorgrams, showing dose-dependent binding of tumor-associated receptor-targeting antibody. Each line on the graph represents the binding kinetics at a particular bsAb concentration (10, 5, 2.5 nM from left to right). FIG2J Binding of bsAb with heavy polypeptide SEQ ID NO: 10 to recombinant NRP1 measured by Octet Red 96 sensorgrams, showing dose-dependent binding of tumor-associated receptor-targeting antibody. Each line on the graph represents binding kinetics at a particular bsAb concentration (10, 5, and 2.5 nM from left to right). FIG2K Binding of bsAb with heavy polypeptide SEQ ID NO: 11 to recombinant NRP1 measured by Octet Red 96 sensorgrams, showing dose-dependent binding of tumor-associated receptor-targeting antibody. Each line on the graph represents binding kinetics at a particular bsAb concentration (10, 5, and 2.5 nM from left to right).

[0026] [Diagram 3] Figure 3 Western blot showing that incubation of HUVEC cells with bsAb having the heavy chain polypeptide SEQ ID NO:1 fails to block VEGF-mediated VEGFR2 signal activation as detected by phosphorylated VEGFR2 using anti-phospho-VEGFR2 (Y1175).

[0027] [Figure 4-1]Figure 4A Western blot density shows the dose response of bsAb with heavy chain polypeptide SEQ ID NO:2 to inhibit VEGFR2 signaling. HUVEC cells were pre-incubated with bsAb with heavy chain polypeptide SEQ ID NO:2 (6-fold serial dilution concentration from 1 μM) for 30 min 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 lysates using anti-phosphorylated VEGFR2 (Y1175). The inhibitory effect of bsAb with heavy chain polypeptide SEQ ID NO:2 was calculated by comparing VEGF165 control lysates and band intensity using ImageJ software densitometry analysis (numbers shown under P-VEGFR2 (Y1175) blot).

[0028] [Figure 4-2] FIG. 4B Prism9 was used to calculate the IC50 of bsAb having heavy chain polypeptide SEQ ID NO:2 on VEGFR2 phosphorylation (n=2).

[0029] [Figure 5-1] Figure 5A Western blot density shows the dose response of bsAb with heavy chain polypeptide SEQ ID NO:3 to inhibit VEGFR2 signaling. HUVEC cells were pre-incubated with bsAb with heavy chain polypeptide SEQ ID NO:3 (6-fold serial dilution concentration from 1 μM) for 30 min and treated with VEGF165 (2.2 ng / ml) for another 10 min to induce VEGFR2 activation. VEGFR2 signaling activation was detected by Western blot of lysates using anti-phosphorylated VEGFR2 (Y1175). The inhibitory effect of bsAb with heavy chain polypeptide SEQ ID NO:3 was calculated by comparing band intensity with VEGF165 control lysates using densitometric analysis of ImageJ software (numbers shown under P-VEGFR2 (Y1175) blot).

[0030] [Figure 5-2]Figure 5B IC50 graph (n=2) of VEGFR2 phosphorylation inhibition of bsAb with heavy chain polypeptide SEQ ID NO: 3. IC50 was calculated using Prism9.

[0031] [Figure 6-1] Figure 6A Western blot density shows the dose response of VEGFR2 signaling inhibition by bsAb with heavy chain polypeptide SEQ ID NO:4. HUVEC cells were pre-incubated with bsAb with heavy chain polypeptide SEQ ID NO:4 (6-fold serial dilution concentration from 1 μM) for 30 min 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 lysates with anti-phosphorylated VEGFR2 (Y1175). The inhibitory effect of bsAb with heavy chain polypeptide SEQ ID NO:4 was calculated by comparing band intensity with VEGF165 control lysates using densitometric analysis of ImageJ software (numbers shown under P-VEGFR2 (Y1175) blot).

[0032] [Figure 6-2] Figure 6B IC50 graph of bsAb with heavy chain polypeptide SEQ ID NO: 4 VEGFR2 phosphorylation inhibition (n=2). IC50 was calculated using Prism9.

[0033] [Figure 7-1]Figure 7A Western blot density shows the dose response of bsAb with heavy chain polypeptide SEQ ID NO:5 inhibiting VEGFR2 signaling. HUVEC cells were pre-incubated with bsAb with heavy chain polypeptide SEQ ID NO:5 (6-fold serial dilution concentration from 1 μM) for 30 min and treated with VEGF165 (2.2 ng / ml) for another 10 min to induce VEGFR2 activation. VEGFR2 signaling activation was detected by Western blot of lysates with anti-phosphorylated VEGFR2 (Y1175). The inhibitory effect of bsAb with heavy chain polypeptide SEQ ID NO:5 was calculated by comparing band intensity with VEGF165 control lysates using densitometric analysis of ImageJ software (numbers shown under P-VEGFR2 (Y1175) blot).

[0034] [Figure 7-2] Figure 7B IC50 graph of bsAb with heavy chain polypeptide SEQ ID NO:5 (n=1) in inhibiting VEGFR2 phosphorylation. IC50 was calculated using Prism9.

[0035] [Figure 8-1] Figure 8A Western blot density shows the dose response of bsAb with heavy chain polypeptide SEQ ID NO:6 inhibiting VEGFR2 signaling. HUVEC cells were pre-incubated with bsAb with heavy chain polypeptide SEQ ID NO:6 (6-fold serial dilution concentration from 1 μM) for 30 min and treated with VEGF165 (2.2 ng / ml) for another 10 min to induce VEGFR2 activation. VEGFR2 signaling activation was detected by Western blot of lysates using anti-phosphorylated VEGFR2 (Y1175). The inhibitory effect of bsAb with heavy chain polypeptide SEQ ID NO:6 was calculated by comparing band intensity with VEGF165 control lysate using densitometric analysis of ImageJ software (numbers shown under P-VEGFR2 (Y1175) blot).

[0036] [Figure 8-2]Figure 8B IC50 graph of bsAb with heavy chain polypeptide SEQ ID NO: 6 in inhibiting VEGFR2 phosphorylation (n=2). IC50 was calculated using Prism9.

[0037] [Figure 9-1] Figure 9A Western blot densitometry shows the dose response of bsAb with heavy chain polypeptide SEQ ID NO: 7 inhibiting VEGFR2 signaling. HUVEC cells were pre-incubated with bsAb with heavy chain polypeptide SEQ ID NO: 7 (6-fold serial dilution concentration from 1 μM) for 30 min and treated with VEGF165 (2.2 ng / ml) for another 10 min to induce VEGFR2 activation. VEGFR2 signaling activation was detected by Western blot of lysates using anti-phosphorylated VEGFR2 (Y1175). The inhibitory effect of bsAb with heavy chain polypeptide SEQ ID NO: 7 was calculated by comparing band intensity with VEGF165 control lysates using ImageJ software densitometry analysis (numbers shown under P-VEGFR2 (Y1175) blot).

[0038] [Figure 9-2] Figure 9B IC50 graph (n=2) of bsAb with heavy chain polypeptide SEQ ID NO: 7 in inhibiting VEGFR2 phosphorylation. IC50 was calculated using Prism9.

[0039] [Figure 10-1]Figure 10A Western blot density shows the dose response of bsAb containing heavy chain polypeptide SEQ ID NO: 11 inhibiting VEGFR2 signaling. HUVEC cells were pre-incubated with bsAb containing heavy chain polypeptide SEQ ID NO: 11 (6-fold serial dilution concentration from 1 μM) for 30 minutes and treated with VEGF165 (2.2 ng / ml) for another 10 minutes to induce VEGFR2 activation. VEGFR2 signaling activation was detected by Western blot of lysates using anti-phosphorylated VEGFR2 (Y1175). The inhibitory effect of bsAb with heavy chain polypeptide SEQ ID NO: 11 was calculated by comparing band intensity with VEGF165 control lysate using densitometric analysis of ImageJ software (numbers shown under P-VEGFR2 (Y1175) blot).

[0040] [Figure 10-2] Figure 10B IC50 graph (n=2) of bsAb with heavy chain polypeptide SEQ ID NO: 11 in inhibiting VEGFR2 phosphorylation. IC50 was calculated using Prism9.

[0041] [Figure 11-1] FIG. 11A Percent proliferation of H1975 cells in the presence of bsAb having heavy chain polypeptide SEQ ID NO:1 relative to control.

[0042] [Figure 11-2] FIG. 11B Percent proliferation of H1975 cells in the presence of bsAb having heavy chain polypeptide SEQ ID NO:1 relative to control.

[0043] [Figure 12-1] FIG. 12A Mean tumor growth inhibition in an H1975 xenograft mouse model treated with PBS (control) and bsAb comprising heavy chain polypeptide SEQ ID NO:1 (10 mg / kg, ip, BIW).

[0044] [Figure 12-2]FIG. 12B Mean tumor growth inhibition in H1975 xenograft mouse model treated with PBS (control), panitumumab (5 mg / kg, ip, BIW), or bsAb comprising heavy chain polypeptide SEQ ID NO:1 (5 mg / kg, ip, BIW).

[0045] [Figure 12-3] FIG. 12C Mean tumor growth inhibition in H1975 xenograft mouse model treated with PBS (control), panitumumab (5 mg / kg, ip, BIW), or bsAb comprising heavy chain polypeptide SEQ ID NO:2 (6.825 mg / kg, ip, BIW).

[0046] [Figure 12-4] Figure 12D Mean tumor growth inhibition in H1975 xenograft mouse model treated with PBS (control), panitumumab (5 mg / kg, ip, BIW), or bsAb comprising heavy chain polypeptide SEQ ID NO:3 (6.85 mg / kg, ip, BIW).

[0047] [Figure 12-5] Figure 12E Mean tumor growth inhibition in H1975 xenograft mouse model treated with PBS (control), panitumumab (5 mg / kg, intraperitoneal, BIW), or bsAb comprising heavy chain polypeptide SEQ ID NO:7 (6.85 mg / kg, intraperitoneal, BIW).

[0048] [Figure 12-6] Figure 12F Mean tumor growth inhibition in H1975 xenograft mouse model treated with PBS (control), panitumumab (5 mg / kg, intraperitoneal, BIW), or bsAbs containing heavy chain polypeptides SEQ ID NOs: 7, 9, and 11 (6.86 mg / kg, intraperitoneal, BIW).

[0049] [Figure 13] Figures 13A-B Binding kinetics (KD shown) of construct 12 to NRP1 (Figure 13A) or EGFR (Figure 13B).

[0050] [Figure 14]FIG. 14. Mean tumor growth inhibition in H1975 xenograft mouse model treated with IgG1 isotype control, Construct 11 bsAb, or Construct 12 bsAb.

[0051] [Figure 15] FIG. 15. Schematic diagram of a bispecific antibody of the present disclosure.

[0052] [Figure 16] Figures 16A-16D: Receptor internalization of NRP1 receptor (Figures 16A, 16B) or EGFR receptor (Figures 16C, 16D) in H1975 cells (Figures 16A, 16C) or H1975-NRP1OE cells (Figures 16B, 16D) by construct 11bsAb compared with anti-NRP1 alone, anti-EGFR alone, or the combination of anti-NRP1 + anti-EGFR.

[0053] [Figure 17] FIG. 17 Receptor degradation of NRP1 or EGFR receptors by construct 11bsAb was compared to anti-NRP1 alone, anti-EGFR alone, or the combination of anti-NRP1 + anti-EGFR.

[0054] Detailed Description definition 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. Thus, the following terms are intended to have the following meanings:

[0055] As used in this specification and claims, the singular forms "a," "an," and "the" include the plural forms as well, unless the context clearly dictates otherwise.

[0056] As used herein, "administration" of the disclosed polypeptides encompasses delivery of the polypeptides or compositions of the invention described herein, or a prodrug or other pharma- ceutically acceptable derivative thereof, to a subject using any suitable formulation or route of administration, as described herein.

[0057] As used herein, the term "and / or" when used in a list of two or more items means that any one of the listed items can be used alone, or any combination of two or more of the listed items can be used.

[0058] As used herein, the term "bispecific antibody" refers to an antibody that contains at least two binding sites that bind to at least two different target antigens or target epitopes.

[0059] As used herein, "treatment", "treat" or "treating" are used interchangeably herein and refer to an approach to obtain a therapeutic benefit, which is determined by whether the tumor shrinks, remains the same size, or increases progression-free survival compared to placebo.

[0060] As used herein, "subject" can refer to any animal, such as a mammal, such as a laboratory animal, livestock, or pet, that is afflicted with cancer. 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, quails, or turkeys, or mammals), specifically referring to "mammals," including non-primates (e.g., cows, pigs, horses, sheep, rabbits, guinea pigs, rats, cats, dogs, and mice) and primates (e.g., monkeys, chimpanzees, and humans), more specifically referring to humans. In one embodiment, the subject is a non-human animal, such as a livestock animal (e.g., horses, cows, pigs, or sheep) or a pet (e.g., dog, cat, guinea pig, or rabbit). In a preferred embodiment, the subject is a "human."

[0061] As used herein, the term "fusion" refers to the integration of two molecules with the same or different functions or structures, and the method of fusion includes any physical, chemical or biological method that can bind a peptide to a protein, a small molecule drug, a nanoparticle or a liposome. Preferably, the fusion can be mediated by a linker peptide, for example, a linker peptide can be fused to the C-terminus of a fragment of an antibody light chain variable region (Fc). Alternatively, the two molecules are fused by the integration of multiple domains within the polypeptide sequence.

[0062] As used herein, the term "linker" refers to a molecule or peptide that links two polypeptide subunits together. The linker peptide sequence can include the amino acid sequence (GGGGS)n, where n defines the number of repeats. The number of subunit repeats defines the flexibility of the linker peptide. A flexible peptide linker increases the flexibility between the two binding domains.

[0063] As used herein, an "effective amount" refers to an amount sufficient to induce a desired anti-cancer response. In the present invention, the desired biological response is to inhibit cell proliferation. The exact amount of bispecific antibody administered to a subject depends on the method of administration, the type and severity of the cancer, and characteristics such as the subject's general health, age, sex, weight, and tolerance to drugs. A skilled artisan can determine the appropriate dosage depending on these and other factors. When used in combination with other anti-cancer agents, e.g., in combination with chemotherapy, the "effective amount" of the second agent depends on the type of drug used. Appropriate dosages of approved agents are known and can be adjusted by a skilled artisan depending on the subject's condition, the type of cancer being treated, and the amount of polypeptide administered herein. If an amount is not explicitly stated, it should be considered an effective amount. For example, the bispecific antibodies described herein can be administered to a subject at weekly or biweekly intervals in a dosage range of about 0.01 to 100 mg / kg body weight / day.

[0064] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

[0065] As used herein, the term "mutant" refers to a sequence with a change from a wild-type, conventional, or primary sequence. The change can be in the form of an amino acid or nucleotide deletion, substitution, or insertion. A mutant can contain one or more changes, including a combination of sequence changes.

[0066] The term "reduce", or other forms of the term such as "reducing" or "reduction", generally refers to a decrease in an event or characteristic (e.g., one or more symptoms, or the binding of one protein to another). It is understood that this is usually in relation to some standard or expected value, i.e., relative, but does not necessarily refer to a standard or relative value.

[0067] When used in the context of "binding affinity," the term "affinity" refers to the reduced affinity of one molecule for another. For example, in some embodiments, a protein, domain, or motif can specifically bind to a particular target, e.g., a peptide, polypeptide, protein, carbohydrate, saccharide, polysaccharide, glycosaminoglycan, or any epitope thereof, with a given affinity. The term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule and its binding target or partner (e.g., an antigen). The affinity of a molecule for a target is determined by the dissociation constant (K D ) which can be expressed as the dissociation rate constant and the association rate constant (k off and k on The strength or affinity of a binding interaction is determined by the dissociation constant (K D ) can be expressed as K D A smaller K indicates a higher affinity. The binding properties (affinity) of a selected polypeptide can be quantified using methods well known in the art. One such method is to measure the rates of formation and dissociation of the antigen-binding site / antigen complex. These rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, the "on-rate constant" (K on ) and the "off rate constant" (K off ) can be determined by calculating the concentrations and the actual rates of association and dissociation (see Nature 361:186-87 (1993)).

[0068] K off / K on The ratio of K to K allows to cancel all parameters not related to affinity, and the dissociation constant K D (See generally, Davies et al. (1990) Annual Rev Biochem 59:439-473.) In some embodiments, the recombinant polypeptides of the invention have an equilibrium binding constant (K DIn some embodiments, a recombinant polypeptide of the present invention can specifically bind to an epitope when the equilibrium binding constant (K D In some embodiments, a recombinant polypeptide of the present invention can specifically bind to an epitope when the equilibrium binding constant (K D In some embodiments, a recombinant polypeptide of the invention can specifically bind to an epitope when the equilibrium binding constant (K) is 10 nM or less, as measured by an assay such as surface plasmon resonance (SPR), octet assay, or similar assay known to one of skill in the art. D In some embodiments, the epitope can be specifically bound when K D is 10 -5 M or less (e.g., 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -8 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13 M or less, 10 -14 M or less, 10 -15 M or less, or 10 -16 M or less).

[0069] Thus, equivalent affinities can include different rate constants, so long as the ratio of the rate constants is the same. Thus, in some embodiments, "decreased binding" refers to a decrease in affinity for the respective interaction. Conversely, "increased binding" refers to an increase in binding affinity for the respective interaction.

[0070] As used herein, the term "heavy chain" can be interpreted as including a full-length heavy chain that includes a heavy chain variable region domain (VH) that includes an amino acid sequence with sufficient variable region sequence to confer antigen specificity, and the three heavy chain constant region domains CH1, CH2, and CH3, or a fragment thereof. Also, as used herein, the term "light chain" can be interpreted as including a full-length light chain that includes a light chain variable region domain (VL) that includes an amino acid sequence with sufficient variable region sequence to confer antigen specificity, and a fragment thereof, the light chain constant region domain (CL). As used herein, the term "FAB" refers to a region that binds to an antigen. A FAB is composed of one variable heavy and light chain and one constant heavy and light chain.

[0071] 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 when optimally aligned and compared (with appropriate insertions or deletions for optimal alignment). The percent identity between two sequences is a function of the number of identical positions shared by the two sequences (i.e., % identity = number of identical positions / total number of positions x 100). This takes into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences. The comparison of sequences and the determination of the percent identity between two sequences can be performed using a mathematical algorithm, as described in the non-limiting examples below. Methods and algorithms for determining the percent homology between two protein sequences are well established in the art.

[0072] 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 incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com), using a Blossum62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6. Additionally, protein amino acid sequences can be used as "query sequences" to perform searches against public databases, for example, to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program with score=50 and wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison, 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.

[0073] As used herein, the terms "combination" or "co-administration" can be used interchangeably to refer to the use of more than one treatment modality (e.g., one or more prophylactic and / or therapeutic agents). The use of these terms does not restrict the order in which therapies (e.g., prophylactic and / or therapeutic agents) are administered to a subject.

[0074] As used herein, the term "synergistic" refers to a combination of a polypeptide of the invention with another treatment (e.g., a prophylactic or therapeutic agent) where the combination is more effective than the additive effects of the treatments.

[0075] The term "pharmaceutically acceptable salts" is intended to include salts of active bispecific antibodies prepared with relatively non-toxic acids or bases, depending on the particular substituents found on the bispecific antibodies described herein.

[0076] As used herein, the term "parenteral" includes, but is not limited to, subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0077] The term "carrier" refers to a vehicle used to formulate a composition and may consist of multiple excipients.

[0078] As used herein, the term "excipient" refers to a pharmacologically inactive, natural or synthetic ingredient or substance that is formulated alongside (e.g., simultaneously) or subsequently with the active ingredient of the present invention. In some embodiments, the excipient can be any additive, adjuvant, binder, filler, carrier, coating, diluent, disintegrant, filler, glidant, lubricant, preservative, vehicle, or combination thereof that can administer the recombinant polypeptide of the present invention and / or is useful in preparing the composition of the present invention. Excipients include any such substance known in the art that is non-toxic and does not interact with other components of the composition. In some embodiments, the excipient can be formulated alongside the recombinant polypeptide when preparing the composition for the purpose of bulking the composition (and thus is often referred to as a bulking agent, filler, or diluent). In other embodiments, the excipient can be used to provide enhancements to the active ingredient in the final dosage form, such as facilitating absorption and / or solubility. In still other embodiments, the excipient can be used to provide stability or prevent contamination (e.g., microbial contamination). In other embodiments, excipients can be used to impart physical characteristics to the composition (e.g., a composition that is in the physical form of a dry granule or dry flowable powder). Reference to an excipient includes both one and more of such excipients. Suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences by E.W. Martin, the disclosure of which is incorporated herein by reference in its entirety.

[0079] 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 a stated step, element, or integer, or group of steps, elements, or integers, but not the exclusion of other steps, elements, or integers, or groups of elements, steps, or integers.

[0080] All patent applications, patents, and printed publications mentioned 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, and 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 except to the extent the incorporated material is inconsistent with the express disclosure herein, in which case the language of this disclosure will control.

[0081] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be included within the scope of the appended claims. Moreover, it should be understood that all values ​​are approximate and are provided for illustrative purposes.

[0082] Bispecific antibodies having EGFR-binding domain and NRP1-binding domain Bispecific antibodies have many advantages over monoclonal antibodies. First, it is more efficient to produce one molecule with two effective targets. For example, bispecific antibodies can target multiple 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 a cancer cell antigen, such as EGFR, and an immune cell receptor or immune checkpoint regulator, such as neuropilin-1 (NRP1). Bispecific antibodies for anti-cancer therapy are designed to enhance anti-cancer efficacy.

[0083] EGFR-binding domain The epidermal growth factor receptor (EGFR) is one of the most frequently altered oncogenes in solid tumors. Increased EGFR signaling promotes proliferation and cell survival in many cancer types, including breast, prostate, non-small cell lung cancer (NSCLC), esophagogastric, hepatocellular, glioblastoma, cervical, ovarian, bladder, renal, pancreatic, colon, and rectal cancers. Increased EGFR signaling can occur through overexpression of EGFR, mutation of EGFR, or constitutive activation by mediators within the EGFR signaling pathway, and / or elevated levels of EGFR cognate ligands, such as EGF, tumor necrosis factor alpha (TGF-α), amphiregulin (AREG), epigen, β-cellulin, heparin-binding EGF (HB-EGF), and epiregulin. EGFR signaling activates downstream signaling cascades, including the RAS-RAF-MEK-ERK and PI3K-Akt-mTOR axes, leading to proliferation and survival of cancer cells. Unfortunately, anti-EGFR drugs, such as tyrosine kinase inhibitors, monoclonal antibodies, and radiotherapy, are only effective against a few cancer types, including metastatic colorectal cancer, non-small cell lung cancer (NSCLC), and advanced head and neck cancer. Moreover, the treatment improves survival rates only in a small proportion of people, and initial responses usually end in drug resistance. Therefore, new rational designs are needed to improve therapeutic efficacy.

[0084] Antibodies are proteins that can bind to antigens with high specificity and high affinity and neutralize the activity of the antigen. Anti-EGFR antibodies block ligand-activated EGFR signaling and induce receptor endocytosis, leading to EGFR degradation in the proteasome. If mutations occur at the EGFR binding site of the antibody during treatment, the subject may acquire anti-EGFR antibody resistance. Cetuximab, a mouse / human chimeric monoclonal antibody, and panitumumab, a fully humanized monoclonal antibody, have different binding sites on EGFR, so panitumumab remains effective after cetuximab resistance has developed, and vice versa. Therefore, subjects who have acquired resistance to one anti-EGFR antibody can be administered another anti-EGFR antibody that targets a different EGFR binding site.

[0085] Panitumumab is an IgG2 anti-EGFR humanized monoclonal antibody that binds to EGFR with approximately 8-fold higher affinity than cetuximab, an IgG1 anti-EGFR chimeric human / mouse monoclonal antibody (Garcia-Foncillas et al. 2019). Antibody binding can induce an immune response before inducing endocytosis. Panitumumab binding induces antibody-dependent cellular cytotoxicity (ADCC) and initiates antibody-dependent cellular phagocytosis (ADCP) via activation of neutrophils and monocytes (Schneider-Merk et al. 2010). The IgG1 Fc domain of cetuximab binds to the FcγRIIIA (CD16) receptor on natural killer (NK) cells and induces ADCC. Activated NK cells secrete perforin and granzymes, lyse cancer cells, and release immune stimulatory molecules such as interferon-gamma (IFN-γ), TNF-α, chemokines, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Secretion of cytokines by NK cells stimulates dendritic cell maturation, NK cell crosstalk, and co-expression of CD137. Expression of CD137 recruits anti-EGFR CD8+ T cells, increasing the killing of EGFR-expressing cancer cells. 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 to the first subunit of the C1 complement complex (C1q), initiating antibody-dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC) by the cells, respectively. Similar to cetuximab, the anti-EGFR antibodies cetuximab, necitumumab, and nimotuzumab also have an IgG1 Fc region for inducing ADCC.

[0086] NRP1-binding domain Cancer cells overexpress pro-angiogenic factors that promote the rapid growth of new blood vessels. Blood vessels around tumors are abnormal, causing capillary constriction and reducing overall blood flow to the tumor. The reduced blood flow rate increases tumor interstitial fluid pressure and reduces the flow of drugs from the blood vessels to the tumor (Milosevic et al., 1999). Unlike normal tissue, tumor tissue lacks lymphatic vessels, which also contributes to abnormal angiogenesis and increased tumor interstitial pressure. Vascular endothelial growth factor A (VEGF) 165 Inhibition of angiogenesis by targeting VEGF may normalize blood vessel formation, vascular fluid pressure, and increase accumulation of therapeutic drugs at tumor sites (Marcucci et al. 2013). However, anti-VEGF antibodies such as bevacizumab may cause side effects and are only effective with a limited range of exposure (Kamba and McDonald, 2007).

[0087] 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 regions of the VEGF ligand family and Sema3 ligands that bind to NRP1 and NRP2, respectively, bind 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 an R / KxxR / K (R=arginine, K=lysine, x=any amino acid) motif, which is commonly present in the C-terminal region of NRP-binding ligands and is known as the "C-end rule" (CendR) (Teesalu et al. 2009). Proteins or peptides that contain the C-end rule sequence can bind to NRPs through their C-terminal arginine (Arg) or lysine (Lys) residues (Zanuy et al., 2013).

[0088] Selectively targeting NRP1 is important for anti-EGFR cancer therapy, because 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 colorectal cancer, melanoma, astrocytoma, lung cancer, prostate cancer, and pancreatic ductal adenocarcinoma, and plays an important role in cancer progression (Graziani and Lacal, 2015). NRP1 is not only overexpressed in various cancers, but also in tumor-associated endothelial cells. NRP1 acts as a vascular endothelial growth factor (VEGF) inhibitor. 165 NRP1 functions as a coreceptor for ligands involved in angiogenesis, including 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, contributing to VEGFR signaling leading to increased angiogenesis. NRP1 also binds to secreted class 3 semaphorin ligands (Sema3A, Sema3B, Sema3C, Sema3D, Sema3E, Sema3F, and Sema3G) and functions as a coreceptor for plexin family receptors that regulate angiogenesis.

[0089] Inhibition of NRP1 ligand binding by the anti-NPR1 specific binding domain reduces the expression of the endothelial adhesion molecule VE-cadherin and increases the extravasation of anti-EGFR antibodies from blood vessels, which in turn reduces tumor interstitial pressure, allowing the drug to flow more freely from the blood vessels into the tumor environment.

[0090] The epithelial barrier around solid tumors consists of intercellular spaces densely filled with stromal epithelial cells connected by intercellular adhesion factors, preventing the penetration of therapeutic drugs into the tumor. Overexpression of E-cadherin contributes to cell-cell adhesion. Since a substance that reduces E-cadherin was found in a virus (adenovirus 3), a case has been reported in which the anticancer effect of an antibody was enhanced by coadministering only a part of the virus protein (JO-1) that has the activity of reducing cellular E-cadherin in tight junctions with an antibody (Beyer et al. 2011). Targeting NRP1 reduces the expression of the epithelial barrier adhesion molecule E-cadherin and the integrin β1 subunit, which are overexpressed in many solid tumors, and increases the binding of the extracellular matrix, thereby reducing the permeability of drugs. Integrin β1 subunit is also involved in the activation of cell proliferation via growth factor receptors.

[0091] In summary, targeting NRP1 inhibits VEGF 165 EGFR binding, and expression of VE-cadherin, E-cadherin, and integrin β1, which together reduce angiogenesis, tumor interstitial pressure, and increase anti-EGFR antibody extravasation and tumor penetration.

[0092] Cancer cells produce immune checkpoint molecules that inhibit immune responses. NRP1 is an immune checkpoint molecule whose expression is increased in tumor-associated endothelial cells and cancer cells. Expression of NRP1 induces immune suppression by increasing Treg activity and decreasing tumor-specific CD8+ T cell responses (Chuckran et al. 2020). Thus, besides increasing drug extravasation and penetration, the NRP1 binding domain functions as an immune checkpoint inhibitor. In yet another aspect, the present disclosure provides bispecific antibodies that target NRP1 to relieve cancer-mediated checkpoint molecule immune suppression, enable natural immunity at tumor sites, and increase the likelihood of tumor response.

[0093] The anti-NRP1 antibody MNRP1685A (also known as besencumab) inhibits VEGF receptors on NRP1.165 It functions to competitively bind to and inhibit VEGF signaling through VEGFR2, thereby affecting angiogenesis, cell survival, migration, adhesion, and invasion (Pan Q et al. 2007). However, subjects receiving besencumab in Phase I trials for advanced solid tumors suffered intolerable side effects such as gastrointestinal bleeding, fungemia, duodenal obstruction, thrombocytopenia, proteinuria, alopecia, dysphonia, fatigue, and nausea, leading to discontinuation of further trials (Weekes et al., 2014, Patnaik et al., 2014).

[0094] NRP1 is active as a homodimer or heterodimer, and monomeric peptides such as the truncated penetrating peptide iRGD have a weak ability to control the biological activity of NRP1 (Sugahara et al. 2010). Therefore, peptides that selectively bind to NRP1 as a homodimer and control its biological activity are desirable. Unfortunately, although the Fc heavy chain fused NRP binding peptide A22p is presented as a double peptide (homodimer), A22p binds to both NRP1 and NRP2 (Shin et al. 2014). Therefore, it is important to identify effective and non-toxic NRP1 selective targeting molecules.

[0095] In one aspect, the present disclosure relates to monoclonal antibodies (anti-NRP1 mAbs) that bind to NRP1, which can be used in the bispecific antibodies of the present disclosure. In an embodiment, the present disclosure provides a monoclonal antibody having a heavy chain polypeptide sequence set forth in SEQ ID NOs: 41-47, which includes an N-terminal NRP1 binding domain. In some embodiments, the monoclonal antibody heavy chain polypeptide includes an N-terminal NRP1 binding domain including a variable heavy chain (VH) and a constant heavy chain 1 (CH1), and an Fc domain including a constant heavy chain 2 (CH2) and a constant heavy chain 3 (CH3). In some embodiments, the monoclonal antibody includes a light chain polypeptide sequence set forth in any of SEQ ID NOs: 48-54. Thus, the heavy chain of SEQ ID NOs: 41-47 can pair with the light chain of SEQ ID NOs: 48-54. Polynucleotide sequences encoding the described heavy and light chain polypeptides are set forth in SEQ ID NOs: 55-61 and SEQ ID NOs: 62-68, respectively. Example 8 describes a method for producing a monoclonal antibody.

[0096] In some embodiments, the NRP1 binding domain of the monoclonal antibody has an NRP1 affinity (K) in the range of 70 nM to subnanomolar for a monoclonal antibody having a heavy chain polypeptide sequence set forth in SEQ ID NOs: 41-47 and a corresponding light chain polypeptide sequence set forth in SEQ ID NOs: 48-54. D ).

[0097] Blocking VEGF binding to NRP1 with a monoclonal antibody to the NRP1-binding domain can inhibit VEGFR2 activation as measured by VEGFR2 phosphorylation.

[0098] In some embodiments, the monoclonal antibody inhibits the proliferation of cancer cells. In some embodiments, the monoclonal antibody Fc domain, including constant heavy chains 2 and 3 (CH2 and CH3), is an IgG1 or IgG2 subclass. In some embodiments, the present disclosure provides a monoclonal antibody NRP1 binding domain that potently inhibits cell proliferation in cancer cell line H1975.

[0099] Bispecific antibodies with binding affinity to EGFR and NRP1 The present disclosure provides a bispecific antibody having a heavy chain polypeptide sequence comprising an N-terminal EGFR-binding domain and a C-terminal NRP1-binding domain. The bispecific antibody heavy chain polypeptide comprises an N-terminal EGFR-binding domain comprising a variable heavy chain (VH) and a constant heavy chain 1 (CH1), an Fc domain comprising a constant heavy chain 2 (CH2) and a constant heavy chain 3 (CH3), and a C-terminal short chain variable fragment (scFv) NRP1-binding domain.

[0100] A schematic diagram of a representative bispecific antibody structure of the present disclosure is shown in FIG.

[0101] The ScFv NRP1 binding domain consists of a variable heavy chain and a variable light chain connected by a flexible peptide linker. In some embodiments, the flexible peptide linker is a repeat of the subunit sequence of SEQ ID NO: 13. In some embodiments, the peptide linker connects the ScFv to CH3. In some embodiments, the peptide linker consists of one or more peptide linker subunits of SEQ ID NO: 13, and GGGGS is one subunit.

[0102] The bsAb heavy chain polypeptide sequences are shown in SEQ ID NOs: 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: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 40. In some embodiments, the bispecific antibodies further comprise a light chain polypeptide sequence comprising a variable light chain and a constant light chain paired with the heavy chain polypeptide, as 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 embodiments, the disclosure provides polynucleotide sequences used to encode the GGGGS subunits, representative examples of which are polynucleotides. The sequences are shown in SEQ ID NOs: 26-38. Example 1 describes a method for producing a bispecific antibody.

[0103] In some embodiments, the bispecific antibody comprises an anti-EGFR binding arm comprising (or consisting of) a VH amino acid sequence set forth in SEQ ID NO: 69 and a VL amino acid sequence set forth in SEQ ID NO: 70. In some embodiments, 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 sequence set forth in SEQ ID NO: 71, 72 and 73, respectively. In some embodiments, the bispecific antibody comprises an anti-EGFR binding arm comprising a heavy chain CDR3 region comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 73. In some embodiments, the bispecific antibody comprises an anti-EGFR binding arm comprising a light chain CDR1, CDR2 and CDR3 region comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 74, 75 and 76, respectively. In some embodiments, the bispecific antibody comprises an anti-EGFR binding arm comprising a light chain CDR3 region comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 76. In some embodiments, the anti-EGFR binding arm comprises (or consists of) one or more sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.8% identical to any of the aforementioned VH, VL, HCDR, or LCDR sequences.

[0104] In some embodiments, the bispecific antibody comprises an anti-NRP1 binding arm comprising (or consisting of) a VH amino acid sequence set forth in SEQ ID NO: 77 and a VL amino acid sequence set forth in SEQ ID NO: 78. In some embodiments, the bispecific antibody comprises an anti-NRP1 binding arm comprising a heavy chain CDR1, CDR2 and CDR3 region (HCDR1, HCDR2 and HCDR3, respectively), where HCDR1 comprises (or consists of) the sequence set forth in SEQ ID NO: 79, HCDR2 comprises (or consists of) the sequence set forth in SEQ ID NO: 80 and HCDR3 comprises (or consists of) the sequence set forth in any one of SEQ ID NOs: 81 to 84. In some embodiments, the bispecific antibody comprises an anti-NRP1 binding arm comprising a light chain CDR1, CDR2 and CDR3 region (LCDR1, LCDR2 and LCDR3, respectively), where LCDR1 comprises (or consists of) the sequence set forth in any one of SEQ ID NOs: 85 to 87, LCDR2 comprises (or consists of) the sequence set forth in SEQ ID NO: 88 and LCDR3 comprises (or consists of) the sequence set forth in SEQ ID NO: 89. In some embodiments, the bispecific antibody comprises an anti-NRP1 binding arm comprising a heavy chain CDR3 region comprising (or consisting of) the amino acid sequence set forth in any one of SEQ ID NOs: 81-84. In some embodiments, 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 set forth in SEQ ID NOs: 79, 80 and 84, respectively, and a light chain CDR1, CDR2 and CDR3 region comprising (or consisting of) the amino acid sequence set forth in SEQ ID NOs: 85, 88, 89, respectively. 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 aforementioned VH, VL, HCDR, or LCDR sequences.

[0105] In some embodiments, the EGFR binding domain of the bispecific antibody has an EGFR affinity (K DIn some embodiments, the NRP1-binding domain of the bispecific antibody has an NRP1 affinity (K) ranging from 70 nM for a bispecific antibody construct having the control polypeptide sequence of SEQ ID NO: 1 to subnanomolar for bispecific antibodies having the polypeptide sequences set forth in SEQ ID NOs: 2, 3, 5, 7, 10, and 11. D ). Thus, in one embodiment, the disclosure provides a bispecific antibody having an EGFR binding domain with affinity for EGFR in the subnanomolar range and an NRP1 binding domain with affinity for NRP1 in the subnanomolar range, as shown in Table 1 of Example 2. Figures 1A-1H show binding affinity curves of the bispecific antibody to immobilized huEGFR. Figures 2A-2H show binding affinity curves of the bispecific antibody to immobilized huEGFR. Figures 2A-2I show binding affinity curves of the bispecific antibody to immobilized huNRP1.

[0106] One aspect of the present disclosure provides a bispecific antibody having asymmetric binding affinity for EGFR and NRP1. See Examples. In some embodiments, the K of the EGFR-binding domain for EGFR is D is the K of the NRP1-binding domain for NRP1 D The asymmetric affinity is at least 2-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 100-fold, or more than 100-fold greater than NRP1. The asymmetric affinity reduces cytotoxicity of targeting NRP1 while improving homing to cancer cells expressing EGFR.

[0107] Blocking VEGF binding to NRP1 with the NRP1-binding domain of a bispecific antibody may inhibit VEGFR2 activation as measured by VEGFR2 phosphorylation. However, a bispecific antibody having a heavy chain of SEQ ID NO: 1 inhibits NRP1 K D69.7 nM and does not inhibit VEGFR phosphorylation (FIG. 3). In some embodiments, the bispecific antibody inhibits VEGFR2 phosphorylation and prevents angiogenesis. See Table 2 and FIGS. 4A, 4B-10A, 10B. In some embodiments, the inventors have determined that the dissociation constant (K D ) does not reflect the ability of the bispecific antibody to inhibit VEGFR2 phosphorylation. For example, a bispecific antibody having a heavy chain SEQ ID NO:6 inhibits 50% of VEGFR2 phosphorylation with an IC50 of 10130 nM, but has a K D On the other hand, the bispecific antibody with heavy chain SEQ ID NO:4 inhibited 50% of VEGFR2 phosphorylation with an IC50 of 410 nM, but the K D is 8.8 nM. Thus, in some embodiments, the disclosure provides bispecific antibodies with imbalanced NRP1-binding domain affinity and biological function with respect to inhibition of VEGFR2 phosphorylation.

[0108] In some embodiments, the bispecific antibody inhibits the proliferation of cancer cells. In some embodiments, the bispecific antibody Fc domain, including constant heavy chains 2 and 3 (CH2 and CH3), is of IgG1 or IgG2 subclass. In some embodiments, the present disclosure provides a bispecific antibody having an EGFR binding domain and an NRP1 scFv binding domain that potently inhibits cell proliferation in cancer cell line H1975 (Table 3 and Figures 11A and 11B). Example 4 describes the method used to measure the bispecific antibody IC50 for cell proliferation inhibition in H1975 cancer cell line.

[0109] Additional Binding Arms of Bispecific Antibodies In other embodiments, an anti-NRP1 binding arm (described herein) can be paired in a bispecific antibody with a second binding arm that binds to a second target protein. Antibodies that bind to these second target proteins are known in the art and can be used in the bispecific antibodies of the present disclosure, as described herein.

[0110] In some embodiments, the second target protein comprises a receptor tyrosine kinase (RTK). In some embodiments, the tyrosine kinase comprises a platelet-derived growth factor receptor (PDGFR), a fibroblast growth factor receptor (FGFR), a receptor tyrosine kinase Met (MET), and a receptor vascular endothelial growth factor (VEGFR). In some embodiments, the receptor tyrosine kinase comprises 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.

[0111] In some embodiments, the target protein comprises EGFR. In some embodiments, EGFR is selected from the group consisting of EGFR / HER1 / Erb1, HER2 / ErbB2, HER3 / ErbB3, and HER4 / ErbB4. In some embodiments, the target protein is EGFR.

[0112] In some embodiments, the target protein comprises an FGFR. In some embodiments, the FGFR1 is selected from the group consisting of FGFR1, FGFR2, FGFR3, and FGFR4.

[0113] 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).

[0114] In some embodiments, the target protein comprises a PDGFR. In some embodiments, the PDGFR is selected from the group consisting of PDGFR, PDGFRα, PDGFRβ, CSF-1R, Kit, and FLT-3.

[0115] In some embodiments, the target protein comprises a VEGFR. In some embodiments, the VEGFR is selected from the group consisting of VEGFR1, VEGFR2, and VEGFR3.

[0116] In some embodiments, the receptor tyrosine kinase is selected from the group consisting of EGFR / HER1 / Erb1, HER2 / ErbB2, HER3 / ErbB3, HER4 / ErbB4 VEGFR1, VEGFR2, and VEGFR3.

[0117] In some embodiments, the receptor tyrosine kinase is selected from the group consisting of EGFR / HER1 / Erb1, HER2 / ErbB2, HER3 / ErbB3, HER4 / ErbB4, VEGFR1, VEGFR2, and VEGFR3.

[0118] In some embodiments, target proteins include receptor serine / threonine kinases (RSTKs), G protein-coupled receptors (GPCRs), immune checkpoint receptors, and ion channel receptors.

[0119] In some embodiments, the target protein comprises a receptor serine / threonine kinase (RSTK). In some embodiments, the RSTK comprises ACVRL1, ACVR1, ACVR1B, ACVR1C, BMPR1A, BMPR1B, TGFBR1, ACVR2A, ACVR2B, AMHR2, BMPR2, TGFBR2.

[0120] In some embodiments, the target protein comprises a G protein-coupled receptor (GPCR). In some embodiments, the GPCR is selected from the group consisting of CXCR4, CCR5, FFAR2, GLP2R, 5-HT1A receptor, 5-HT2A receptor, 5-HT4 receptor, 5-HT5A receptor, M1 receptor, M2 receptor, A1 receptor, A2A receptor, alpha1A adrenergic receptor, alpha2A adrenergic receptor, beta1 adrenergic receptor, beta3 adrenergic receptor, AT1 receptor, BB1 receptor, B1 receptor, CB1 receptor, CB2 receptor, chemerin receptor 1, CCR1, CX3CR1, ACKR3, CCK1 receptor, GPR3, GPR12, GPR17, GPR32, GPR35.

[0121] In some embodiments, the target protein comprises an immune checkpoint receptor, hi some embodiments, the immune checkpoint receptor is selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, NOX2, PD-1, TIM-3, VISTA, SIGEC7, and PD-L1.

[0122] In some embodiments, the target protein comprises an ion channel receptor. In some embodiments, the ion channel receptor is selected from the group consisting of KCa1.1, KCa2.1, CatSper1, TPC1, CNGA1, HCN1, Kir1.1, Kir3.2, RyR1, TRPA1, TRPC3, TRPM1, TRPP1, TRPV1, K2P1.1, K2P10.1, Cav1.1, Cav2.1, Kv1.1, Kv1.8, Kv11.2, Hv1, Nav1.1, and Nav1.2.

[0123] In some embodiments, the target protein comprises a membrane-associated target protein and the target protein binding domain of the bispecific binding molecule binds to a cell surface receptor. In some embodiments, the target protein binding domain of the bispecific binding molecule binds to an extracellular epitope of the membrane-associated target protein. In some embodiments, the target cell comprises a tumor cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the cancer cell is selected from the group consisting of lung cancer, breast cancer, colon and rectal cancer, head and neck cancer, esophagogastric cancer, liver cancer, glioblastoma, prostate cancer, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, and pancreatic cancer. In some embodiments, the target cell comprises an immune cell.

[0124] Tumor growth inhibition by bispecific antibodies in tumor mouse models In some embodiments of the disclosure, treatment with bispecific antibodies reduces tumor growth. In some embodiments, a bispecific antibody having heavy chain SEQ ID NO: 11 and light chain SEQ ID NO: 12 (also referred to herein as construct 11) significantly reduces tumor growth. In some embodiments, a bispecific antibody having heavy chain SEQ ID NO: 39 and light chain SEQ ID NO: 12 (also referred to herein as construct 12) significantly reduces tumor growth. In some aspects, the addition of an scFv NRP1 binding domain significantly improves the efficacy of tumor growth inhibition. Example 5 describes the methods and results of treating H1975 xenograft mouse models with bispecific antibodies for tumor growth inhibition. Table 4 shows the percent tumor growth inhibited, and Figures 12A-12G and 17 show the tumor growth over days after implantation.

[0125] Summary of the biological effects of bispecific antibodies In summary, the present disclosure provides bispecific antibodies comprising an NRP1 binding domain, such as a bispecific antibody that combines an EGFR binding domain with an NRP1 binding domain to promote homing to tumors with high EGFR expression by increasing EGFR affinity. The bispecific antibody has multiple biological effects that combine to reduce cancer cell proliferation and survival, such as reducing aberrant angiogenesis, increasing extravasation and penetration by reducing VE-cadherin, E-cadherin, and integrin β1 expression. Targeting NRP1 further reduces EGFR surface pooling, promotes EGFR downregulation, and inhibits NRP1 checkpoint immune suppression. The combination of these features results in a bispecific antibody that effectively inhibits EGFR-mediated cancer cell proliferation and survival.

[0126] FcRn binding for transcytotic recycling The size of the antibody is approximately 150 kD, resulting in a long serum half-life and long-lasting therapeutic effect. In addition, the antibody IgGFc portion, consisting of heavy chain constant regions 2 and 3 (CH2 and CH3), binds to the neonatal Fc receptor (FcRn) and Fcγ receptor (FcγR) on the cell surface, and is endocytosed and recycled to the serum through transcytosis, further prolonging the antibody serum half-life. Because cancers with low expression of FcRn or FcγR are associated with poor prognosis (Pyzik et al., 2019), recycling via FcRn or FcγR plays an important role in maintaining antibody serum concentrations. IgG1 antibodies have more efficient FcRn and FcγR recycling processes than IgG2 antibodies. Thus, having an IgG1Fc domain in a therapeutic antibody helps maintain drug therapeutic levels and reduce dosing frequency, while the shortened half-life is ideal for diagnostic testing or toxicity control.

[0127] In some embodiments, the polypeptides of the invention comprise an immunoglobulin domain, including a polypeptide comprising an immunoglobulin domain that comprises an Fc domain selected from the IgG1 subclass.

[0128] Expression constructs As used herein, the term "vector" refers to a means for expressing a target gene in a host cell.For example, vectors can include plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors such as adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors.Recombinant vectors can be produced by manipulating plasmids (e.g., pSC101, pGV1106, pACYC177, ColEl, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), phages (e.g., Agt4AB, A-Charon, AAz1, M13, etc.), or viruses (e.g., CMV, SV40, etc.) commonly used in the art.

[0129] In another embodiment, the present invention provides an isolated polynucleotide encoding the heavy and light chain amino acid sequences of a bispecific antibody. The polynucleotide encoding in the recombinant vector can be operably linked to a promoter. As used herein, the term "operably linked" refers to a functional linkage between a nucleotide expression control sequence (such as a promoter sequence) and a second nucleotide sequence. Thus, the regulatory sequence can control the transcription and / or translation of the second nucleotide sequence. The recombinant vector can generally be constructed as a cloning vector or an expression vector. As an expression vector, a vector commonly used in the art to express foreign proteins from plants, animals or microorganisms can be used. The recombinant vector can be constructed by various methods known in the art. The recombinant vector can be constructed using, but is not limited to, a eukaryotic cell as a host having a fl origin of replication, an SV40 origin of replication, a pMB1 origin of replication, an adeno origin of replication, an AAV origin of replication, a CMV origin of replication, a BBV origin of replication, and the like.

[0130] Also, a promoter derived from the genome of a mammalian cell (e.g., a metallothionine promoter) or a promoter derived from a virus of a mammalian cell (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a cytomegalovirus (CMV) promoter, or a tk promoter of HSV) can be used, and the promoter usually has a polyadenylation sequence as a transcription termination sequence. The vector can express not only the peptide domain specifically binding to NRP1 of the present invention, but also an antibody fused with a peptide and a linker peptide. In the case of an antibody fused with a peptide, both a vector system expressing a peptide and an antibody or a fragment thereof in one vector and a vector system expressing a peptide and an antibody or a fragment thereof in separate vectors can be used. In the latter case, the two vectors can be introduced into a host cell by co-transformation and target transformation. Thus, in another aspect, the present disclosure includes a vector comprising a nucleic acid disclosed in any one of SEQ ID NOs: 14 to 38 and 40. Example 1 describes the characteristics of a vector that can be used for the production of a bispecific antibody.

[0131] Another aspect of the present disclosure provides a host cell transformed with a recombinant vector. Any type of host cell known in the relevant art can be used as the 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 X1776, E. coli W3110, or strains belonging to the genus Bacillus, such as Bacillus subtilus and Basillus thuringiensis, enteric flora and strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas Spp. Transformation of prokaryotic cells is useful for cloning plasmids on a large scale. Because prokaryotic host cells may lack post-translational modifications necessary for antibody assembly and structure, a preferred embodiment is vector transformation in eukaryotic host cells, such as yeast (Saccharomyce cerevisiae), insect cells, plant cells, mammalian cells, e.g., SP2 / 0, CHO (Chinese Hamster Ovary) K1, CHODG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RN, and MDCK cell lines.

[0132] 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. The polynucleotide and the recombinant vector containing the polynucleotide can be inserted into the host cell using an insertion method well known in the relevant art. For example, if the host cell is a prokaryotic cell, the transfer can be performed according to the CaCl2 method or the electroporation method, and the like, and if the host cell is a eukaryotic cell, the vector can be transferred into the host cell by various methods, such as microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transformation, and gene bombardment, but the transfer method is not limited thereto. The method for selecting the transformed host cell can be easily performed according to a method well known in the relevant art using the phenotype expressed by the selected marker. For example, if the selected marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

[0133] Pharmaceutical Compositions The polypeptides (bsAbs and mAbs) described herein can be formulated into pharmaceutical compositions further comprising a pharma- ceutically acceptable carrier, diluent, adjuvant or vehicle. In one embodiment, the present invention relates to a pharmaceutical composition comprising the disclosed polypeptide and a pharma- ceutically acceptable carrier, diluent, adjuvant or vehicle. In one embodiment, the present invention is a pharmaceutical composition comprising an effective amount of the polypeptide of the present invention or a pharma- ceutically acceptable salt thereof and a pharma- ceutically acceptable carrier, diluent, adjuvant or vehicle. Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients or carriers that are appropriately selected with respect to the intended form of administration and consistent with conventional pharmaceutical practice.

[0134] Pharmaceutically acceptable carriers or excipients may contain inactive ingredients that do not excessively inhibit the biological activity of the polypeptide. Pharmaceutically acceptable carriers must be biocompatible, e.g., non-toxic, non-inflammatory, non-immunogenic, or have no other undesirable reactions or side effects when administered to a subject. Standard pharmaceutical formulation techniques can be used.

[0135] As used herein, pharma- ceutically acceptable carriers, adjuvants, or vehicles include any solvents, diluents, or other liquid vehicles, dispersing or suspending agents, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, suitable for a particular mode of administration. Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) discloses a variety of carriers used in formulating pharma- ceutical acceptable compositions and known techniques for their preparation. Except where a conventional carrier medium is incompatible with the polypeptides described herein (such as producing undesirable biological effects or interacting in a deleterious manner with other components of the pharma- ceutical acceptable composition), its use is considered to be within the scope of the present invention. As used herein, the phrase "side effects" includes undesirable side effects of treatment.

[0136] Substances that can function as pharma- ceutically acceptable carriers for antibodies are those that increase conformational stability, reduce protein dynamics, inhibit aggregation, and protect protein adsorption to the liquid-air interface, and include cyclodextrin hydrogels, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., Tween 80, phosphates, glycine, sorbic acid, potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, methylcellulose, hydroxypropyl methylcellulose, wool fat, sugars such as lactose, glucose, and sucrose, corn starch, and potato starch. Starches such as starch, cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, powdered tragacanth, malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; propylene glycol or polyethylene glycol, and the like; 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; ethyl alcohol, and phosphate buffers, as well as other non-toxic 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 can also be present in the composition, according to the judgment of the formulator.

[0137] In some embodiments, the composition of the present invention comprises a pharmaceutically acceptable salt.When the polypeptide of the present invention comprises a relatively acidic functional group, a base addition salt can be obtained by contacting a neutral form of such polypeptide with a sufficient amount of a desired base, either directly 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 polypeptide of the present invention comprises a relatively basic functional group, an acid addition salt can be obtained by contacting a neutral form of such polypeptide with a sufficient amount of a desired acid, either directly or in a suitable inert solvent. Examples of pharma- ceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphate, diphosphoric, sulfuric, monohydrogensulfate, hydroiodic, or phosphorous, as well as salts derived from relatively non-toxic organic acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, etc. Also included are salts of amino acids such as arginic acid, and salts of organic acids such as glucuronic and galacturonic acids (see, e.g., Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain polypeptides of the present disclosure contain both basic and acidic functionalities that allow the polypeptides to be converted into either base or acid addition salts.

[0138] Thus, the disclosed polypeptides may exist as salts with pharma- ceutically acceptable acids, etc. The present invention includes such salts. Non-limiting examples of such salts include hydrochloride, hydrobromide, phosphate, sulfate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, propionate, tartrate (e.g., (+)-tartrate, (-)-tartrate, or mixtures thereof, including racemic mixtures), succinate, benzoate, and salts with amino acids such as 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.

[0139] The neutral form of the polypeptide is 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.

[0140] Certain polypeptides of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, solvated forms are equivalent to unsolvated forms and are included within the scope of the present invention. Certain polypeptides of the present invention can exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses envisioned by the present invention and are intended to be within the scope of the present invention.

[0141] In some embodiments, the subcutaneous formulation may contain recombinant human PH20 hyaluronidase (rHuPH20) to facilitate dispersion of the antibody from the injection site.

[0142] Method of administration The compositions of the present invention can 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 present invention, such as a polypeptide or a pharma- ceutically acceptable salt thereof, to a subject in need of cancer treatment.

[0143] As used herein, "intermittent administration" involves the administration of an agent for 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 an "off-period"), followed by a period during which the composition is again administered (which may be considered a "second administration period"). Generally, during the second administration phase, the dosage level of the agent will be consistent with that administered during the first administration period, but may be increased or decreased as medically necessary.

[0144] In some embodiments, the compositions of the present invention can be administered orally, by suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or by implantation of a sustained release device, such as a mini-osmotic pump, into a subject. Thus, administration can be by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palate, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular, and intravenous. Other delivery methods include, but are not limited to, the use of liposomal formulations, intravenous infusion, or via an implanted reservoir. Specifically, the compositions are administered orally, intraperitoneally, or intravenously.

[0145] In some embodiments, the composition (e.g., bispecific antibody composition) is administered by systemic intravenous (IV) or oral route for intestinal cancer, such as gastric or intestinal cancer (Tashima et al., 2021). Formulations for delivery can be optimized by routine conventional methods well known in the art. Liquid dosage forms for oral administration include, but are not limited to, pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, elixirs, and the like. In addition to the active polypeptide, the liquid dosage form may contain an inert diluent commonly used in the art, such as, for example, water or other solvents, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, 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.

[0146] In some embodiments, the composition (e.g., bispecific antibody composition) is administered by subcutaneous injection. Injectable bispecific antibody formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents dispersed in sterile water or other sterile injectable medium prior to use. In order to prolong the effect of the polypeptides described herein, it is often desirable to slow the absorption of the polypeptide from subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of crystalline or amorphous material with poor water solubility. In this case, the rate of absorption of the polypeptide will depend upon its rate of dissolution, which may depend upon the size and crystalline form of the crystals. Alternatively, to delay absorption of a parenterally administered polypeptide form, the polypeptide is dissolved or suspended in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the polypeptide in biodegradable polymers, such as polylactide-polyglycolide. Depending on the ratio of polypeptide to polymer and the nature of the particular polymer employed, 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 tissue.

[0147] Sterile injectable forms of the compositions described herein may be aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectables may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example as a solution 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 fixed oils are conventionally used as solvents or suspending media. For this purpose, any bland fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants, which are commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween, Span, and other emulsifiers or bioavailability enhancers, commonly used in the manufacture of pharma-ceutically acceptable dosage forms, may also be used for formulation purposes.

[0148] In some embodiments, the formulation may include agents such as excipients, buffers, isotonicity agents, preservatives, surfactants, preferably zinc. The formulation may also include excipients or agents for polypeptide stabilization, such as buffers, reducing agents, bulk proteins, or carbohydrates. Bulk proteins useful for formulating at least one polypeptide composition include albumin, protamine, and the like. Exemplary carbohydrates useful for formulating at least one polypeptide include sucrose, mannitol, lactose, trehalose, glucose, and the like. The bispecific antibody formulation may also include a surfactant that can reduce or prevent surface-induced aggregation of at least one polypeptide caused by atomizing the solution in forming an aerosol. A variety of conventional surfactants can be used, such as polyoxyethylene fatty acid esters and alcohols, polyoxyethylene sorbital fatty acid esters, and the like. The amount generally ranges from about 0.001% to 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 formulation of a polypeptide, such as an antibody protein, can also be included in the formulation.

[0149] Cell Treatment Methods In another aspect, the present disclosure provides a method of treating cells with a bispecific antibody of the present disclosure, for example for the purpose of receptor internalization or receptor degradation. As demonstrated herein, the bsAbs of the present disclosure can mediate internalization and degradation of the receptors to which they bind (e.g., NRP1, EGFR).

[0150] Thus, in one embodiment, the disclosure provides a method of internalizing one or more receptors by a cell, the method comprising contacting the cell with a bispecific antibody of the disclosure that binds to a receptor on the cell and internalizing the receptor. In one embodiment, the bsAb mediates internalization of the receptors NRP1 and EGFR.

[0151] In another embodiment, the disclosure provides a method of degrading one or more receptors by a cell, the method comprising contacting the cell with a bispecific antibody of the disclosure that binds to the receptor on the cell to degrade the receptor. In one embodiment, the bsAb mediates degradation of the receptors NRP1 and EGFR.

[0152] Tumor growth inhibition In another aspect, the present disclosure provides a method for inhibiting tumor cell proliferation using the bispecific antibody of the present disclosure. As demonstrated herein, the bsAb of the present disclosure exhibits tumor growth inhibition ability. Thus, the bsAb of the present disclosure can be used to inhibit tumor cell proliferation, such as in the treatment of cancer. In one aspect, a method for treating cancer is provided herein, comprising administering to a subject in need thereof an effective amount of the bsAb of the present disclosure, such that the proliferation of cancerous tumors is inhibited or reduced, and / or regression of cancerous tumors and / or prolongation of survival is achieved. In some embodiments, the bsAb described herein may be administered in combination with additional cytotoxic or therapeutic agents described herein.

[0153] Cancers whose proliferation can be inhibited using the bsAbs described herein include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. In one embodiment, the cancer is associated with abnormal EGFR expression and / or function (e.g., overexpression of EGFR, expression of mutant EGFR), non-limiting examples of which include lung cancer (e.g., non-small cell lung cancer), kidney cancer, breast cancer (e.g., invasive ductal carcinoma), glioblastoma, head and neck cancer, prostate cancer, and ovarian cancer. In one embodiment, the cancer is non-small cell lung cancer (NSCLC). In one embodiment, the cancer is selected from the group consisting of lung cancer (e.g., NSCLC), colon cancer, and head and neck cancer.

[0154] In another embodiment, the cancer is associated with abnormal NRP1 expression and / or function (e.g., overexpression of NRP1, expression of mutant NRP1), non-limiting examples of which include lung cancer, breast cancer, leukemia, malignant melanoma, glioma, osteosarcoma, gastric cancer, esophageal cancer and colon cancer.

[0155] In another embodiment, cancer is associated with the abnormal expression of both EGFR and NRP1 (e.g., overexpression of both EGFR and NRP1).Non-limiting examples of cancer associated with overexpression of both EGFR and NRP1 include pancreatic cancer, lung cancer, head and neck cancer, cervical cancer, kidney cancer, ovarian cancer and colon cancer.The gene expression profile analysis of the Cancer Genome Atlas database of patient samples shows that 75%, 70%, 65%, 65%, 60%, 55% and 40% of the patient tumor population of pancreatic cancer, lung cancer, head and neck cancer, cervical cancer, kidney cancer, ovarian cancer and colon cancer, respectively, overexpress both EGFR and NRP1.

[0156] In one embodiment, the bsAb of the present disclosure is used to treat metastatic squamous non-small cell lung cancer, optionally in combination with gemcitabine and / or cisplatin. In certain embodiments, the use of the bsAb is not indicated for the treatment of non-squamous non-small cell lung cancer.

[0157] In one embodiment, the bsAb of the present disclosure is used in the treatment of wild-type RAS metastatic colon cancer (defined as both KRAS and NRAS being wild-type by a test approved by the FDA for this use), for example as first-line therapy, optionally in combination with FOLFOX (folinic acid, fluorouracil and oxaliplatin) or FOLFIRI (folinic acid, fluorouracil and irinotecan). In one embodiment, the bsAb of the present disclosure is used in the treatment of wild-type RAS metastatic colon cancer (defined as both KRAS and NRAS being wild-type by a test approved by the FDA for this use), for example as monotherapy after disease progression following previous chemotherapy treatment with a chemotherapy including a fluoropyrimidine, oxaliplatin and / or irinotecan. In certain embodiments, the use of bsAb is not indicated for the treatment of patients with RAS-mutated metastatic colon cancer or patients with unknown RAS mutation status.

[0158] In one embodiment, the bsAb of the present disclosure is used to treat locally or locally advanced squamous cell carcinoma of the head and neck, optionally in combination with radiation therapy. In one embodiment, the bsAb of the present disclosure is used to treat locoregionally recurrent disease or metastatic squamous cell carcinoma of the head and neck, optionally in combination with platinum-based therapy with fluorouracil. In one embodiment, the bsAb of the present disclosure is used to treat recurrent or metastatic squamous cell carcinoma of the head and neck that progresses after platinum-based therapy.

[0159] Additional non-limiting examples of suitable cancers include basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer (e.g., estrogen receptor positive breast cancer, HER2 positive breast cancer; triple negative breast cancer); peritoneal cancer; cervical cancer; bile duct cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer (e.g., hepatocellular carcinoma; hepatocellular carcinoma). );intraepithelial neoplasia;kidney or renal cancer;laryngeal cancer;leukemia;lung cancer (e.g., small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma);lymphoma, including Hodgkin's lymphoma and non-Hodgkin's lymphoma;melanoma;myeloma;neuroblastoma;oral cancer (e.g., lips, tongue, mouth, and pharynx);ovarian cancer;pancreatic cancer;prostate cancer;retinoblastoma;rhabdomyosarcoma;rectal cancer;cancer of the respiratory system;salivary gland cancer;sarcoma;skin cancer;squamous cell carcinoma;teratocarcinoma;testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; and other carcinomas and sarcomas; and B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with nematoses, edema (such as that associated with brain tumors), tumors of primitive origin, and Meigs' syndrome.

[0160] Other cancers that can be treated using the bsAbs described herein include metastatic pancreatic cancer, metastatic adenocarcinoma of the pancreas, gastric cancer, fibrous carcinoma, glioma, malignant glioma, diffuse intrinsic pontine glioma, recurrent pediatric brain tumor renal cell carcinoma, clear cell metastatic renal cell carcinoma, metastatic castration-resistant prostate cancer, stage IV prostate cancer, metastatic melanoma, malignant melanoma, recurrent melanoma of the skin, melanoma brain metastases, malignant melanoma of the head and neck, squamous non-small cell lung cancer, metastatic breast cancer, follicular lymphoma, aggressive B-cell NHL, HL including diffuse large B-cell lymphoma (DLBCL), multiple myeloma, chronic myelogenous leukemia, adult acute myelogenous leukemia in remission, adult acute myelogenous leukemia with Inv(16)(p13.1q22), CBFB-MYH11, t(16:16)(p13.1 Adult acute myeloid leukemia with t(8:21)(d22:q22), CBFB-MYH11, Adult acute myeloid leukemia with t(8:21)(d22:q22), RUNX1-RUNX1T1, Adult acute myeloid leukemia with t(9:11)(p22:q23), MLLT3-MLL, Adult acute promyelocytic leukemia with tO15:17)(q22:q12), PML-RARA, Alzheimer's disease Killer-associated acute myeloid leukemia, Richter's syndrome, adult glioblastoma, adult gliosarcoma, recurrent glioblastoma, recurrent childhood rhabdomyosarcoma, recurrent Ewing's sarcoma / peripheral primitive neuroectodermal tumor, recurrent neuroblastoma, recurrent osteosarcoma, colorectal cancer, MSI-positive colorectal cancer, MSI-negative colorectal cancer, nasopharyngeal nonkeratinizing carcinoma, recurrent nasopharyngeal undifferentiated carcinoma, cervical adenocarcinoma, cervical adenosquamous carcinoma. Includes cervical squamous cell carcinoma, recurrent cervical cancer, anal canal squamous cell carcinoma, metastatic anal canal cancer, recurrent anal canal cancer, recurrent head and neck cancer, head and neck squamous cell carcinoma, head and neck squamous cell carcinoma (HNSCC), ovarian cancer, colorectal cancer, advanced gastrointestinal cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, bone tumor, soft tissue sarcoma, osteosarcoma, thymic carcinoma, urothelial carcinoma, Merkel cell carcinoma, recurrent Merkel cell carcinoma, mycosis fungoides, Sézary syndrome, neuroendocrine carcinoma, nasopharyngeal carcinoma, basal cell skin carcinoma, squamous cell skin carcinoma, dermatofibrosarcoma, glioma, mesothelioma, myelodysplastic syndrome (MDS), myelofibrosis (MF), myeloproliferative neoplasms, and acute myeloid leukemia (AML).

[0161] The cancer can be, for example, a metastatic or primary cancer, a desmoplastic or non-desmoplastic cancer, or a recurrent cancer.

[0162] The cancer growth inhibition ability of the bsAbs described herein can be evaluated in suitable animal models or human xenograft models predicting efficacy in human tumors, as described in the Examples. In one embodiment, the xenograft model is the H1975 model, as described in the Examples. Alternatively, this property of the composition can be evaluated by examining the inhibitory ability of the compound using in vitro assays known to the skilled practitioner. A therapeutically effective amount of one or more treatments can reduce tumor size or alleviate the subject's symptoms. Those skilled in the art will be able to determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0163] In one embodiment, the cancer patient to be treated with the bsAb of the present disclosure is selected before treatment based on the expression of one or more biomarkers that indicate that bsAb treatment may be effective.For example, in the case of NRP1 x EGFR bispecific antibody, the cancer patient to be treated can be selected based on the overexpression of EGFR, NRP1, or both in the patient's tumor cells.The method of examining the surface expression of the biomarker of interest (e.g., the expression of NRP1 and / or EGFR) in the patient's tumor cells before bsAb treatment is well established in the art (e.g., FACS analysis by flow cytometry, etc.).

[0164] Combination therapy In some embodiments, in the methods or pharmaceutical compositions of the present invention, an effective amount of the disclosed composition (e.g., bispecific antibody) can be achieved by using the polypeptide or a pharma- ceutically acceptable salt or solvate (e.g., hydrate) alone or in combination with an additional appropriate chemotherapeutic agent, such as oxaliplatin, irinotecan, or FOLFOX. When "combination therapy" is employed, an effective amount can be achieved using a first amount of the polypeptide or a pharma- ceutically acceptable salt or solvate (e.g., hydrate) and a second amount of an additional appropriate therapeutic agent, such as a chemotherapeutic agent.

[0165] Anti-cancer agents suitable for use in combination therapy with the bsAbs described herein include surgery, chemotherapeutic agents, growth inhibitors, cytotoxic agents, radiation therapy and agents used in radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for the treatment of cancer, such as anti-HER-2 antibodies (e.g., HERCEPTIN®), anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (TARCEVA®)), platelet-derived growth factor inhibitors (e.g., Gleevec (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines; antagonists (interferons) that bind to and neutralize the activity of one or more of the following targets: and / or MEDI4736); CTLA4 (e.g., tremelimumab (PFIZER) and ipilimumab); LAG3 (e.g., BMS-986016); CD103; TIM-3 and / or other TIM family members; CEACAM1, CEACAM6, and / or other CEACAM family members; ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA or VEGF receptors, TRAIL / Apo2, PARP inhibitors (e.g., AZD-2281, Lynparza (Zejula) Niraparib), DNA damage repair inhibitors (e.g., ATMi, ATRi, DNAPKi), and other biologically active and organic chemicals (including those described in Section VII). Combinations thereof are also specifically contemplated for the methods described herein.

[0166] Suitable chemotherapeutic agents for use in combination therapy with the bsAbs described herein include alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; temozolomide; alkylsulfonates such as busulfan, improsulfan, piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylmelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); irinotecan (camptosar, CPT-11) (including combination therapy of irinotecan with 5-FU and leucovorin); the topoisomerase inhibitors RFS2000; bryostatin; kallistatin; CC-1065 (its adzelosin, calystatin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (especially the synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictine; spongistatins; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediyne antibiotics (e.g. calicheamicins, especially calicheamicin gamma 1I and calicheamicin omega 1I (e.g. Agnew, See Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemycins (including dynemycin A); bisphosphonates (such as clodronate); esperamicin;Neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolino doxorubicin, deoxy ... including sidoxorubicin), mitomycins such as epirubicin, esorubicin, idarubicin, marcelomycin, and mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, keramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; metabolic inhibitors such as methotrexate and 5-fluorouracil (5-FU); folates such as denopterin, methotrexate, pteropterin, and trimetrexate. purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calstarone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; aceglatone; and aldophosphamide glycosides. ;Aminolevulinic acid;Eniluracil;Amsacrine;Bestravcil;Bisantrene;Edatraxate;Defofamine;Demecolcine;Diazicon;Elformitin;Elliptinium acetate;Epothilone;Etoglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidynin;Maytansinoids such as maytansine and ansamitocin;Mitoguazone;Mitoxantrone;Mopidammol;Nitraerin;Pentostatin;Fenamet;Pirarubicin;Rosoxantrone;Podophyllic acid;2-Ethylhydrazide;Procarbazine;PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; schizofuran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® cremophor free, an albumin-processed paclitaxel nanoparticle formulation (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE® doxetaxel (Rhone- Poulenc Rorer, Antony, France); chlorambucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; platinum analogs such as cisplatin, oxaliplatin, carboplatin, vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE, vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; difluoromethylornithine (DM FO); retinoids such as retinoic acid; capecitabine; combretastatins; leucovorin (LV); oxaliplatin (including oxaliplatin treatment regimens (FOLFOX)), CEACAM5 atinib (TYKERB), inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g. erlotinib (TARCEVA®)) and VEGF-A that inhibit cell proliferation, and pharmacologic acceptable salts, acids or derivatives of any of the above;

[0167] In some embodiments, the additional therapeutic agent is an immune checkpoint inhibitor. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-LAG3 antibody or antigen-binding portion thereof, an anti-VISTA antibody or antigen-binding fragment thereof, an anti-BTLA antibody or antigen-binding fragment thereof, an anti-TIM3 antibody or antigen-binding fragment thereof, an anti-CTLA4 antibody or antigen-binding fragment thereof, an anti-HVEM antibody or antigen-binding fragment thereof, an anti-CD27 antibody or antigen-binding fragment thereof, an anti-CD137 antibody or antigen-binding fragment thereof, an anti-OX40 antibody or antigen-binding fragment thereof, an anti-CD28 antibody or antigen-binding fragment thereof, an anti-PDL1 antibody or antigen-binding fragment thereof, an anti-PDL2 antibody or antigen-binding fragment thereof, an anti-GITR antibody or antigen-binding fragment thereof, an anti-ICOS antibody or antigen-binding fragment thereof, an anti-SIRPα antibody or antigen-binding fragment thereof, an anti-ILT2 antibody or antigen-binding fragment thereof, an anti-ILT3 antibody or antigen-binding fragment thereof, an anti-ILT4 antibody or antigen-binding fragment thereof, an anti-ILT5 antibody or antigen-binding fragment thereof, or an anti-4-1BB antibody or antigen-binding fragment thereof. In some embodiments, the anti-PD1 antibody or antigen-binding fragment thereof is pembrolizumab or an antigen-binding fragment thereof.

[0168] Co-administration encompasses administering a first and second amount of a polypeptide and a chemotherapeutic agent essentially simultaneously, such as in a single pharmaceutical composition, e.g., a capsule having a fixed ratio of the first and second amounts, or in multiple separate capsules for each. Additionally, such co-administration also encompasses the sequential use of each polypeptide in any order.

[0169] When co-administration involves separate administration of a first amount of polypeptide and a second amount of additional therapeutic agent, the polypeptide is administered close enough in time to achieve desired therapeutic effect.For example, the time between each administration to achieve desired therapeutic effect can range from a few minutes to a few hours, and can be determined by considering the properties of each agent, such as efficacy, solubility, bioavailability, plasma half-life, and kinetic profile.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.

[0170] More specifically, a bispecific antibody 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 prior to), concomitantly with, or subsequent 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 prior to) administration of a second anti-cancer agent to a subject.

[0171] It is understood that the method of co-administration of a first amount of a bispecific antibody and a second amount of an additional therapeutic agent results in an enhanced or synergistic therapeutic effect, which combined effect is greater than the additive effect resulting from separate administration of the first amount of the polypeptide and the second amount of the additional therapeutic agent.

[0172] The synergistic effect of a combination of therapies (e.g., a combination of prophylactic or therapeutic agents) allows for the use of lower dosages of one or more treatments and / or less frequent administration of said treatments to a subject. The ability to use lower dosages of a therapy (e.g., a prophylactic or therapeutic agent) and / or administer said therapy less frequently can reduce the toxicity associated with administering said therapy to a subject without reducing the efficacy of said therapy in treating cancer. Furthermore, synergistic effects can improve the efficacy of drugs in preventing, managing or treating disease. Finally, synergistic effects of a combination of therapies (e.g., a combination of prophylactic or therapeutic agents) can avoid or reduce adverse or undesirable side effects that occur when either treatment is used alone.

[0173] When combination therapy using the bispecific antibodies of the invention is combined with another anti-cancer drug, both treatments can be administered, potentially with a long period between each administration (e.g., days, weeks, or months).

[0174] The presence of synergistic effect can be determined using suitable methods 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 additive equation (Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114: 313-326 (1926)), and the median effect equation (Chou, TC and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). When each of the above equations is applied to experimental data, a corresponding graph is generated that is useful for evaluating the effect of drug combination. The corresponding graphs associated with the above equations are the concentration-effect curve, the isobologram curve, and the combination index curve, respectively.

[0175] Exemplary embodiments The examples herein are not intended, and should not be used, to limit the invention, they are provided only to illustrate the invention. Example 1

[0176] Example 1. Production of bispecific antibodies The designed proteins were generated by codon-optimized gene synthesis and inserted into pcDNA3.4 as an expression vector using NotI and HindIII restriction enzymes. Exemplary amino acid sequences of bispecific antibody heavy chain polypeptides are shown in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 39, combined with the light chain polypeptide sequence shown in SEQ ID NO: 12. SEQ ID NO: 1 (encoded by SEQ ID NO: 14) shows a control heavy chain containing an NRP1-binding peptide. Exemplary polynucleotide sequences encoding the bispecific antibody heavy chain polypeptides are shown in SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 40 (encoding the heavy chain polypeptides of SEQ ID NOs: 2-11 and 39, respectively). A polynucleotide sequence encoding a light chain polynucleotide that binds to the bispecific antibody heavy chain is shown in SEQ ID NO: 25 (encoding the light chain polypeptide of SEQ ID NO: 12). The sequences shown in SEQ ID NOs: 26-38 disclose polynucleotide sequences used to encode the GGGGS peptide linker subunit (the amino acid sequence of which is shown in SEQ ID NO: 13). The constructed expression vector contains a signal peptide and may contain a Kozak sequence in the 5' untranslated region for optimized transcription.

[0177] To obtain the amount of plasmid construct for transfection, the plasmid construct was transformed into One Shot™ Top10 E. coli competent cells and grown overnight. The construct plasmid was obtained with the PureLink™ HiPure Expi Plasmid Megaprep Kit.

[0178] Fusion proteins were transiently expressed in the CHO-S system (Thermo Fisher Scientific Inc.). Proteins were expressed individually according to the manufacturer's instructions. Briefly, a total of 0.8 μg of plasmid DNA with a 1:1 ratio of light to heavy chains per mL of CHO-S culture was prepared using OPTIPRO® SFM and ExpiFectamine®. The mixture was incubated at a viable cell density of 6 × 10 6 cells / mL, viability ≥98%. Cell cultures were incubated overnight at 37°C, 80% humidity, and 8% CO2 in Nalgene® disposable PETG Erlenmeyer flasks with shaking at 125 RPM in a 19 mm orbit. The next day, cultures were enriched (ExpiCHO® Enhancer, Thermo Fisher Scientific Inc.) and fed (ExpiCHO® Feed, Thermo Fisher Scientific Inc.) and transferred to an environment of 32°C, 80% humidity, and 5% CO2 with shaking at 125 RPM in a 19 mm orbit. A second feeding was performed on day 5, and cultures were returned to 32°C until harvest on day 12. Harvesting was performed by centrifugation at 4000×g for 20 min. 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.

[0179] All antibody sterile supernatants were purified using MabSelect prismA® resin (GE Healthcare Life Sciences) on an AKTApure (GE Healthcare Life Sciences). The resin was equilibrated using 50 mM sodium phosphate, 150 mM NaCl, pH 7.0 buffer. The antibody supernatant was then loaded onto the column. The resin was washed with 50 mM sodium phosphate, 150 mM NaCl, pH 7.0 buffer until the chromatographic baseline returned to the column equilibrium level. Elution was then performed using 100 mM sodium acetate, 20% glycerol, pH 3.0, and fractions were collected. Fractions were immediately neutralized with 1 M Tris, pH 9. Fractions containing the main absorbance at a wavelength of 280 nm were pooled into an Amicon 10-kDa ultrafiltration device for buffer exchange. The elution buffer was removed by diluting 7-fold using storage buffer (phosphate-buffered saline) and centrifugation in an Amicon concentrator. The material was submitted to the SEC and then stored at 4°C.

[0180] Cation exchange chromatography was used for antibody purification. A cation exchange chromatography column (Capto S ImpAct) was sanitized with 1M NaOH and rinsed with MQ. Equilibration was performed with 50mM NaAc pH5.5 (start buffer) and 50mM NaAc pH5.5, 1M NaCl (elution buffer). Protein A purified antibodies were 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 over 25 column volumes. Each peak in the CEX purification was collected separately and concentrated by centrifugation at 4000xg using an Amicon® Ultra-15 Centrifugal Filter Unit, after which the buffer was exchanged into PBS.

[0181] Size exclusion chromatography (SEC) analysis was performed on an Agilent Infinity 1260 II Quatenary Pump high performance liquid chromatography (HPLC) system equipped with a diode array UV detector WR. 20 μg of antibody material was injected onto an aXBridge Protein BEH SEC column, 200 Å, 2.5 μm, 4.6 mm × 150 mm column. The mobile phase was 100 mM phosphate, 300 mM sodium chloride pH 7.0, 50 °C, and a flow rate of 0.3 mL / min. Antibody material was detected at wavelengths of 220, 280, and 330 nm with a sampling rate of 1 Hz during a 10 min acquisition. Example 2

[0182] Example 2. Binding affinity of bispecific antibodies to EGFR and NRP1 BLI binding studies using the Octet Red 96 system were performed to evaluate the binding of the bispecific antibodies to recombinant huEGFR and huNRP1. Briefly, commercially available biotinylated huEGFR and huNRP1 were immobilized on streptavidin (Sa) biosensors and the generated constructs were used for binding and characterization. BLI technology was used to characterize the binding to the constructs in terms of kinetics and binding affinity (equilibrium binding constant, K D ) and in a bivalent format. These studies were performed to determine whether tumor-associated essential receptor-targeting antibodies (TARE-TABs) bind to cancer targets (EGFR and NRP1) and to evaluate affinity for the targets. Constructs and K binding to huEGFR and huNRP1 were D Determination was performed using the Octet Red 96 system.

[0183] huEGFR and huNRP1 (in their respective immobilization columns) were immobilized on the Sa biosensor at a concentration of 0.2 μg / ml (loading signal range 0.2-0.4 nm) in 2X Kinetic buffer with a loading time of 180 s each. Post-baseline loading was performed for 60 s. Binding of constructs started at 100 nM (10 nM for high affinity binding), followed by two 1:1 serial dilutions, then buffer only (blank) in the fourth well. Dissociation of constructs was performed in baseline wells. Reference sensors were generated by applying constructs to blank AMC biosensor or streptavidin biosensor surfaces. Binding and dissociation steps were each 600 s. Data were analyzed using Octet analysis software to determine the dissociation constant K D (M) Reported 1:1 and 2:1 model fits were applied.

[0184] All constructs including SEQ ID NO:1 (control) and SEQ ID NO:11 bind to EGFR with double-digit picomolar affinity (see Table 1). For NRP-1 binding, the bsAb with the heavy chain of SEQ ID NO:7 showed subnanomolar affinity, whereas SEQ ID NO:1 binds to NRP-1 with double-digit nanomolar affinity. Figures 1A-1K show the binding affinity curves of the bispecific antibodies to immobilized huEGFR. Figures 2A-2K show the binding affinity curves of the bispecific antibodies to immobilized huNRP1. [Table 1] Example 3

[0185] Example 3. Inhibition of VEGFR2 signaling in HUVEC cells Bispecific antibody inhibition of VEGFR2 signaling in HUVEC cells was tested by probing electrophoresed cell lysates with anti-phospho-VEGFR2 on Western blot. HUVEC cells were cultured overnight in 6-well plates in EBM-2 medium supplemented with EGM-2 SingleQuots. The next day, cells were cultured for 4 h in 2 ml of F-12K medium containing 0.1 mg / ml heparin, endothelial cell growth supplement, and 10% FBS. To confirm dose-dependent responses to antibodies, cells were treated with 6-fold serial dilutions of bsAb from a highest concentration of 1 μM to a lowest concentration of 0.005 μM in serum-free medium for 30 min, followed by treatment with 2.2 ng / ml VEGF165 or control for 10 min. Cell lysates were prepared by collecting cells with a cell scraper and incubating with 100 μl of NP-40 lysis buffer containing protease / phosphatase inhibitors for 20 min on ice. The supernatant lysates were collected in new tubes after centrifugation at 13,000 rpm for 10 min, protein quantification was performed using the BCA protein assay, and then mixed with NuPAGE LDS sample buffer and LDS sample reducing buffer, followed by denaturation at 70 °C for 10 min.

[0186] Cell lysates equivalent to 10 μg were loaded with 4 μl of PageRuler Plus prestained protein ladder onto a 4–12% Bis-Tris Gel and run at 200 V for 45 min. 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 h at room temperature and incubated with anti-phosphorylated VEGFR2 (Y1175) in 5% milk in 1x TBST overnight at 4 °C. The next day, the membrane was washed 3 times for 10 min with TBST and incubated with secondary antibody in 5% milk for 1 h at room temperature. The membrane was washed 3 times for 10 min with 1x TBST. The membrane was then incubated with SuperSignal Femto Chemiluminescent substrate for 1–2 min and imaged on an Amersham ImageQuant 800. The blots were then stripped with Restore Plus Western Blot stripping buffer for 15 min at room temperature on a rocker, followed by 3 washes with 1xTBST, and the procedure was repeated from milk blocking to anti-VEGFR2 primary antibody incubation.

[0187] The intensities of phosphorylated VEGFR2 (Y1175) and VEGFR2 western blot bands were analyzed using ImageJ software. The resulting phosphorylated VEGFR2 (Y1175) band densities were normalized to VEGFR2, and the inhibition of VEGFR2 phosphorylation by bsAb was calculated as a ratio to VEGF165 control cell lysate (positive control). The IC50 of VEGFR2 phosphorylation by bsAb was measured using Prism9 software. The western blot in Figure 3 shows that incubation of HUVEC cells with bispecific antibody of SEQ ID NO:1 did not inhibit VEGF165-mediated VEGFR phosphorylation. Figures 4-10 show the western blot and IC50 plots of VEGFR2 phosphorylation inhibition after incubation with bispecific antibodies. The IC50 of the bispecific antibodies is shown in Table 2. [Table 2] Example 4

[0188] Example 4: Effect of bispecific antibodies on cell viability Inhibition of cell viability by bispecific antibodies was tested in the H1975 lung cancer cell line. Briefly, H1975 lung cancer cells were cultured in RPMI1640 supplemented with 1% penicillin / streptomycin and 10% FBS. Cells were trypsinized and seeded (3,000 cells / 120 μl / well) in round-bottom 96-well 3D culture plates and cultured for 3–4 h until they aggregated and formed 3D spheroids. Stock master plates were prepared with 6-fold serial dilutions of bsAbs from 5 μM to 0.6 pM. H1975 cells were treated with 30 ul of bsAb and cultured for 72 h (in duplicate). CellTiter-Glo 3D reagent and 25 μl of serum-free medium were then added to each well, wrapped in foil, and incubated on a rocker for 7 min. Plates were read on a Varioskan Lux Multimode Plate Reader and analyzed with Prism9 software.

[0189] Table 3 shows the IC50 of the bispecific antibodies. The bispecific antibody with heavy chain SEQ ID NO: 1 (control) shows a higher IC50 (1.96 μM) as shown in Figure 11A compared to the other bispecific antibodies (Figure 11B). The IC50 of cell growth inhibition is much lower for the bispecific antibody with the NRP1 short chain variable fragment (scFv) binding domain, resulting in improved cell growth inhibition. [Table 3] Example 5

[0190] Example 5: Tumor growth inhibition in H1975 xenograft mouse model cell line The tumor growth inhibitory ability of bsAbs was tested in 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.

[0191] Xenograft model Experiments and procedures using mice were performed in accordance with the United States Department of Agriculture, Department of Health and Human Services, and NIH policies for the humane care and use of laboratory animals. Female athymic (nu / nu) mice, 6-8 weeks of age, from Charles River Laboratories (Wilmington, MA, USA) were housed in a pathogen-free environment and provided laboratory chow and water ad libitum. All cell lines were screened for infectious agents (e.g., mycoplasma) prior to tumor cell line implantation. Xenografts were performed at 1x10 cells per mouse. 7 Tumors were established by subcutaneous injection of 100 μl of NCI-H1975 cells mixed 1:1 with Matrigel (Corning, Corning, NY, USA) into the right flank. In the efficacy study, tumors were 150–300 mm before randomization and treatment initiation. 3 (7-10 mice per group).

[0192] Treatment and Tumor Measurements Antibodies were diluted in sterile phosphate-buffered saline (Corning, Corning, NY, USA) and injected intraperitoneally in a total volume of 100 μl per mouse at the indicated dose on the day of randomization and twice weekly thereafter.

[0193] Digital caliper measurements were used to measure tumor size, and tumor volume was calculated as V = (W 2Calculations were performed using the formula: W = tumor width, L = tumor length.

[0194] statistical analysis GraphPad Prism 7 software (La Jolla, CA, USA) was used for statistical analysis. Results are presented as means. Data between control and experimental groups at individual time points or end points were compared using Student's t test. Type II analysis of variance was used for growth curve analysis, including longitudinal data with repeated measures. Statistical differences of p<0.05 were considered significant. TGI% was calculated as TGI(%)=(V c1 -V t1 ) / (V c0 -V t0 )X100, where V c1 and V t1 is the mean tumor volume of the control and treated groups at the end of the study, and V c0 and V t0 is the mean tumor volume of the control and treatment groups at the start of the experiment.

[0195] Table 4 shows tumor growth inhibition of bsAbs in xenograft mouse models and percent tumor volume of treated vs. control. The curves in Figures 12A-12F show the average tumor volume several days after implantation in mice treated with bsAbs. bsAbs with scFv NRP1 binding domains showed the highest tumor growth inhibition, e.g., treatment with bsAb with heavy chain SEQ ID NO:11 inhibited tumor growth by 100%. [Table 4] Example 6

[0196] Example 6: Functional activities of additional bispecific antibodies An additional bispecific antibody was prepared that is composed of a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:39 and a light chain comprising the amino acid sequence of SEQ ID NO:12. This bispecific antibody is referred to herein as Construct 12. The binding kinetics of Construct 12 for binding to NRP1 and EGFR are shown in Figures 13A and 13B, respectively. Construct 12 has a K D is 24.5 nM, and the K D is less than 0.1 nM.

[0197] The anti-tumor activity of construct 12 was tested in the H1975 xenograft mouse model (described in Example 5) in comparison to construct 11 and an isotype control. Mice were treated with bsAb (15 mg / kg, i.p., QW) or isotype control (10.97 mg / kg, i.p., QW) and the mean tumor volume was measured over time. The results are shown in Figure 14 and demonstrate that construct 12 is as effective as construct 11 in suppressing tumor growth in the xenograft model. Example 7

[0198] Example 7: Receptor internalization and degradation activity of construct 11 In this example, the ability of construct 11bsAb to internalize NRP1 or EGFR and mediate receptor degradation was tested in comparison with anti-NRP1 or anti-EGFR alone or the combination of the two mAbs.

[0199] For receptor internalization studies, H1975 or H1975-NRP1OE cells (overexpressing NRP1) were treated with construct 11, anti-NRP1 mAb alone, anti-EGFR mAb alone, or a combination of anti-NRP1 + anti-EGFR. For each sample, 200,000 cells were treated with 150 nM bs Ab or mAb. Cell surface levels of NRP1 or EGFR (% of control) were measured 5 min, 1 h, 5 h, and 24 h after treatment. Results for NRP1 expression are shown in Figure 16A and Figure 16B. Results for EGFR expression are shown in Figure 16C and Figure 16D. Results for H1975 cells are shown in Figure 16A and Figure 16C. Results for H1975-NRP1OE cells are shown in Figure 16B and Figure 16D. For EGFR, the results showed that anti-NRP1 antibody alone did not internalize EGFR (as expected), whereas anti-EGFR mAb (alone or in combination with anti-NRP1) and construct 11 were able to fully internalize EGFR within 5 minutes of treatment. For NRP1, the results showed that anti-EGFR antibody alone did not internalize NRP1 (as expected), whereas NRP1 mAb alone or in combination with anti-EGFR mAb was able to partially internalize NRP1, but construct 11 was able to internalize NRP1 more effectively over time than anti-NRP1 mAb (alone or in combination with anti-EGFR).

[0200] For receptor degradation studies, cells were treated with construct 11, anti-NRP1 mAb alone, anti-EGFR mAb alone, or the combination of anti-NRP1 + anti-EGFR, and the degradation of NRP1 and EGFR was measured. Briefly, H1975 cells (300,000 cells / sample) were cultured overnight in 6-well plates and then treated with bsAb or mAb (150 nM) and incubated for 6 hours. Cell lysates were prepared and analyzed by standard Western blotting to detect protein levels of EGFR, NRP1, and beta-actin (used as a loading control). The results are shown in Figure 17. The results showed that construct 11 was more effective at degrading both NRP1 and EGFR than either the single mAb treatment or the combination treatment of anti-EGFR + anti-NRP1. Example 8

[0201] Example 8: Generation of anti-NRP1 monoclonal antibodies The designed anti-NRP1 proteins were generated by codon-optimized gene synthesis and inserted into pcDNA3.4 as an expression vector using NotI and HindIII restriction enzymes. Exemplary monoclonal antibody heavy chain polypeptide amino acid sequences are shown in SEQ ID NOs: 41, 42, 43, 44, 45, 46 and 47, which are combined with the light chain polypeptide sequences shown in SEQ ID NOs: 48, 49, 50, 51, 52, 53 and 54, respectively.

[0202] Polynucleotide sequences encoding the heavy chain polypeptides of the monoclonal antibodies are set forth in SEQ ID NOs: 55, 56, 57, 58, 59, 60, and 61 (encoding SEQ ID NOs: 41-47, respectively). Polynucleotide sequences encoding the light chain polypeptides that bind to the heavy chains of the monoclonal antibodies are set forth in SEQ ID NOs: 62, 63, 64, 65, 66, 67, and 68 (encoding SEQ ID NOs: 48-54, respectively). The constructed expression vectors contain a signal peptide and can include a Kozak sequence in the 5' untranslated region to optimize transcription.

[0203] To obtain the amount of plasmid construct for transfection, the plasmid construct was transformed into One Shot™ Top10 E. coli competent cells and cultured overnight. The construct plasmid was obtained by PureLink™ HiPure Expi Plasmid Megaprep Kit.

[0204] Fusion proteins were transiently expressed in the CHO-S system (Thermo Fisher Scientific Inc.). Proteins were expressed individually according to the manufacturer's instructions. Briefly, a total of 0.8 μg of plasmid DNA with a 1:1 ratio of light to heavy chains per mL of CHO-S culture was prepared using OPTIPRO® SFM and ExpiFectamine®. The mixture was added to CHO-S cells at a cell density of 6 × 106 cells / mL and viability of ≥98%. Cell cultures were grown overnight at 37 °C, 80% humidity, and 8% CO2 in Nalgene® disposable PETG Erlenmeyer flasks with 19 mm orbital shaking at 125 RPM. The next day, cultures were enriched (ExpiCHO® Enhancer, Thermo Fisher Scientific Inc.) and fed (ExpiCHO® Feed, Thermo Fisher Scientific Inc.) and transferred to an environment of 32 °C, 80% humidity, and 5% CO2 with shaking at 125 RPM in a 19 mm orbit. A second feed was performed on day 5 and cultures were returned to 32 °C until harvest on day 12. Harvesting was performed by centrifugation at 4000 × g for 20 min. 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.

[0205] All antibody sterile supernatants were purified using MabSelect prismA® resin (GE Healthcare Life Sciences) on an AKTApure (GE Healthcare Life Sciences). The resin was equilibrated using 50 mM sodium phosphate, 150 mM NaCl, pH 7.0 buffer. The antibody supernatant was then loaded onto the column. The resin was washed with 50 mM sodium phosphate, 150 mM NaCl, pH 7.0 buffer until the chromatographic baseline returned to the column equilibrium level. Elution was then performed using 100 mM sodium acetate, 20% glycerol, pH 3.0, and fractions were collected. Fractions were immediately neutralized with 1 M Tris, pH 9. Fractions with predominant absorbance at a wavelength of 280 nm were pooled into an Amicon 10-kDa ultrafiltration device for buffer exchange. The elution buffer was removed by diluting in half and centrifuging seven times in an Amicon concentrator using storage buffer (phosphate buffered saline). The material was submitted to SEC and stored at 4 °C.

[0206] Cation exchange chromatography was used for antibody purification. A cation exchange chromatography column (Capto S ImpAct) was sanitized with 1M NaOH and washed with MQ. Equilibration was performed with 50mM NaAc pH5.5 (start 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 over 25 column volumes. Each peak of the CEX purification was collected separately and concentrated by centrifugation at 4000xg using Amicon® Ultra-15 centrifugal filter units and resuspended in PBS.

[0207] Size exclusion chromatography (SEC) analysis is performed on an Agilent Infinity 1260 II Quatenary Pump high performance liquid chromatography (HPLC) system equipped with a diode array UV detector WR. 20 μg of antibody material was injected onto an XBridge Protein BEH SEC column, 200 Å, 2.5 μm, 4.6 mm × 150 mm column. The mobile phase was 100 mM phosphate, 300 mM sodium chloride pH 7.0, 50 °C, and a flow rate of 0.3 mL / min. Antibody material was detected at wavelengths of 220, 280, and 330 nm with a sampling rate of 1 Hz during a 10 min acquisition.

[0208] The activity of anti-NRP1 monoclonal antibodies can be assessed by methods established in the art. For example, binding affinity, inhibition of VEGFR2 signaling, and / or effects on in vitro cell viability or in vivo tumor growth can be assessed as described in the Examples of U.S. Provisional Application No. 63 / 325,317, the entire contents of which are specifically incorporated herein by reference.

[0209] Sequence Listing Overview [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8]

Table 6-9

Table 6-10

Table 6-11

Table 6-12

Table 6-13

Table 6-14

Table 6-15

Table 6-16

Table 6-17

Table 6-18

Table 6-19

Table 6-20

Table 6-21

Table 6-22

Table 6-23

Table 6-24

Table 6-25

Claims

1. 1. A bispecific antibody comprising a first binding domain that binds to the human neuropilin-1 receptor (NRP1) and a second binding domain that binds to a target other than NRP1, wherein the first binding domain: (i) an antibody heavy chain variable (VH) domain comprising CDR1, CDR2 and CDR3 regions (HCDR1, HCDR2 and HCDR3, respectively), wherein HCDR1 consists of the sequence set forth in SEQ ID NO:79, HCDR2 consists of the sequence set forth in SEQ ID NO:80, and HCDR3 consists of the sequence set forth in any one of SEQ ID NOs:81-84; and (ii) an antibody light chain variable (VL) domain comprising CDR1, CDR2 and CDR3 regions (LCDR1, LCDR2 and LCDR3, respectively), wherein LCDR1 consists of the sequence set forth in any one of SEQ ID NOs: 85-87, LCDR2 consists of the sequence set forth in SEQ ID NO: 88, and LCDR3 consists of the sequence set forth in SEQ ID NO:

89. A bispecific antibody comprising:

2. (i) HCDR1 consists of the sequence set forth in SEQ ID NO:79, HCDR2 consists of the sequence set forth in SEQ ID NO:80, and HCDR3 consists of the sequence set forth in SEQ ID NO:84; and (ii) LCDR1 consists of the sequence shown in SEQ ID NO: 85, LCDR2 consists of the sequence shown in SEQ ID NO: 88, and LCDR3 consists of the sequence shown in SEQ ID NO: 89; The bispecific antibody of claim 1.

3. 1. A bispecific antibody comprising a first binding domain that binds to the human neuropilin-1 receptor (NRP1) and a second binding domain that binds to a target other than NRP1, wherein the first binding domain: (i) an antibody heavy chain variable (VH) domain comprising CDR1, CDR2 and CDR3 regions (HCDR1, HCDR2 and HCDR3, respectively), wherein HCDR1 comprises the sequence set forth in SEQ ID NO:79, HCDR2 comprises the sequence set forth in SEQ ID NO:80, and HCDR3 comprises the sequence set forth in any one of SEQ ID NOs:81-84; and (ii) an antibody light chain variable (VL) domain comprising CDR1, CDR2 and CDR3 regions (LCDR1, LCDR2 and LCDR3, respectively), wherein LCDR1 comprises the sequence set forth in any one of SEQ ID NOs: 85-87, LCDR2 comprises the sequence set forth in SEQ ID NO: 88, and LCDR3 comprises the sequence set forth in SEQ ID NO:

89. A bispecific antibody comprising:

4. The bispecific antibody of any one of claims 1 to 3, wherein the second binding domain targets EGFR.

5. 5. The bispecific antibody of claim 4, wherein the second binding domain is composed of an N-terminal EGFR-binding heavy chain variable domain (VH) and a heavy chain constant 1 domain (CH1), and the first binding domain is composed of a C-terminal NRP1-binding single chain variable fragment (scFv), wherein the N-terminal variable heavy chain (VH) and the C-terminal scFv binding domain are at opposite ends of the contiguous sequence.

6. 6. The bispecific antibody of claim 5, wherein the second binding domain further comprises a heavy chain constant 2 domain (CH2) and a heavy chain constant 3 domain (CH3).

7. 7. The bispecific antibody of claim 6, wherein the C-terminal NRP1-binding scFv comprises a light chain variable domain (VL) connected to a heavy chain variable domain (VH) by a flexible linker peptide.

8. 8. The bispecific antibody of claim 7, further comprising a corresponding EGFR-binding light chain variable domain (VL) and a light chain constant domain (CL), wherein the bispecific antibody is composed of two polypeptides.

9. The second binding domain that targets EGFR comprises: (i) an antibody heavy chain variable (VH) domain comprising CDR1, CDR2 and CDR3 regions (HCDR1, HCDR2 and HCDR3, respectively), wherein HCDR1 consists of the sequence set forth in SEQ ID NO:71, HCDR2 consists of the sequence set forth in SEQ ID NO:72, and HCDR3 consists of the sequence set forth in SEQ ID NO:73; and (ii) an antibody light chain variable (VL) domain comprising CDR1, CDR2 and CDR3 regions (LCDR1, LCDR2 and LCDR3, respectively), wherein LCDR1 consists of the sequence set forth in SEQ ID NO:74, LCDR2 consists of the sequence set forth in SEQ ID NO:75, and LCDR3 consists of the sequence set forth in SEQ ID NO:76; The bispecific antibody of claim 4, comprising:

10. 4. The bispecific antibody of claim 1 , wherein the first binding domain comprises a VH domain comprising the sequence set forth in SEQ ID NO: 77 and a VL domain comprising the sequence set forth in SEQ ID NO:

78.

11. 5. The bispecific antibody of claim 4, wherein the first binding domain comprises a VH domain comprising the sequence set forth in SEQ ID NO: 77 and a VL domain comprising the sequence set forth in SEQ ID NO: 78; and the second binding domain comprises a VH domain comprising the sequence set forth in SEQ ID NO: 69 and a VL domain comprising the sequence set forth in SEQ ID NO:

70.

12. 5. The bispecific antibody of claim 4, wherein the first binding domain and the second binding domain comprise a bispecific antibody heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 39.

13. 13. The bispecific antibody of claim 12, wherein the bispecific antibody heavy chain is paired with an antibody light chain comprising the amino acid sequence set forth in SEQ ID NO:

12.

14. 14. The bispecific antibody of claim 13, wherein the bispecific antibody heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 11 or 39, and the bispecific antibody light chain comprises the amino acid sequence set forth in SEQ ID NO:

12.

15. The binding affinity of the EGFR binding domain (K Ｄ 5. The bispecific antibody of claim 4, wherein the IgG antibody has a IgG binding affinity of less than 0.1 nM.

16. Binding affinity (K Ｄ 5. The bispecific antibody of claim 4, wherein the mAb concentration is in the range of 0.1 nM to 100 nM.

17. The EGFR-binding domain binds to NRP1 via the NRP1-binding domain. Ｄ a K for EGFR that is at least 2-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 100-fold, or more than 100-fold Ｄ The bispecific antibody of claim 4, comprising:

18. 1. A bispecific antibody comprising a first binding domain that binds to the human neuropilin-1 receptor (NRP1) and a second binding domain that binds to the human epidermal growth factor receptor (EGFR), wherein the first binding domain is: (i) an antibody heavy chain variable (VH) domain comprising CDR1, CDR2, and CDR3 regions; and (ii) an antibody light chain variable (VL) domain comprising CDR1, CDR2, and CDR3 regions; A bispecific antibody comprising:

19. A pharmaceutical composition comprising the bispecific antibody of claim 4.

20. A polynucleotide encoding the bispecific antibody of claim 4.

21. 21. The polynucleotide of claim 20, wherein the bispecific antibody comprises a heavy chain polypeptide encoded by a polynucleotide sequence set forth in any one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 40.

22. 22. The polynucleotide of claim 21, wherein the polynucleotide sequence is inserted into a vector for protein expression.

23. A method for internalizing the neuropilin-1 (NRP1) receptor by a cell, comprising contacting the cell in vitro with the bispecific antibody of claim 1 to cause internalization of NRP1 by the cell.

24. 10. A method for internalizing epidermal growth factor receptor (EGFR) by a cell, comprising contacting the cell in vitro with the bispecific antibody of claim 4 to cause EGFR to be internalized by the cell.

25. A pharmaceutical composition for treating cancer associated with aberrant NRP1 expression, comprising the bispecific antibody of claim 1.

26. A pharmaceutical composition for treating cancer associated with aberrant EGFR expression, comprising the bispecific antibody of claim 4.

27. 27. The pharmaceutical composition of claim 26, wherein the cancer is non-small cell lung cancer.

28. 27. The pharmaceutical composition of claim 26, wherein the pharmaceutical composition further comprises or is administered in combination with at least one additional chemotherapeutic agent for combination therapy.

29. 10. Use of the bispecific antibody of claim 1 in the manufacture of a medicament for use in treatment.

30. 5. Use of the bispecific antibody of claim 4 in the manufacture of a medicament for treating cancer associated with aberrant EGFR expression.

31. A bispecific antibody comprising a first binding domain that binds to the human neuropilin-1 receptor (NRP1) and a second binding domain that binds to the human epidermal growth factor receptor (EGFR), wherein the first binding domain comprises a single-chain fragment variable (scFv) antibody.

32. 32. The bispecific antibody of claim 31 , wherein the second binding domain comprises an N-terminal EGFR-binding heavy chain variable domain (VH) and a heavy chain constant 1 domain (CH1), and the first binding domain comprises a C-terminal NRP-1-binding single chain variable fragment (scFv), wherein the N-terminal VH and the C-terminal scFv are at opposite ends of the contiguous sequence.

33. 33. The bispecific antibody of claim 32, wherein the second binding domain further comprises a heavy chain constant 2 domain (CH2) and a heavy chain constant 3 domain (CH3).

34. 33. The bispecific antibody of claim 32, wherein the C-terminal NRP1-binding scFv comprises a light chain variable domain (VL) connected to a heavy chain variable domain (VH) by a flexible linker peptide.

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