Bispecific antibodies comprising NRP1 binding domains and methods of use thereof

Abstract

The present disclosure provides bispecific antibodies and compositions and methods of treating cancer that incorporate the bispecific antibodies. The bispecific antibody comprises a polypeptide comprising an EGFR binding domain and a neurociliin (NRP1) specific binding domain at the C-terminal end. A monoclonal antibody that specifically binds to NRP1 is also provided.

Description

Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 325,312 filed on March 30, 2022 and U.S. Provisional Application No. 63 / 325,317 filed on March 30, 2022, both of which are incorporated herein by reference in their entirety. Background Art

[0002] Therapeutic antibodies are used to treat solid tumors and blood cancers (such as leukemia and lymphoma). Antibody treatments for solid tumors and blood cancers show different response rates. For example, the response rate of antibody treatment in blood cancers reaches 30 to 51%, while the response rate of solid tumors is only 8 to 15%. This is because cancer cells in the blood are easily targeted by antibodies, while the efficacy of solid tumors depends on extravasation and tumor penetration.

[0003] Antibodies have reduced tumor penetration for a number of reasons, including: 1) the intrinsic nature of the antibody size of approximately 150 kDa hinders penetration across the endothelial barrier, 2) high tumor interstitial fluid pressure; and 3) abnormal microstructural / physiological properties of tumor tissue, including reduced perfusion and abnormal angiogenesis, low lymphangiogenesis, high cell density, and high extracellular density (Jain and Stylianopoulos, 2010). For these reasons, the amount of antibody accumulated in tumor tissue is very small (0.01% to 0.0001% of the injected dose per gram of tumor tissue), resulting in a low response rate (Thurber et al., 2008). Therefore, the development of antibody technology that enables antibodies to selectively accumulate in tumor tissue and have the ability to highly penetrate tumor tissue will reduce toxicity and improve therapeutic efficacy.

[0004] Efforts are being made to improve tumor penetration by engineering smaller antibodies, adjusting antigen binding specificity, and combining administered antibodies with molecules that promote 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 reduced sizes that overcome the endothelial barrier. However, antibody fragments do not have Fc fragments, and due to their small size, the kidneys quickly filter the fragments from the serum after in vivo administration, reducing the circulation half-life and therapeutic effect (Behr et al., 1998). Antibodies can be equipped with features that improve tumor penetration. Summary of the invention

[0005] The present disclosure provides a bispecific antibody 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, for example, an epidermal growth factor receptor (EGFR) binding domain. Therefore, in one aspect, 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 NRP1 binding domains from anti-NRP1 monoclonal antibodies that form bispecific antibodies with beneficial properties. For example, compared to anti-EGFR mAbs or anti-NRP1 mAbs alone or in combination, the NRP1×EGFR bispecific antibodies of the present disclosure exhibit enhanced ability to internalize and degrade NRP1 and EGFR receptors. The present disclosure provides EGFR×NRP1 bispecific antibodies with high affinity for EGFR to provide high homing ability to tumors expressing EGFR. The bispecific antibody has asymmetric binding affinity for EGFR and NRP1, and the affinity for EGFR is at least 10 times that for NRP1. The NRP1 targeting portion of the antibody contains an NRP1 binding domain that reduces the NRP1 occupancy time to reduce toxicity in healthy tissues. The combination of these features in bispecific antibodies significantly increases the potential for delivering high therapeutic efficacy for cancer treatment while reducing toxic side effects.

[0007] In one aspect, the present disclosure relates to a bispecific antibody comprising a first binding domain that binds to 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 shown in SEQ ID NO:79, HCDR2 consists of the sequence shown in SEQ ID NO:80, and HCDR3 consists of the sequence shown in any one of SEQ ID NOs:81-84; and ii. an antibody light chain variable (VL) domain comprising CDR1, CDR2 and CDR3 regions (LCDR1, LCDR2 and LCDR3, respectively), wherein LCDR1 consists of the sequence shown in any one of SEQ ID NOs:85-87, LCDR2 consists of the sequence shown in SEQ ID NO:88, and LCDR3 consists of the sequence shown in SEQ ID NO:89.

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

[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), wherein HCDR1 comprises the sequence shown in SEQ ID NO:79, HCDR2 comprises the sequence shown in SEQ ID NO:80, and HCDR3 comprises the sequence shown in any one of SEQ ID NOs:81-84; and (ii) an antibody light chain variable (VL) domain comprising CDR1, CDR2, and CDR3 regions (LCDR1, LCDR2, and LCDR3, respectively), wherein LCDR1 comprises the sequence shown in any one of SEQ ID NOs:85-87, LCDR2 comprises the sequence shown in SEQ ID NO:88, and LCDR3 comprises the sequence shown in SEQ ID NO:89.

[0010] In certain embodiments, the second binding domain targets EGFR. For example, in one embodiment, 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 NRP1-binding single-chain variable fragment (scFv), wherein the N-terminal variable heavy chain (VH) and the C-terminal scFv binding domain are located at opposite ends of the continuous 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), wherein the bispecific antibody comprises 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), wherein HCDR1 consists of the sequence shown in SEQ ID NO:71, HCDR2 consists of the sequence shown in SEQ ID NO:72, and HCDR3 consists of the sequence shown 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 shown in SEQ ID NO:74, LCDR2 consists of the sequence shown in SEQ ID NO:75, and LCDR3 consists of the sequence shown in SEQ ID NO:76.

[0012] In certain embodiments, the bispecific antibody comprises a first binding domain (ie, an NRP1 binding domain) comprising a VH domain comprising the sequence shown in SEQ ID NO:77 and a VL domain comprising the sequence shown 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 shown in SEQ ID NO:77 and a VL domain comprising the sequence shown in SEQ ID NO:78; and a second binding domain (i.e., an EGFR binding domain) comprising a VH domain comprising the sequence shown in SEQ ID NO:69 and a VL domain comprising the sequence shown 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 shown in SEQ ID NO: 12. In certain embodiments, the bispecific antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 11 or 39, and the bispecific antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 12.

[0015] In certain embodiments of the bispecific antibodies, the binding affinity (K) of the EGFR binding domain D ) is <0.1 nM. In certain embodiments, the binding affinity (KD ) ranges from 0.1 nM to 100 nM. In certain embodiments, the EGFR binding domain comprises a K of the NRP1 binding domain to NRP1. D The K of the EGFR of 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 is greater than that of the EGFR of D .

[0016] In another aspect, the present disclosure relates to pharmaceutical compositions comprising the bispecific antibodies of the present disclosure.

[0017] In another aspect, the present disclosure relates to polynucleotides encoding the bispecific antibodies of the present disclosure. In one embodiment, the bispecific antibody comprises a heavy chain polypeptide encoded by a polynucleotide sequence shown 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 present disclosure relates to a method for internalizing a neuropilin-1 (NRP1) receptor by a cell, the method comprising contacting a cell with a bispecific antibody of the present disclosure, such that NRP1 is internalized by the cell. In another embodiment, the present disclosure provides a method for internalizing an epidermal growth factor receptor (EGFR) by a cell, the method comprising contacting a cell with an NRP1×EGFR bispecific antibody of the present disclosure, such that EGFR is internalized by the cell.

[0019] On the other hand, the present disclosure relates to a method for treating a cancer associated with abnormal NRP1 expression in a subject, the method 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 a cancer associated with abnormal EGFR expression in a subject, the method comprising administering to the subject an NRP1×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 present disclosure relates to the use of the bispecific antibodies of the present disclosure in the manufacture of a medicament for therapy, e.g., for the treatment of cancer described herein. In one embodiment, the present disclosure relates to the use of the NRP1×EGFR bispecific antibodies of the present disclosure in the manufacture of a medicament for treating cancer associated with aberrant EGFR expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following drawings are provided by way of illustration and are not intended to limit the scope of the present invention.

[0022] Figure 1 A Binding of bsAb with heavy chain polypeptide SEQ ID NO: 1 to recombinant human EGFR measured by Octet Red 96 sensorgram depicts the dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0023] Figure 1 B Binding of bsAb with heavy chain polypeptide SEQ ID NO: 2 to recombinant human EGFR measured by Octet Red 96 sensorgram depicts the dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0024] Figure 1 C Binding of bsAb with heavy chain polypeptide SEQ ID NO: 3 to recombinant human EGFR measured by Octet Red 96 sensorgram depicts the dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0025] Figure 1 D Binding of bsAb with heavy chain polypeptide SEQ ID NO: 4 to recombinant human EGFR measured by Octet Red 96 sensorgram depicts the dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0026] Figure 1 E Binding of bsAb with heavy chain polypeptide SEQ ID NO: 5 to recombinant human EGFR measured by Octet Red 96 sensorgram depicts the dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0027] Figure 1 F Binding of bsAb with heavy chain polypeptide SEQ ID NO: 6 to recombinant human EGFR measured by Octet Red 96 sensorgram depicts dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0028] Figure 1G Binding of bsAb with heavy chain polypeptide SEQ ID NO:7 to recombinant human EGFR measured by Octet Red 96 sensorgram depicts the dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0029] Figure 1 H Binding of bsAb with heavy chain polypeptide SEQ ID NO: 8 to recombinant human EGFR measured by Octet Red 96 sensorgram depicts dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0030] Figure 1 1 Binding of bsAb with heavy chain polypeptide SEQ ID NO: 9 to recombinant human EGFR measured by Octet Red 96 sensorgram depicts the dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0031] Figure 1 J Binding of bsAb with heavy chain polypeptide SEQ ID NO: 10 to recombinant human EGFR measured by Octet Red 96 sensorgram depicts the dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0032] Figure 1 K Binding of bsAb with heavy chain polypeptide SEQ ID NO: 11 to recombinant human EGFR measured by Octet Red 96 sensorgram depicts the dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0033] Figure 2 A Binding of bsAb with heavy chain polypeptide SEQ ID NO: 1 to recombinant NRP1 measured by Octet Red 96 sensorgram, depicting dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0034] Figure 2B Binding of bsAb with heavy chain polypeptide SEQ ID NO: 2 to recombinant NRP1 measured by Octet Red 96 sensorgram, depicting dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0035] Figure 2 C Binding of bsAb with heavy chain polypeptide SEQ ID NO:3 to recombinant NRP1 measured by Octet Red 96 sensorgram, which depicts dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0036] Figure 2 D Binding of bsAb with heavy chain polypeptide SEQ ID NO: 4 to recombinant NRP1 measured by Octet Red 96 sensorgram, depicting dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0037] Figure 2 E Binding of bsAb with heavy chain polypeptide SEQ ID NO: 5 to recombinant NRP1 measured by Octet Red 96 sensorgram, depicting dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0038] Figure 2 F Binding of bsAb with heavy chain polypeptide SEQ ID NO: 6 to recombinant NRP1 measured by Octet Red 96 sensorgram, depicting dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0039] Figure 2 G Binding of bsAb with heavy chain polypeptide SEQ ID NO:7 to recombinant NRP1 measured by Octet Red 96 sensorgram, which depicts dose-dependent binding of T tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0040] Figure 2H Binding of bsAb with heavy chain polypeptide SEQ ID NO: 8 to recombinant NRP1 measured by Octet Red 96 sensorgram, depicting dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0041] Figure 2 1 Binding of bsAb with heavy chain polypeptide SEQ ID NO: 9 to recombinant NRP1 measured by Octet Red 96 sensorgram, depicting dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0042] Figure 2 J Binding of bsAb with heavy chain polypeptide SEQ ID NO: 10 to recombinant NRP1 measured by Octet Red 96 sensorgram, depicting dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0043] Figure 2 K Binding of bsAb with heavy chain polypeptide SEQ ID NO: 11 to recombinant NRP1 measured by Octet Red 96 sensorgram, which depicts dose-dependent binding of tumor-associated receptor targeting antibodies. Each line on the graph represents the binding kinetics at a specific bsAb concentration (10, 5, and 2.5 nM from left to right).

[0044] Figure 3 Western blot showing that incubation of HUVEC cells with bsAb having heavy chain polypeptide SEQ ID NO: 1 failed to prevent VEGF-mediated activation of VEGFR2 signaling by detecting phosphorylated VEGFR2 using anti-phospho-VEGFR2 (Y1175).

[0045] Figure 4AWestern blot density shows that bsAb with heavy chain polypeptide SEQ ID NO:2 inhibits the dose response of VEGFR2 signaling. HUVEC cells were pre-incubated with bsAb with heavy chain polypeptide SEQ ID NO:2 (6-fold serial dilution concentration starting from 1 μM) for 30 minutes and treated with VEGF165 (2.2 ng / ml) for another 10 minutes to induce VEGFR2 activation. Using anti-phosphorylated VEGFR2 (Y1175), VEGFR2 signaling activation was detected by Western blot of lysate. The inhibitory effect of bsAb with heavy chain polypeptide SEQ ID NO:2 was calculated by comparing the band intensity with VEGF165 control lysate using ImageJ software densitometry analysis (digital display below P-VEGFR2 (Y1175) blot).

[0046] Figure 4B The IC50 of bsAb with heavy chain polypeptide SEQ ID NO: 2 (n=2) in VEGFR2 phosphorylation was calculated using Prism9.

[0047] Figure 5A Western blot density shows that bsAb with heavy chain polypeptide SEQ ID NO:3 inhibits the dose response of VEGFR2 signaling. HUVEC cells were pre-incubated with bsAb with heavy chain polypeptide SEQ ID NO:3 (6-fold serial dilution concentration starting from 1 μM) for 30 minutes and treated with VEGF165 (2.2 ng / ml) for another 10 minutes to induce VEGFR2 activation. Using anti-phosphorylated VEGFR2 (Y1175), VEGFR2 signaling activation was detected by Western blot of lysate. The inhibitory effect of bsAb with heavy chain polypeptide SEQ ID NO:3 was calculated by comparing the band intensity with VEGF165 control lysate using ImageJ software densitometry analysis (digital display below P-VEGFR2 (Y1175) blot).

[0048] Figure 5B IC50 graph for inhibition of VEGFR2 phosphorylation by bsAbs (n=2) with heavy chain polypeptide SEQ ID NO: 3. IC50 was calculated using Prism9.

[0049] Fig. 6AWestern blot density shows the dose response of VEGFR2 signaling inhibition mediated 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 starting from 1 μM) for 30 minutes and treated with VEGF165 (2.2 ng / ml) for another 10 minutes to induce VEGFR2 activation. Using anti-phosphorylated VEGFR2 (Y1175), VEGFR2 signaling activation was detected by Western blot of lysate. The inhibitory effect of bsAb with heavy chain polypeptide SEQ ID NO:4 was calculated by comparing the band intensity with VEGF165 control lysate using Image J software densitometry analysis (digital display below P-VEGFR2 (Y1175) blot).

[0050] Figure 6B IC50 graph for inhibition of VEGFR2 phosphorylation by bsAb (n=2) with heavy chain polypeptide SEQ ID NO: 4. IC50 was calculated using Prism9.

[0051] Fig. 7A Western blot density shows that bsAb with heavy chain polypeptide SEQ ID NO:5 inhibits the dose response of VEGFR2 signal transduction. HUVEC cells were pre-incubated with bsAb with heavy chain polypeptide SEQ ID NO:5 (6-fold serial dilution concentration starting from 1 μM) for 30 minutes and treated with VEGF165 (2.2 ng / ml) for another 10 minutes to induce VEGFR2 activation. Using anti-phosphorylated VEGFR2 (Y1175), VEGFR2 signal transduction activation was detected by Western blot of lysate. The inhibitory effect of bsAb with heavy chain polypeptide SEQ ID NO:5 was calculated by comparing the band intensity with VEGF165 control lysate using ImageJ software densitometry analysis (digital display below P-VEGFR2 (Y1175) blot).

[0052] Figure 7B IC50 graph of bsAb with heavy chain polypeptide SEQ ID NO: 5 (n=1) in inhibition of VEGFR2 phosphorylation. IC50 was calculated using Prism9.

[0053] Fig. 8AWestern blot density shows that bsAb with heavy chain polypeptide SEQ ID NO:6 inhibits the dose response of VEGFR2 signal transduction. HUVEC cells were pre-incubated with bsAb with heavy chain polypeptide SEQ ID NO:6 (6-fold serial dilution concentration starting from 1 μM) for 30 minutes and treated with VEGF165 (2.2 ng / ml) for another 10 minutes to induce VEGFR2 activation. Using anti-phosphorylated VEGFR2 (Y1175), VEGFR2 signal transduction activation was detected by Western blot of lysate. The inhibitory effect of bsAb with heavy chain polypeptide SEQ ID NO:6 was calculated by comparing the band intensity with VEGF165 control lysate using ImageJ software densitometry analysis (digital display below P-VEGFR2 (Y1175) blot).

[0054] Figure 8B IC50 graph of bsAb with heavy chain polypeptide SEQ ID NO: 6 (n=2) in inhibition of VEGFR2 phosphorylation. IC50 was calculated using Prism9.

[0055] Fig.9A Western blot density shows that bsAb with heavy chain polypeptide SEQ ID NO:7 inhibits the dose response of VEGFR2 signaling. HUVEC cells were pre-incubated with bsAb with heavy chain polypeptide SEQ ID NO:7 (6-fold serial dilution concentration starting from 1 μM) for 30 minutes and treated with VEGF165 (2.2 ng / ml) for another 10 minutes to induce VEGFR2 activation. Using anti-phosphorylated VEGFR2 (Y1175), VEGFR2 signaling activation was detected by Western blot of lysate. The inhibitory effect of bsAb with heavy chain polypeptide SEQ ID NO:7 was calculated by comparing the band intensity with VEGF165 control lysate using ImageJ software densitometry analysis (digital display below P-VEGFR2 (Y1175) blot).

[0056] Fig. 9B IC50 graph of bsAb with heavy chain polypeptide SEQ ID NO: 7 (n=2) in VEGFR2 phosphorylation inhibition. IC50 was calculated using Prism9.

[0057] Fig. 10AProtein blot density shows that bsAb with heavy chain polypeptide SEQ ID NO:11 inhibits the dose response of VEGFR2 signaling. HUVEC cells were pre-incubated with bsAb with heavy chain polypeptide SEQ ID NO:11 (6-fold serial dilution concentration starting from 1 μM) for 30 minutes and treated with VEGF165 (2.2 ng / ml) for another 10 minutes to induce VEGFR2 activation. Using anti-phosphorylated VEGFR2 (Y1175), VEGFR2 signaling activation was detected by protein blot of lysate. The inhibitory effect of bsAb with heavy chain polypeptide SEQ ID NO:11 was calculated by comparing the band intensity with VEGF165 control lysate using Image J software densitometry analysis (digital display below P-VEGFR2 (Y1175) blot).

[0058] Fig. 10B IC50 graph of bsAb with heavy chain polypeptide SEQ ID NO: 11 (n=2) in VEGFR2 phosphorylation inhibition. IC50 was calculated using Prism9.

[0059] Fig.11A 1 Percent growth rate of H1975 cells in the presence of bsAb with heavy chain polypeptide SEQ ID NO: 1 relative to control.

[0060] Fig. 11B Percent growth rate of H1975 cells in the presence of bsAbs having heavy chain polypeptides of SEQ ID NOs: 3, 7, 9, 10, and 11 relative to control.

[0061] Fig. 12A Mean tumor growth inhibition in H1975 xenograft mouse model treated with PBS (control) and bsAb with heavy chain polypeptide SEQ ID NO: 1 (10 mg / kg, ip, BIW).

[0062] Fig. 12B Mean tumor growth inhibition in the H1975 xenograft mouse model treated with PBS (control), panitumumab (5 mg / kg, ip, BIW), or bsAb having the heavy chain polypeptide SEQ ID NO: 1 (5 mg / kg, ip, BIW).

[0063] Fig. 12C Mean tumor growth inhibition in H1975 xenograft mouse model treated with PBS (control), panitumumab (5 mg / kg, ip, BIW), or bsAb having heavy chain polypeptide SEQ ID NO: 2 (6.825 mg / kg, ip, BIW).

[0064] Fig.12DMean tumor growth inhibition in the H1975 xenograft mouse model treated with PBS (control), panitumumab (5 mg / kg, ip, BIW), or bsAb having the heavy chain polypeptide SEQ ID NO: 3 (6.85 mg / kg, ip, BIW).

[0065] Fig.12E Mean tumor growth inhibition in the H1975 xenograft mouse model treated with PBS (control), panitumumab (5 mg / kg, ip, BIW), or bsAb having the heavy chain polypeptide SEQ ID NO: 7 (6.85 mg / kg, ip, BIW).

[0066] Fig.12F Mean tumor growth inhibition in H1975 xenograft mouse model treated with PBS (control), panitumumab (5 mg / kg, ip, BIW), or bsAbs having heavy chain polypeptides of SEQ ID NOs: 7, 9, and 11 (6.86 mg / kg, ip, BIW).

[0067] Figure 13A-13B Construct 12 and NRP1 ( Fig.13A ) or EGFR( Fig. 13B ) binding kinetics, where K is shown D .

[0068] Fig.14 Mean tumor growth inhibition in the H1975 xenograft mouse model treated with IgG1 isotype control, Construct 11 bsAb, or Construct 12 bsAb.

[0069] Fig.15 Schematic diagram of the bispecific antibodies of the present disclosure.

[0070] Figures 16A-16D Compared with anti-NRP1 alone, anti-EGFR alone, or the combination of anti-NRP1 + anti-EGFR, the expression of construct 11bsAb in H1975 cells ( Fig.16A , Fig. 16C ) or H1975-NRP1 OE cells ( Fig. 16B , Fig.16D ) in the NRP1 receptor ( Fig.16A , Fig. 16B ) or EGFR receptor ( Fig. 16C , Fig.16D ) receptor internalization.

[0071] Fig.17 Receptor degradation of NRP1 receptor or EGFR receptor mediated by construct 11bsAb compared to anti-NRP1 alone, anti-EGFR alone, or anti-NRP1 + anti-EGFR combined. DETAILED DESCRIPTION definition

[0072] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Therefore, the following terms are intended to have the following meanings: As used in the specification and claims, the singular form "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0073] As used herein, "administration" of a disclosed polypeptide encompasses delivering a polypeptide or composition of the invention as described herein, or a prodrug or other pharmaceutically acceptable derivative thereof, to a subject using any suitable formulation or route of administration (e.g., as described herein).

[0074] 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 may be employed alone or any combination of two or more of the listed items may be employed.

[0075] As used herein, the term "bispecific antibody" refers to an antibody that comprises at least two binding sites that bind to at least two different target antigens or target epitopes.

[0076] As used herein, "treatment," "treat," or "treating" are used interchangeably herein and refer to methods for obtaining a therapeutic benefit. Therapeutic benefit is determined by whether the tumor shrinks, remains the same size, or whether progression-free survival is improved compared to a placebo.

[0077] As used herein, "subject" can refer to any animal suffering from cancer, for example, a mammal, such as an experimental animal, a farm animal, a pet, etc. In some embodiments, the animal is a primate, preferably a person. As used herein, the terms "subject" and "subject" are used interchangeably. The terms "subject" and "subject" refer to animals (e.g., birds, such as chickens, quail or turkeys, or mammals), particularly "mammals", including non-primates (e.g., cattle, pigs, horses, sheep, rabbits, guinea pigs, rats, cats, dogs and mice) and primates (e.g., monkeys, chimpanzees and humans), and more specifically humans. In one embodiment, the subject is a non-human animal, such as a farm animal (e.g., horse, cattle, pigs or sheep) or a pet (e.g., dog, cat, guinea pig or rabbit). In a preferred embodiment, the subject is a "person".

[0078] As used herein, the term "fusion" refers to the merging of two molecules with the same or different functions or structures, and the fusion method can include any physical, chemical or biological method that can combine peptides with proteins, small molecule drugs, nanoparticles or liposomes. Preferably, the fusion can be mediated by a linker peptide, and for example, the linker peptide can be fused to the C-terminus of a fragment of an antibody light chain variable region (Fc). Alternatively, two molecules are fused by integrating multiple domains within the polypeptide sequence.

[0079] As used herein, the term "joint" is a molecule or peptide that connects two polypeptide subunits together. The joint peptide sequence can include an amino acid sequence (GGGGS)n, where n defines the number of repetitions. The number of subunit repetitions defines the joint peptide flexibility. A flexible peptide joint allows greater flexibility between the two binding domains.

[0080] As used herein, "effective amount" refers to an amount sufficient to induce the desired anticancer response. In the present invention, the desired biological response is to inhibit cell proliferation. The exact amount of the bispecific antibody administered to the subject will depend on the mode of administration, the type and severity of the cancer, and the characteristics of the subject, such as general health, age, sex, body weight, and tolerance to the drug. Those skilled in the art will be able to determine the appropriate dose based on these and other factors. When co-administered with other anticancer agents, such as co-administered with chemotherapy, the "effective amount" of the second agent will depend on the type of drug used. For approved agents, suitable doses are known and can be adjusted by those skilled in the art according to the subject's condition, the type of cancer being treated, and the amount of the polypeptide administered herein. In the absence of a clear indication of the amount, it should be assumed to be an effective amount. For example, the bispecific antibodies described herein can be administered to the subject at weekly or biweekly intervals at a dose ranging from about 0.01 to 100 mg / kg body weight / day.

[0081] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

[0082] As used herein, the term "variant" describes a sequence with changes from a wild-type sequence, a conventional sequence, or a primary sequence. The changes can take the form of deletions, substitutions, or insertions of amino acids or nucleotides. A variant can contain one or more sequence changes, including combinations of sequence changes.

[0083] The term "reduce" or other forms of the word, 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 will be understood that this is generally related to some standard or expected value, in other words, it is relative, but it is not always necessary to mention a standard or relative value.

[0084] The term "affinity" as used in the context of "binding affinity" refers to the reduced affinity of one molecule for another molecule. For example, in some embodiments, a protein, domain or motif can specifically bind to a particular target, such as a peptide, polypeptide, protein, carbohydrate, sugar, 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 its target can be measured by the dissociation constant (K D ) represents the dissociation constant (K D ) are the dissociation rate constant and the association rate constant (k off and k on The strength or affinity of a binding interaction can be expressed as the dissociation constant (K D ) is used to represent the smaller K D =K represents greater affinity. The binding properties (affinity) of a selected polypeptide can be quantified using methods known in the art. One such method requires measuring the rates of formation and dissociation of antigen binding sites / antigen complexes, where those rates depend on the concentration of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates equally in both directions. Thus, the "association rate constant" (K on ) and the “dissociation rate constant” (K off ) Both can be determined by calculating the concentration and the actual rates of association and dissociation, (see Nature 361: 186-87 (1993)).

[0085] K off / K on The ratio of 2 / 3 allows the elimination of all affinity-independent parameters and is equal to the dissociation constant K D (See generally, Davies et al., (1990) Annual Rev Biochem 59:439-473.) In some embodiments, when the equilibrium binding constant (K D )≤1 μM, the recombinant polypeptide of the present invention can specifically bind to the epitope. In some embodiments, when the equilibrium binding constant (K D)≤100 nM, the recombinant polypeptide of the present invention can specifically bind to the epitope. In some embodiments, when the equilibrium binding constant (K D )≤10 nM, the recombinant polypeptide of the present invention can specifically bind to the epitope. In some embodiments, when the equilibrium binding constant (K) is less than or equal to 10 nM, as measured by an assay such as surface plasmon resonance (SPR), Octet assay, or a similar assay known to those skilled in the art, D ) is ≤100 pM to about 1 pM, the recombinant polypeptide of the present invention can specifically bind to the epitope. In some embodiments, K D Can be 10 -5 M or smaller (e.g. 10 -6 M or smaller, 10 -7 M or smaller, 10 -8 M or smaller, 10 -8 M or smaller, 10 -10 M or smaller, 10 -11 M or smaller, 10 -12 M or smaller, 10 -13 M or smaller, 10 -14 M or smaller, 10 -15 M or smaller, or 10 -16 M or smaller).

[0086] Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Thus, in some embodiments, "reduced binding" refers to a decrease in the affinity of the corresponding interaction. Conversely, "increased binding" refers to an increase in the binding affinity of the corresponding interaction.

[0087] As used herein, the term "heavy chain" can be interpreted as including a full-length heavy chain, including a heavy chain variable region domain (VH), which includes an amino acid sequence with a variable region sequence sufficient to confer antigen specificity, and three heavy chain constant region domains CH1, CH2 and CH3, or fragments thereof. In addition, as used herein, the term "light chain" can be interpreted as including a full-length light chain, including a light chain variable region domain (VL), which includes an amino acid sequence with a variable region sequence sufficient to confer antigen specificity, and a light chain constant region domain (CL), fragments thereof. As used herein, the term "FAB" refers to an antigen-binding region. FAB consists of a variable heavy chain and a light chain and a constant heavy chain and a light chain.

[0088] As used herein, the term "percent identity" between two sequences (e.g., amino acid or nucleotide sequences) refers to the percentage (out of a possible 100%) of positions that are identical (with appropriate insertions or deletions for optimal alignment) when optimally aligned and compared. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions / total # of positions x 100), taking into account the number of spaces that must be introduced and the length of each space in order to perform optimal alignment of the two sequences. Comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below. Methods and algorithms for determining the % homology between two protein sequences are well established in the art.

[0089] 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, using a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6, which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com). In addition, a protein amino acid sequence can be used as a "query sequence" to search 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, score = 50, word length = 3, to obtain amino acid sequences homologous to protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used 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 (eg, XBLAST and NBLAST) can be used.

[0090] As used herein, the terms "combination" or "co-administration" are used interchangeably to refer to the use of more than one therapy (e.g., one or more prophylactic and / or therapeutic agents). The use of this term does not limit the order in which the therapies (e.g., prophylactic and / or therapeutic agents) are administered to a subject.

[0091] As used herein, the term "synergistic" refers to a combination of a polypeptide of the invention and another therapy (eg, a prophylactic or therapeutic agent) that is more effective than the additive effects of the therapies.

[0092] The term "pharmaceutically acceptable salts" is meant to include salts of the active bispecific antibodies which are prepared with relatively nontoxic acids or bases, depending on the particular substituents present on the bispecific antibodies described herein.

[0093] The term "parenteral" as used herein includes, but is not limited to, subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0094] The term "carrier" refers to a vehicle used to formulate a composition and may be composed of a variety of excipients.

[0095] As used herein, the term "excipient" refers to any pharmacologically inactive, natural or synthetic component or material prepared together with (e.g., simultaneously) or after the active ingredient of the present invention. In some embodiments, the excipient can be any additive, adjuvant, adhesive, extender, carrier, coating, diluent, disintegrant, filler, glidant, lubricant, preservative, vehicle or a combination thereof, and the recombinant polypeptide of the present invention can be used together with it, and or it can be used to prepare the composition of the present invention. Excipients include any such material known in the art that is nontoxic and does not interact with other components of the composition. In some embodiments, when preparing the composition, the excipient can be prepared together with the recombinant polypeptide for the purpose of increasing the composition (therefore generally referred to as extender, filler or diluent). In other embodiments, the excipient can be used to enhance the active ingredient in the final dosage form, such as promoting absorption and / or solubility. In further embodiments, the excipient can be used to provide stability or prevent contamination (e.g., microbial contamination). In other embodiments, the excipient can be used to impart physical properties to the composition (e.g., a composition in the physical form of dry particles or dry flowable powder). Reference to an excipient includes both one and more than one such excipient. Suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences by EW Martin, the disclosure of which is incorporated herein by reference in its entirety.

[0096] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step, element or integer, or groups of steps, elements or integers, but not the exclusion of any other step, element or integer, or groups of elements, steps or integers.

[0097] All patent applications, patents, and printed publications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Furthermore, all patent applications, patents, and printed publications cited herein are incorporated by reference in their entirety, except for any definitions, subject matter disclaimers, or disclaimers, and except to the extent the incorporated material is inconsistent with the explicit disclosure herein, in which case the language of the present disclosure controls.

[0098] The present invention is not limited to the scope of the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will become apparent to those skilled in the art based on the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. It should also be understood that all values ​​are approximate and are provided for illustration. Bispecific antibodies having an EGFR binding domain and an NRP1 binding domain

[0099] Bispecific antibodies have many advantages over monoclonal antibodies. First, it is more effective to manufacture a molecule with two effective targets. For example, bispecific antibodies can target a variety of immune cell receptors (CD3, CD16 or CD47) or immune checkpoint proteins (PD1, LAG-3 or CTLA-4), or both. For example, bispecific antibodies can target cancer cell antigens, such as EGFR, and immune cell receptors or immune checkpoint regulators, such as neuropilin 1 NRP1. Bispecific antibodies designed for anticancer therapy are used to improve anticancer efficacy. EGFR binding domain

[0100] Epidermal growth factor receptor (EGFR) is one of the most frequently altered oncogenes in solid tumors. The increased EGFR signaling drives the proliferation and cell survival of many cancer types, including breast cancer, prostate cancer, non-small cell lung cancer (NSCLC), esophageal cancer, liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, pancreatic cancer, colon cancer and rectal cancer. The increased EGFR signaling can be caused by EGFR overexpression, the mutation of EGFR or transduction factors in the EGFR signaling pathway leading to constitutive activation and / or the level of increased EGFR cognate ligands (such as EGF, tumor necrosis factor-α (TGF-α), amphiregulin (AREG), epigen, β-cells, heparin binding EGF (HB-EGF) and epiregulin). EGFR signaling activates downstream signaling cascades, including RAS-RAF-MEK-ERK and PI3K-Akt-mTOR axes leading to proliferation and cancer cell survival. Unfortunately, anti-EGFR agents (including tyrosine kinase inhibitors, monoclonal antibodies and radiotherapy) are only effective in a few cancer types, such as metastatic colorectal cancer, non-small cell lung cancer (NSCLC) and advanced head and neck cancer. In addition, treatment only improves survival in some individuals, and initial responses often end in drug resistance. Therefore, new rational designs are needed to improve therapeutic efficacy.

[0101] Antibodies are proteins that bind antigens with high specificity and high affinity and can neutralize antigen activity. Anti-EGFR antibodies block ligand-activated EGFR signaling and induce receptor endocytosis, resulting in EGFR degradation in the proteasome. When mutations occur in the EGFR binding site of the antibody, the subject can obtain anti-EGFR antibody resistance during the course of treatment. Cetuximab (a chimeric mouse / human monoclonal antibody) and panitumumab (a fully humanized monoclonal antibody) have different binding sites on EGFR, and therefore panitumumab is still effective after resistance to cetuximab, and vice versa. Therefore, another anti-EGFR antibody targeting different EGFR binding sites can be administered to a subject who obtains resistance to a kind of anti-EGFR antibody.

[0102] Panitumumab is an IgG2 anti-EGFR humanized monoclonal antibody that binds to EGFR with approximately 8 times greater affinity than cetuximab, an IgG1 anti-EGFR chimeric human / mouse monoclonal antibody (Garcia-Foncillas et al., 2019). Antibody binding can induce an immune response prior to inducing endocytosis. Panitumumab binding induces antibody-dependent cell-mediated cytotoxicity (ADCC) and initiates antibody-dependent cellular phagocytosis (ADCP) by activating neutrophils and monocytes (Schneider-Merk et al., 2010). The IgG1 Fc domain of cetuximab binds to the FcγRIIIA (CD16) receptor on natural killer (NK) cells to induce ADCC. Activated NK cells secrete perforins and granzymes, leading to cancer cell lysis and release of immunostimulatory molecules such as interferon-γ (IFN-γ), TNF-α, chemokines, and granulocyte macrophage colony stimulating factor (GM-CSF). Cytokines secreted by NK cells stimulate dendritic cell maturation, NK cell cross-talk, and co-expression of CD137. CD137 expression recruits anti-EGFR CD8+T cells, resulting in increased killing of cancer cells expressing EGFR. Mature dendritic cells further activate NK cells and present tumor antigens to cytotoxic CD8+T cells. The IgG1Fc region also binds to Fc receptors on macrophages or plasmacytoid dendritic effector cells or to the first subunit of the C1 complement complex (C1q) to initiate antibody-dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC) of the cell, respectively. Like cetuximab, the anti-EGFR antibodies cetuximab, nemotuzumab, and nimotuzumab also have an IgG1 Fc region for ADCC induction. NRP1 binding domain

[0103] Cancer cells overexpress pro-angiogenic factors that promote rapid growth of new blood vessels. The blood vessels surrounding the tumor are abnormal, with capillaries constricted, which reduces total blood flow to the tumor. Reduced blood flow increases tumor interstitial fluid pressure and reduces drug flow from blood vessels to the tumor (Milosevic et al., 1999). Unlike in normal tissues, the lack of lymphatic vessels in tumor tissue also leads to abnormal angiogenesis and high tumor interstitial pressure. By targeting vascular endothelial growth factor A (VEGF 165 ) to inhibit angiogenesis can normalize blood vessel formation, vascular fluid pressure, and enhance the accumulation of therapeutic drugs at the tumor site (Marcucci et al., 2013). However, anti-VEGF antibodies, such as bevacizumab, can have adverse side effects and are only effective within a narrow exposure range (Kamba and McDonald, 2007).

[0104] Neuropilin-1 (NRP1) and neuropilin-2 (NRP2) receptors are multifunctional single-pass transmembrane glycoproteins that play an important role in angiogenesis and lymphangiogenesis, respectively. The C-terminal region of each of the VEGF ligand family and Sema3 ligands that bind to NRP1 and NRP2 binds to the arginine binding pocket in the b1 domain of NRP1 and NRP2 (Parker et al., 2012). Binding to the arginine binding pocket occurs through the motif of R / KxxR / K (R = arginine, K = lysine, and x = any amino acid), which is usually present in the C-terminal region of NRP binding ligands and is called "C-end rule" (CendR) (Teesalu et al., 2009). Proteins or peptides containing the C-end rule sequence can bind to NRPs through the C-terminal arginine (Arg) or lysine (Lys) residue (Zanuy et al., 2013).

[0105] Selective targeting of NRP1 is crucial for anti-EGFR cancer therapy, as silencing NRP2 significantly increased EGFR expression in lung and gastric cancer cells (Rizzolio et al., 2017). In addition, NRP1 is overexpressed in many cancer cells, including colon cancer, melanoma, astrocytoma, lung, prostate, and pancreatic ductal adenocarcinoma, and plays a key role in cancer progression (Graziani and Lacal, 2015). In addition to NRP1 being overexpressed in a variety of cancers, NRP1 is also overexpressed in tumor-associated endothelial cells. NRP1 acts as a ligand involved in angiogenesis, such as vascular endothelial growth factor (VEGF 165 ), class 3 semaphorin ligands, integrin β1, TGF-β, HGF, FGF, PDGF, and galectin-1. NRP1 interacts with receptor tyrosine kinases (RTKs) such as VEGFR1 and VEGFR2, and thereby contributes to VEGFR signaling, leading to increased angiogenesis. NRP1 also binds to secreted class 3 semaphorin ligands (Sema3A, Sema3B, Sema3C, Sema3D, Sema3E, Sema3F, Sema3G) to act as a co-receptor for plexin family receptors (which also regulate angiogenesis).

[0106] Blocking NRP1 ligand binding with an anti-NPR1 specific binding domain reduced the expression of the endothelial adhesion molecule VE-cadherin, leading to increased extravasation of anti-EGFR antibodies from blood vessels. Increased extravasation also reduced tumor interstitial pressure, allowing the drug to flow more freely from the blood vessels to the tumor environment.

[0107] The epithelial barrier around solid tumors is composed of intercellular spaces densely filled with interstitial epithelial cells connected by intercellular adhesion factors, and it hinders therapeutic agents from penetrating tumors. E-cadherin overexpression contributes to intercellular adhesion. Since substances that reduce E-cadherin have been found in viruses (adenovirus-3), a situation has been reported, in which only a part (JO-1) with the activity of reducing the cell E-cadherin in tight junctions is co-administered with antibodies in the proteins constituting the virus, thereby improving the anticancer effect of antibodies (Beyer et al., 2011). Targeting NRP1 reduces the expression of epithelial barrier adhesion molecule E-cadherin and integrin β1 subunits, which are overexpressed in many solid tumors and increase extracellular matrix connections, resulting in reduced drug penetration. In addition, integrin β1 subunits participate in activating growth factor receptor-mediated cell proliferation.

[0108] In conclusion, targeting NRP1 can reduce VEGF 165 Binding, as well as VE-cadherin, E-cadherin, and integrin β1 expression, which together serve to reduce angiogenesis, tumor interstitial pressure, and increase extravasation and tumor penetration of anti-EGFR antibodies.

[0109] Cancer cells produce immune checkpoint molecules that inhibit immune responses. NRP1 is an immune checkpoint molecule, and its expression is increased in tumor-associated endothelial cells and cancer cells. NRP1 expression induces immunosuppression by increasing Treg activity and reducing tumor-specific CD8+T cell responses (Chuckran et al., 2020). Therefore, in addition to increasing drug extravasation and penetration, the NRP1 binding domain also acts as an immune checkpoint inhibitor. On the other hand, the present disclosure provides a bispecific antibody that targets NRP1 to reduce cancer-mediated checkpoint molecule immunosuppression, allowing natural immunity at the tumor site, and increasing the possibility of tumor response.

[0110] The anti-NRP1 antibody MNRP1685A (also known as vesentrumab) competitively binds to VEGF on NRP1 165 Binds to and is used to inhibit VEGF signaling through VEGFR2, thereby affecting angiogenesis, cell survival, migration, adhesion and invasion (PanQ et al., 2007). However, subjects treated with vesentumab in a Phase I trial of advanced solid tumors suffered intolerable side effects, including gastrointestinal bleeding, fungemia, duodenal obstruction, thrombocytopenia, proteinuria, alopecia, dysphonia, fatigue and nausea, which led to the discontinuation of further trials (Weekes et al., 2014; Patnaik et al., 2014).

[0111] NRP1 is active as a homodimer or heterodimer, and monomeric peptides (such as cleaved penetrating peptide iRGD) have only a weak ability to modulate NRP1 biological activity (Sugahara et al., 2010). Therefore, peptides that selectively bind to NRP1 as a homodimer to modulate biological activity are preferred. Unfortunately, although the Fc heavy chain-fused NRP binding peptide A22p appears as a dipeptide (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.

[0112] 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 monoclonal antibodies having heavy chain polypeptide sequences shown in SEQ ID NOs: 41-47, which include an N-terminal NPR1 binding domain. In some embodiments, the monoclonal antibody heavy chain polypeptide comprises an N-terminal NRP1 binding domain and an Fc domain, wherein the N-terminal NRP1 binding domain comprises a variable heavy chain (VH) and a constant heavy chain 1 (CH1), and the Fc domain comprises a constant heavy chain 2 (CH2) and a constant heavy chain 3 (CH3). In some embodiments, the monoclonal antibody comprises a light chain polypeptide sequence as shown in any one of SEQ ID NOs: 48-54. Thus, the heavy chains of SEQ ID NOs: 41-47 can be paired with the light chains of SEQ ID NOs: 48-54. The polynucleotide sequences encoding the heavy and light chain polypeptides are shown in SEQ ID NOs: 55-61 and 62-68, respectively. Example 8 describes a method for producing monoclonal antibodies.

[0113] In some embodiments, the NRP1 affinity (K) of the NRP1 binding domain of the monoclonal antibody is D ) ranged from 70 nM to the subnanomolar range for monoclonal antibodies having the heavy chain polypeptide sequences shown in SEQ ID NOs: 41-47 and their corresponding light chain polypeptide sequences shown in SEQ ID NOs: 48-54.

[0114] Blocking VEGF binding to NRP1 with a monoclonal antibody to the NRP1 binding domain can inhibit VEGFR2 activation as measured by VEGFR2 phosphorylation.

[0115] In some embodiments, the monoclonal antibody inhibits cancer cell growth. In some embodiments, the monoclonal antibody Fc domain comprising constant heavy chains 2 and 3 (CH2 and CH3) belongs to the IgG1 or IgG2 subclass. In some embodiments, the present disclosure provides a monoclonal antibody NRP1 binding domain that robustly inhibits cell proliferation in the cancer cell line H1975. With Bispecific antibodies with binding affinity for EGFR and NRP1

[0116] The present disclosure provides a bispecific antibody having a heavy chain polypeptide sequence, the heavy chain polypeptide sequence including 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 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), and a C-terminal short chain variable fragment (scFv) NRP1 binding domain.

[0117] A schematic diagram of a representative bispecific antibody structure of the present disclosure is shown in Fig.15 middle.

[0118] The ScFv NRP1 binding domain is composed of a variable heavy chain and a variable light chain connected by a flexible peptide linker. In some aspects, the flexible peptide linker is a repetition of the subunit sequence of SEQ ID NO: 13. In some aspects, the peptide linker connects ScFv to CH3. In some aspects, the peptide linker is composed of one or more peptide linker subunits of SEQ ID NO: 13, wherein GGGGS is a subunit.

[0119] 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 aspects, the bispecific antibody further comprises a light chain polypeptide sequence comprising a variable light chain and a constant light chain, the light chain polypeptide sequence being paired with the heavy chain polypeptide and shown in SEQ ID NO: 12. The polynucleotide sequence encoding the light chain polypeptide of SEQ ID NO: 12 is shown in SEQ ID NO: 25. In some aspects, the present disclosure provides polynucleotide sequences for encoding the GGGGS subunit, representative examples of which are shown in SEQ ID NOs: 26-38. Example 1 describes a method for producing a bispecific antibody.

[0120] In some aspects, the bispecific antibody comprises an anti-EGFR binding arm comprising a VH amino acid sequence as shown in SEQ ID NO: 69 and a VL amino acid sequence as shown in SEQ ID NO: 70 (or consisting thereof). In some aspects, the bispecific antibody comprises an anti-EGFR binding arm comprising a heavy chain CDR1, CDR2, and CDR3 region, and the heavy chain CDR1, CDR2, and CDR3 region respectively comprise the amino acid sequences shown in SEQ ID NO: 71, 72, and 73 (or respectively consist thereof). In some aspects, the bispecific antibody comprises an anti-EGFR binding arm comprising a heavy chain CDR3 region, and the heavy chain CDR3 region comprises the amino acid sequence shown in SEQ ID NO: 73 (or consists thereof). In some aspects, the bispecific antibody comprises an anti-EGFR binding arm comprising a light chain CDR1, CDR2, and CDR3 region, and the light chain CDR1, CDR2, and CDR3 region respectively comprise the amino acid sequences shown in SEQ ID NO: 74, 75, and 76 (or respectively consist thereof). In some aspects, the bispecific antibody comprises an anti-EGFR binding arm comprising a light chain CDR3 region comprising (or consisting of) the amino acid sequence shown in SEQ ID NO: 76. In some embodiments, the anti-EGFR binding arm comprises (or consists of) one or more sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.8% identical to any of the aforementioned VH, VL, HCDR, or LCDR sequences.

[0121] In some aspects, the bispecific antibody comprises an anti-NRP1 binding arm comprising (or consisting of) a VH amino acid sequence as shown in SEQ ID NO: 77 and a VL amino acid sequence as shown in SEQ ID NO: 78. In some aspects, the bispecific antibody comprises an anti-NRP1 binding arm comprising a heavy chain CDR1, CDR2, and CDR3 region (HCDR1, HCDR2, and HCDR3, respectively), wherein HCDR1 comprises (or consists of) a sequence shown in SEQ ID NO: 79, HCDR2 comprises (or consists of) a sequence shown in SEQ ID NO: 80, and HCDR3 comprises (or consists of) a sequence shown in any one of SEQ ID NOs: 81-84. In some aspects, the bispecific antibody comprises an anti-NRP1 binding arm comprising a light chain CDR1, CDR2, and CDR3 region (LCDR1, LCDR2, and LCDR3, respectively), wherein LCDR1 comprises (or consists of) a sequence shown in any one of SEQ ID NOs: 85-87, LCDR2 comprises (or consists of) a sequence shown in SEQ ID NO: 88, and LCDR3 comprises (or consists of) a sequence shown in SEQ ID NO: 89. In some aspects, the bispecific antibody comprises an anti-NRP1 binding arm comprising a heavy chain CDR3 region comprising (or consisting of) an amino acid sequence shown in any one of SEQ ID NOs: 81-84. In some aspects, the bispecific antibody comprises an anti-NRP1 binding arm comprising a heavy chain CDR1, CDR2, and CDR3 region comprising (or consisting of) the amino acid sequences shown 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 sequences shown in SEQ ID NOs: 85, 88, and 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.

[0122] In some embodiments, the EGFR binding domain of the bispecific antibody has an EGFR affinity (K) in the subnanomolar range (<0.1 nM). D In some embodiments, the NRP1 affinity (K) of the NRP1 binding domain of the bispecific antibody is D) ranged from 70 nM for a bispecific antibody construct having a control polypeptide sequence of SEQ ID NO: 1 to the subnanomolar range for bispecific antibodies having polypeptide sequences shown in SEQ ID NOs: 2, 3, 5, 7, 10, and 11. Thus, in one aspect, the present disclosure provides bispecific antibodies having an EGFR binding domain with an affinity for EGFR in the subnanomolar range and an NRP1 binding domain with an affinity for NRP1 in the subnanomolar range, as shown in Table 1 of Example 2. Figure 1 A-1H shows the binding affinity curve of the bispecific antibody to immobilized huEGFR. Figure 2 A-2I shows the binding affinity curve of the bispecific antibody to immobilized huNRP1.

[0123] One aspect of the present disclosure provides bispecific antibodies with asymmetric binding affinity for EGFR and NRP1, see Examples. In some embodiments, the K of the EGFR binding domain to EGFR D is the K of the NRP1 binding domain to NRP1 D The asymmetric affinity provides better homing to cancer cells expressing EGFR while reducing NRP1 on-target cytotoxicity.

[0124] Blocking VEGF binding to NRP1 with the NRP1 binding domain of the bispecific antibody can inhibit VEGFR2 activation, as measured by VEGFR2 phosphorylation. However, the bispecific antibody with the heavy chain of SEQ ID NO: 1 had an NRP1 K of 69.7 nM. D , does not inhibit VEGFR phosphorylation ( Figure 3 In some embodiments, the bispecific antibody inhibits VEGFR2 phosphorylation to prevent angiogenesis, see Table 2 and Figure 4A , 4B -10A, 10B. In some embodiments, the present inventors have found that the dissociation constant (K D ) does not reflect the ability of the bispecific antibody to inhibit VEGFR2 phosphorylation. For example, the bispecific antibody with heavy chain SEQ ID NO: 6 inhibited 50% of VEGFR2 phosphorylation at 10130 nM (IC50), but NRP1 K D The bispecific antibody with heavy chain SEQ ID NO: 4 inhibited 50% of VEGFR2 phosphorylation at 410 nM (IC50), but NRP1 K DThus, in some aspects, the present disclosure provides bispecific antibodies in which the affinity and biological function of the NRP1 binding domains are disproportionate in terms of inhibiting VEGFR2 phosphorylation.

[0125] In some aspects, the bispecific antibodies inhibit cancer cell growth. In some embodiments, the bispecific antibody Fc domain comprising constant heavy chains 2 and 3 (CH2 and CH3) belongs to the IgG1 or IgG2 subclass. In some aspects, the present disclosure provides bispecific antibodies having an EGFR binding domain and an NRP1 scFv binding domain that robustly inhibit cell proliferation in the cancer cell line H1975, Tables 3 and Fig.11A and 11B Example 4 describes a method for measuring the IC50 of bispecific antibodies for cell growth inhibition in the H1975 cancer cell line. Additional binding arms for bispecific antibodies

[0126] In other embodiments, an anti-NRP1 binding arm (as described herein) can be paired in a bispecific antibody with a second binding arm that binds 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.

[0127] In some embodiments, the second target protein comprises a receptor tyrosine kinase (RTK). In some embodiments, the tyrosine kinase comprises platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), receptor tyrosine kinase Met (MET), and receptor vascular endothelial growth factor (VEGFR). In some embodiments, receptor tyrosine kinases include EGFR / HER1 / Erb1, HER2 / ErbB2, HER3 / ErbB3, HER4 / ErbB4, FGFR1, FGFR2, FGFR3, FGFR4, MET, RON, PDGFR, PDGFRα, PDGFRβ, CSF-1R, Kit, FLT-3, VEGFR1, VEGFR2, and VEGFR3.

[0128] In some embodiments, the target protein comprises EGFR. In some embodiments, EGFR is selected from EGFR / HER1 / Erb1, HER2 / ErbB2, HER3 / ErbB3, and HER4 / ErbB4. In some embodiments, the target protein is EGFR.

[0129] In some embodiments, the target protein comprises FGFR.In some embodiments, FGFR1 is selected from FGFR1, FGFR2, FGFR3 and FGFR4.

[0130] 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).

[0131] In some embodiments, the target protein comprises PDGFR.In some embodiments, the PDGFR is selected from PDGFR, PDGFRα, PDGFRβ, CSF-1R, Kit, and FLT-3.

[0132] In some embodiments, the target protein comprises a VEGFR. In some embodiments, the VEGFR is selected from VEGFR1, VEGFR2, and VEGFR3.

[0133] In some embodiments, the receptor tyrosine kinase is selected from EGFR / HER1 / Erb1, HER2 / ErbB2, HER3 / ErbB3, HER4 / ErbB4 VEGFR1, VEGFR2, and VEGFR3.

[0134] In some embodiments, the target protein comprises a receptor serine / threonine kinase (RSTK), a G protein-coupled receptor (GPCR), an immune checkpoint receptor, and an ion channel receptor.

[0135] 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.

[0136] In some embodiments, the target protein comprises a G protein coupled receptor (GPCR). In some embodiments, the GPCR is selected from CXCR4, CCR5, FFAR2, GLP2R, 5-HT1A receptor, 5-HT2A receptor, 5-HT4 receptor, 5-HT5A receptor, M1 receptor, M2 receptor, A1 receptor, A2A receptor, α1A-adrenergic receptor, α2A-adrenergic receptor, β1-adrenergic receptor, β3-adrenergic receptor, AT1 receptor, BB1 receptor, B1 receptor, CB1 receptor, CB2 receptor, chemerin receptor 1, CCR1, CX3CR1, ACKR3, CCK1 receptor, GPR3, GPR12, GPR17, GPR32, GPR35.

[0137] In some embodiments, the target protein comprises an immune checkpoint receptor. In some embodiments, the immune checkpoint receptor is selected from CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, NOX2, PD-1, TIM-3, VISTA, SIGEC7, and PD-L1.

[0138] In some embodiments, the target protein comprises an ion channel receptor. In some embodiments, the ion channel receptor is selected from KCa1.1, KCa2.1, CatSper1, TPC1, CNGA1, HCN1, Kir1.1, Kir3.2, RyR1, TRPA1, TRPC3, TRPM1, TRPP1, TRPV1, K2P1.1, K2P10.1, Cav1.1, Cav2.1, Kv1.1, Kv1.8, Kv11.2, Hv1, Nav1.1 and Nav1.2.

[0139] In some embodiments, the target protein includes 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 a 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 lung cancer, breast cancer, colon cancer and rectal cancer, head and neck cancer, esophageal 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. Bispecific Antibody Tumor Growth Inhibition in Tumor Mouse Models

[0140] In some aspects of the present disclosure, treatment with bispecific antibodies reduces tumor growth. In some aspects, a bispecific antibody having a heavy chain of SEQ ID NO: 11 and a light chain of SEQ ID NO: 12 (also referred to herein as construct 11) significantly reduced tumor growth. In some aspects, a bispecific antibody having a heavy chain of SEQ ID NO: 39 and a light chain of SEQ ID NO: 12 (also referred to herein as construct 12) significantly reduced tumor growth. In some aspects, the addition of a scFv NRP1 binding domain significantly increased the efficacy of tumor growth inhibition. Example 5 describes methods and results for treating an H1975 xenograft mouse model with a bispecific antibody to inhibit tumor growth. Table 4 shows the percentage of tumor growth inhibited, and Figure 12A-1 2G and Fig.17 Tumor growth within days after implantation is shown. Summary of the biological effects of bispecific antibodies

[0141] In summary, the present disclosure provides bispecific antibodies comprising NRP1 binding domains, such as bispecific antibodies that combine EGFR binding domains with NRP1 binding domains to promote homing to high EGFR expressing tumors through increased EGFR affinity. The bispecific antibody will have a variety of biological effects that combine to reduce cancer cell proliferation and survival, including reducing abnormal angiogenesis, increasing extravasation and penetration by reducing VE-cadherin, E-cadherin, and integrin β1 expression. NRP1 targeting further reduces EGFR surface aggregation to enhance EGFR downregulation and inhibit NRP1 checkpoint immunosuppression. The combination of these features provides a bispecific antibody with an effective ability to reduce EGFR-mediated cancer cell proliferation and survival. FcRn binding for endocytic recycling

[0142] The antibody size of about 150kD ensures a long serum half-life, and therefore ensures a lasting therapeutic effect. In addition, the antibody IgG Fc portion composed of heavy chain constant regions 2 and 3 (CH2 and CH3) binds to the neonatal Fc receptor (FcRn), and the Fcγ receptor (FcγR) at the cell surface is endocytosed and recycled back to the serum by endocytosis, which further increases the antibody serum half-life. Cancers with low FcRn or FcγR expression are associated with poor prognosis (Pyzik et al., 2019), and thus FcRn or FcγR-mediated recycling plays an important role in maintaining antibody serum concentrations. IgG1 antibodies have more effective FcRn and FcγR recycling processes than IgG2 antibodies. Thus, having an IgG1 Fc domain in a therapeutic antibody helps maintain drug treatment levels and reduce the frequency of administration, and a reduced half-life is ideal for diagnostic testing or toxicity control.

[0143] In some embodiments, a polypeptide of the invention comprises an immunoglobulin domain, including a polypeptide comprising an immunoglobulin domain comprising an Fc domain selected from the IgG1 subclass. Expression constructs

[0144] The term "vector" as used herein refers to a means for expressing a target gene in a host cell. For example, a vector may include a plasmid vector, a cosmid vector, a phage vector, and a viral vector, such as an adenoviral vector, a retroviral vector, and an adeno-associated viral vector. Recombinant vectors can be produced by manipulating plasmids commonly used in related fields (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), phages (e.g., Agt4AB, A-Charon, AAz1, and M13, etc.), or viruses (e.g., CMV, SV40, etc.).

[0145] On the other hand, the present disclosure provides isolated polynucleotides encoding bispecific antibody heavy chain and light chain amino acid sequences. The polynucleotide encoded in the recombinant vector can be operably connected to a promoter. As used herein, the term "operably connected" refers to the functional connection between a nucleotide expression control sequence (such as a promoter sequence) and a second nucleotide sequence. Therefore, the regulatory sequence can control the transcription and / or translation of the second nucleotide sequence. Recombinant vectors can generally be constructed as vectors for cloning or for expression. As vectors for expression, vectors commonly used in the relevant art for expressing exogenous proteins from plants, animals or microorganisms can be used. Recombinant vectors can be constructed by various methods known in the relevant art. Eukaryotic cells with fl replication origin, SV40 replication origin, pMB1 replication origin, glandular replication origin, AAV replication origin, CMV replication origin and BBV replication origin, etc. can be used as hosts to construct recombinant vectors, but are not limited thereto.

[0146] In addition, a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian cell virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a cytomegalovirus (CMV) promoter, or the tk promoter of HSV) can be used, and the promoter generally has a polyadenylation sequence as a transcription termination sequence. The vector can express not only the peptide domain that specifically binds to NRP1 according to the present disclosure, but also an antibody having a peptide fused thereto and a linker peptide. In the case where the antibody has a peptide fused thereto, the vector can use a vector system that expresses the peptide and the antibody or a fragment thereof in one vector, as well as a vector system that expresses the peptide and the antibody or a fragment thereof in separate vectors. For the latter, the two vectors can be introduced into a host cell by co-transformation and targeted transformation. Thus, on the other hand, the present disclosure includes a vector containing a nucleic acid disclosed in any one of SEQ ID NOs: 14-38 and 40. Example 1 describes the properties of vectors that can be used to produce bispecific antibodies.

[0147] 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 a host cell. Examples of prokaryotic cells include strains such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, or strains belonging to the genus Bacillus such as Bascillus subtilus and Bascillus thuringiensis, Salmonella typhimurium, Serratia marcescens and intestinal flora and strains such as various Pseudomonas Spp., etc. Prokaryotic cell transformation can be used for large-scale cloning of plasmids. Prokaryotic host cells may lack the necessary post-translational modifications required for antibody assembly and structure, and thus, a preferred embodiment is vector transformation in eukaryotic host cells, such as yeast (Saccharomyce cerevisiae), insect cells, plant cells, mammalian cells, for example, SP2 / 0, CHO (Chinese Hamster Ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RN and MDCK cell lines, etc.

[0148] Another aspect of the present disclosure provides a method for preparing a peptide that specifically binds to NRP1, the method comprising culturing the above-mentioned host cells. Polynucleotides and recombinant vectors comprising polynucleotides can be inserted into host cells using insertion methods known in the relevant art. For example, when the host cell is a prokaryotic cell, the transfer can be performed according to the CaCl2 method or the electroporation method, and when the host cell is a eukaryotic cell, the vector can be transferred into the host cell by various methods (including microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transformation and gene bombardment, etc., but the transfer method is not limited thereto). The method for selecting transformed host cells can be easily performed according to methods known in the relevant art, using the phenotype expressed by the selected marker. For example, when the selected marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a culture medium containing the antibiotic. Pharmaceutical composition

[0149] The polypeptides described herein (bsAb and mAb) can be formulated into pharmaceutical compositions, which further comprise a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. In one embodiment, the present invention relates to a pharmaceutical composition comprising a disclosed polypeptide and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. In one embodiment, the present invention is a pharmaceutical composition comprising an effective amount of a polypeptide of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients or carriers, which are appropriately selected according to the intended form of administration and are consistent with conventional pharmaceutical practice.

[0150] Pharmaceutically acceptable carriers or excipients may contain inert ingredients that do not excessively inhibit the biological activity of the polypeptide. Pharmaceutically acceptable carriers should be biocompatible, for example, non-toxic, non-inflammatory, non-immunogenic, or have no other undesirable reactions or side effects when administered to a subject. Standard pharmaceutical preparation techniques may be used.

[0151] As used herein, pharmaceutically acceptable carriers, adjuvants or vehicles include any and all solvents, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid adhesives, lubricants, etc. suitable for the desired specific dosage form. Remington's Pharmaceutical Sciences, the sixteenth edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers for preparing pharmaceutically acceptable compositions and known techniques for their preparation. Unless any conventional carrier medium is incompatible with the polypeptide described herein, such as by producing any undesirable biological effect or interacting with any other component of the pharmaceutically acceptable composition in a harmful manner, its use is expected to be within the scope of the present invention. As used herein, the phrase "side effect" covers the unwanted and unfavorable effects of therapy.

[0152] Materials that can serve as pharmaceutically acceptable carriers for antibodies increase conformational stability, reduce protein dynamics, inhibit aggregation, and protect proteins adsorbed to liquid-air interfaces, and include, but are not limited to, cyclodextrin hydrogels, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as Tween 80, phosphates, glycine, sorbic acid or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride or zinc salts), colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, methylcellulose, hydroxypropyl methylcellulose, lanolin, sugars such as lactose, glucose and sucrose; starches Flours such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol and phosphate buffered solutions, and other nontoxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the composition, according to the judgment of the formulator.

[0153] In some embodiments, the compositions of the present invention include pharmaceutically acceptable salts. When the polypeptides of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting such polypeptides in a neutral form with a sufficient amount of the desired base (pure or in a suitable inert solvent). Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts, or similar salts. When the polypeptides of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting such polypeptides in a neutral form with a sufficient amount of the desired acid (pure or in a suitable inert solvent). Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid or phosphorous acid, and those derived from relatively nontoxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, oxalic acid, methanesulfonic acid, and the like. Also included are salts of amino acids such as arginine salts, and salts of organic acids such as glucuronic acid or galacturonic acid, and the like (see, e.g., Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific polypeptides disclosed herein contain both basic and acidic functional groups, which allow the polypeptides to be converted into base addition salts or acid addition salts.

[0154] Thus, the disclosed polypeptides may exist as salts, such as salts with pharmaceutically acceptable acids. The present invention includes such salts. Non-limiting examples of such salts include hydrochlorides, hydrobromides, phosphates, sulfates, mesylates, nitrates, maleates, acetates, citrates, fumarates, propionates, tartrates (e.g., (+)-tartrates, (-)-tartrates, or mixtures thereof, including racemic mixtures), succinates, benzoates, and salts with amino acids such as glutamic acid, and quaternary ammonium salts (e.g., methyl iodide, ethyl iodide, etc.). These salts can be prepared by methods known to those skilled in the art.

[0155] 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.

[0156] Certain polypeptides of the present invention may exist in unsolvated forms as well as solvated forms (including hydrated forms). Typically, solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain polypeptides of the present invention may exist in a variety of crystalline or amorphous forms. Typically, all physical forms are equivalent for the intended uses of the present invention and are intended to be within the scope of the present invention.

[0157] In some embodiments, subcutaneous formulations may contain recombinant human PH20 hyaluronidase (rHuPH20) to facilitate dispersion of the antibody from the injection site. Application method

[0158] The composition 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 (eg, a polypeptide or a pharmaceutically acceptable salt thereof) to a subject in need of cancer treatment.

[0159] As used herein, "intermittent administration" includes administering a pharmaceutical agent for a period of time (which may be considered a "first administration period"), followed by a period of time when the composition is not taken or is taken at a lower maintenance dose (which may be considered a "drug-free period"), followed by a period of time when the composition is administered again (which may be considered a "second administration period"). Typically, during the second administration phase, the dosage level of the pharmaceutical agent will match the dosage level administered during the first administration period, but may be increased or decreased as medically necessary.

[0160] In some embodiments, the composition of the present invention can be applied to the subject by: oral administration, as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or implantation of a slow release device, such as a miniature osmotic pump. Therefore, it can be administered by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palate, gum, nose, vagina, rectum or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular. Other delivery methods include, but are not limited to, using liposome preparations, intravenous infusion or via implanted reservoirs, etc. In particular, oral, intraperitoneal or intravenous administration of the composition.

[0161] In some embodiments, the composition (e.g., a bispecific antibody composition) is administered systemically intravenously (IV) or by oral route for intestinal cancer, such as gastric cancer or intestinal cancer (Tashima et al., 2021). The formulation for delivery can be optimized by routine conventional methods known in the art. Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active polypeptide, the liquid dosage form may contain an inert diluent commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0162] In some embodiments, the composition (e.g., bispecific antibody composition) is administered by subcutaneous injection. Injectable bispecific antibody preparations can be, for example, filtered through a bacteria-retaining filter, or sterilized by incorporating a sterilant dispersed in sterile water or other sterile injectable media before use. In order to prolong the effect of the polypeptide described herein, it is generally desirable to slow down the absorption of the polypeptide from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous material with poor water solubility. The absorption rate of the polypeptide then depends on its dissolution rate, which in turn can depend on crystal size and crystalline form. Alternatively, delayed absorption of the polypeptide form administered parenterally is achieved by dissolving or suspending the polypeptide in an oil vehicle. Injectable reservoir forms are prepared by forming a microcapsule matrix of the polypeptide in a biodegradable polymer such as polylactic acid-polyglycolide. According to the ratio of polypeptide to polymer and the properties of the specific polymer used, the rate of polypeptide release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the polypeptide in liposomes or microemulsions that are compatible with body tissues.

[0163] The sterile injectable form of the composition described herein can be an aqueous or oily suspension. These suspensions can be prepared using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. Sterile injectable preparations can also be sterile injectable solutions or suspensions in nontoxic parenteral acceptable diluents or solvents, for example as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives can be used to prepare injections, as can natural pharmaceutically acceptable oils such as olive oil or castor oil, especially their polyoxyethylated forms. These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersants, such as carboxymethyl cellulose or similar dispersants, which are generally used to prepare pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in preparing pharmaceutically acceptable dosage forms may also be used for formulation purposes.

[0164] In some embodiments, the formulation may include agents such as excipients, buffers, isotonic agents, preservatives, surfactants, and preferably zinc. The formulation may also include excipients or agents for polypeptide stabilization, such as buffers, reducing agents, bulk proteins, or carbohydrates. Bulk proteins that can be used to prepare at least one polypeptide composition include albumin, protamine, and the like. Typical carbohydrates that can be used to prepare 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 atomization of the solution when forming an aerosol. Various conventional surfactants may be used, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. The amount range is generally between about 0.001% by weight and 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. Other agents known in the art for formulating polypeptides, such as antibody proteins, may also be included in the formulation. Cell therapy approaches

[0165] In another aspect, the disclosure provides methods of treating cells with the bispecific antibodies of the disclosure, e.g., for receptor internalization or receptor degradation purposes. As demonstrated herein, the bsAbs of the disclosure can mediate internalization and degradation of receptors to which the bsAbs bind (e.g., NRP1, EGFR).

[0166] Thus, in one embodiment, the disclosure provides a method for 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, such that the receptor is internalized. In one embodiment, the bsAb mediates internalization of receptors NRP1 and EGFR.

[0167] In another embodiment, the disclosure provides a method for degrading 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, such that the receptor is degraded. In one embodiment, the bsAb mediates degradation of receptors NRP1 and EGFR. Tumor growth inhibition

[0168] On the other hand, the present disclosure provides a method for inhibiting tumor cell growth using the bispecific antibodies of the present disclosure. As confirmed herein, the bsAb of the present disclosure exhibits tumor growth inhibition ability. Therefore, the bsAb of the present disclosure can be used to inhibit the growth of tumor cells, such as inhibiting the growth of tumor cells in the treatment of cancer. On the one hand, a method for treating cancer is provided herein, the method comprising administering the bsAb of the present disclosure to a subject in need in an effective amount, thereby inhibiting or reducing the growth of cancerous tumors and / or achieving regression and / or extended survival. In some embodiments, the bsAb described herein can be administered in combination with another cytotoxic agent or therapeutic agent as described herein.

[0169] The cancers whose growth can be suppressed using bsAb described herein include but are not limited to cancer, lymphoma, blastoma, sarcoma and leukemia. In one embodiment, cancer is related to abnormal EGFR expression and / or function (e.g., overexpression of EGFR, expression of mutant EGFR), and its non-limiting examples include lung cancer (e.g., non-small cell lung cancer), renal cancer, breast cancer (e.g., invasive breast ductal carcinoma), glioblastoma, head and neck cancer, prostate cancer and ovarian cancer. In one embodiment, cancer is non-small cell lung cancer (NSCLC). In one embodiment, cancer is selected from lung cancer (e.g., NSCLC), colon cancer and head and neck cancer.

[0170] In another embodiment, the cancer is associated with aberrant 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.

[0171] In another embodiment, cancer is associated with abnormal expression of both EGFR and NRP1, such as overexpression of both EGFR and NRP1. Non-limiting examples of cancers 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. Gene expression profiling of the Cancer Genome Atlas database of patient samples showed that pancreatic cancer, lung cancer, head and neck cancer, cervical cancer, kidney cancer, ovarian cancer and colon cancer had 75%, 70%, 65%, 65%, 60%, 55% and 40% of the patient tumor populations that overexpressed both EGFR and NRP1, respectively.

[0172] In one embodiment, the bsAbs of the present disclosure, optionally in combination with gemcitabine and / or cisplatin, are used to treat metastatic squamous non-small cell lung cancer. In certain embodiments, the use of the bsAb is not indicated for the treatment of non-squamous non-small cell lung cancer.

[0173] In one embodiment, the bsAbs of the present disclosure, optionally in combination with FOLFOX (folinic acid, fluorouracil, and oxaliplatin) or FOLFIRI (folinic acid, fluorouracil, and irinotecan), are used to treat wild-type RAS metastatic colorectal cancer (defined as wild-type in both KRAS and NRAS, as determined by an FDA-approved test for this use), e.g., for first-line therapy. In one embodiment, the bsAbs of the present disclosure are used to treat wild-type RAS metastatic colorectal cancer (defined as wild-type in both KRAS and NRAS, as determined by an FDA-approved test for this use) as monotherapy following disease progression following prior chemotherapy treatment (e.g., chemotherapy treatment with a chemotherapeutic agent containing fluoropyrimidine, oxaliplatin, and / or irinotecan). In certain embodiments, the use of the bsAb is not indicated for the treatment of patients with RAS mutant metastatic colorectal cancer or whose RAS mutation status is unknown.

[0174] In one embodiment, the bsAbs of the present disclosure, optionally in combination with radiation therapy, are used to treat local or regional advanced squamous cell carcinoma of the head and neck. In one embodiment, the bsAbs of the present disclosure, optionally in combination with platinum-based therapy employing fluorouracil, are used to treat recurrent locoregional disease or metastatic squamous cell carcinoma of the head and neck. In one embodiment, the bsAbs of the present disclosure are used to treat recurrent or metastatic squamous cell carcinoma of the head and neck that has progressed following platinum-based therapy.

[0175] Additional non-limiting examples of suitable cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS 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; colorectal 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; hepatoma); intraepithelial neoplasia; kidney cancer 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., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; teratocarcinoma; testicular cancer; thyroid cancer; uterine or endometrial cancer; urological cancer; vulvar cancer; and other cancers and sarcomas; and B-cell lymphomas (including low-grade / follicular non-Hodgkin 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 lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with keloids, edema (such as that associated with brain tumors), tumors of primitive origin, and Meigs' syndrome.

[0176] Additional cancers that can be treated using the bsAbs described herein include metastatic pancreatic cancer, metastatic pancreatic adenocarcinoma, gastric cancer, fibrotic carcinoma, glioma, malignant glioma, diffuse intrinsic pontine glioma, recurrent childhood brain neoplasms renal cell carcinoma, clear cell metastatic renal cell carcinoma, metastatic castration-resistant prostate cancer, stage IV prostate cancer, metastatic melanoma, malignant melanoma, recurrent cutaneous melanoma, melanoma brain metastasis, head and neck malignant melanoma, squamous cell non-small cell lung cancer, metastatic breast cancer, follicular lymphoma, advanced B-cell NHL, HL, including diffuse large B-cell lymphoma (DLBCL) ), multiple myeloma, chronic myeloid leukemia, adult acute myeloid leukemia in remission, adult acute myeloid leukemia with Inv(16)(p13.1q22), CBFB-MYH11, adult acute myeloid leukemia with t(16:16)(p13.1: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), adult acute myeloid leukemia with MLLT3-MLL, and tO15:17)(q22: q12), PML-RARA in adults with acute myeloid leukemia, alkylating agent-associated acute myeloid leukemia, Richter's syndrome, glioblastoma in adults, gliosarcoma in adults, recurrent glioblastoma, recurrent childhood rhabdomyosarcoma, recurrent Ewing 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; cervical squamous cell carcinoma, recurrent cervical cancer, anal squamous cell carcinoma, metastatic anal carcinoma, recurrent anal cancer carcinoma, recurrent head and neck cancer, head and neck squamous cell, head and neck squamous cell carcinoma (HNSCC), ovarian cancer, colon cancer, advanced GI cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, bone tumors, soft tissue sarcoma, osteosarcoma, thymic carcinoma, urothelial carcinoma, Merkel cell carcinoma, recurrent Merkel cell carcinoma, mycosis fungoides, Sezary syndrome, neuroendocrine carcinoma, nasopharyngeal carcinoma, basal cell skin carcinoma, squamous cell skin carcinoma, dermatofibrosarcoma protuberans, glioma, mesothelioma, myelodysplastic syndrome (MDS), myelofibrosis (MF), myeloproliferative neoplasms, and acute myeloid leukemia (AML).

[0177] The cancer can be, for example, a metastatic or primary cancer; a desmoplastic or non-desmoplastic cancer; or a recurrent cancer.

[0178] As described in the examples, the ability of bsAb described herein to inhibit cancer growth can be evaluated in a suitable animal model or human xenograft model predicting the effectiveness in human tumors. As described in the examples, in one embodiment, the xenograft model is the H1975 model. Alternatively, this property of the composition can be evaluated by using an in vitro assay known to those skilled in the art to check the ability of the compound to inhibit. One or more therapeutic agents of a therapeutically effective amount can reduce tumor size, or otherwise improve the symptoms of a subject. Those of ordinary skill 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 selected specific composition or route of administration.

[0179] In one embodiment, cancer patients to be treated with a bsAb of the present disclosure are selected prior to treatment based on the expression of one or more biomarkers that indicate the likelihood that the bsAb treatment will be effective. For example, for an NRP1×EGFR bispecific antibody, a cancer patient to be treated can be selected based on overexpression of EGFR, NRP1, or both on the patient's tumor cells. Methods for testing the surface expression of target biomarkers (e.g., expression of NRP1 and / or EGFR) on a patient's tumor cells prior to bsAb treatment are well-recognized in the art (e.g., by FACS analysis of flow cytometry, etc.). Combination therapy

[0180] In some embodiments, an effective amount of a composition of the present disclosure (e.g., a bispecific antibody) can be achieved in a method or pharmaceutical composition of the present invention using a polypeptide or a pharmaceutically acceptable salt or solvate (e.g., a hydrate) thereof alone or in combination with another suitable chemotherapeutic agent (e.g., oxaliplatin, irinotecan, or FOLFOX). When a "combination therapy" is employed, an effective amount can be achieved using a first amount of a polypeptide or a pharmaceutically acceptable salt or solvate (e.g., a hydrate) thereof and a second amount of another suitable therapeutic agent (e.g., a chemotherapeutic agent).

[0181] Anti-cancer agents suitable for combination therapy with the bsAbs described herein include, but are not limited to, surgery, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, radiation therapy and agents used for radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for treating cancer, such as anti-HER-2 antibodies (e.g. ), anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib ), platelet-derived growth factor inhibitors (e.g., GLEEVEC (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, and cytokines; antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets and / or neutralize the activity of one or more of the following targets: PD1, PDL1, PDL2 (e.g., pembrolizumab; nivolumab; MK-3475; AMP-224; MPDL3280A; MEDI0680; MSB0010718C; and / or MEDI4736); CTLA4 (e.g., Tesitumumab (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-β, BlyS, APRIL, BCMA or VEGF receptors, TRAIL / Apo2, PARP inhibitors (e.g., AZD-2281, LynparzaOCEACAM5arib, Rubraca Rucaparib; (Zejula) Niraparib), DNA damage repair inhibitors (e.g., ATMi, ATRi, DNAPKi), and other bioactive agents and organic chemical agents, including those described in Section VII. Combinations thereof are also specifically contemplated for use in the methods described herein.

[0182] Chemotherapeutic agents suitable for combination therapy with the bsAbs described herein include, but are not limited to, alkylating agents such as thiotepa and Cyclophosphamide; temozolomide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa and uredopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; polyacetogenins (particularly bratacin and bratacinone); camptothecins (including synthetic analogs the topoisomerase inhibitor RFS2000; bryostatin; callystatin; CC-1065 (including its synthetic analogs adolesin, carzelesin and biszelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycins (including synthetic analogs KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongestatin; nitrogen mustards such as chlorambucil, naphthyl mustard, chlorphosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride), melphalan, new nitrogen mustard, phenylephrine, prednisone, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma 1I and calicheamicin omega 1I (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); danemycins (d ynemicin), including danemycin A; bisphosphonates such as clodronate; esperamicin; and the neocarcinogen chromophore and related chromoprotein enediyne antibiotic chromophores, aclarubicin, actinomycin, authramycin, azaserine, bleomycin, actinomycin C, carabicin, caminomycin, carmophilin, chromomycinis, actinomycin D, daunorubicin, detoxibacin, 6-diazo-5-oxo-L-norleucine, Doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, mexilomycin, mitomycins such as mitomycin C, mycophenolic acid, nogamycin, olivetomycin, pyromycin, potfiromycin, puromycin, triferric doxorubicin, rhodorubicin, streptozotocin, streptozotocin, tuberculocidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as dimethylfolate, methotrexate, pteropterin, trimesastatin; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine ; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calutosterone, drostanolone propionate, cyclothiodine, cyclopentyl cyclothiodine, testolactone; antiadreners such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; acetylglucuronolactone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecil; diacronil; elformithine; elliptinium acetate acetate); epothilones; ethoxydimidine; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansine alkaloids such as maytansine and ansamitocin; mitoguanidine; mitoxantrone; mopidanmol; nitraerine; pentostatin; methaminomustine; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; Polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuisporic acid; triazoquinone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, baculozolin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulanol; pyrimethamine; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), Albumin engineered nanoparticle formulations without cremophor and paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.) and docetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine; 6-thioguanine; mercaptopurine; platinum analogs such as cisplatin, oxaliplatin and carboplatin, vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; navelbine, vinorelbine; nosocomial; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; comprestatin; leucovorin (LV); oxaliplatin, including oxaliplatin-based regimens (FOLFOX); CEACAM5atinib (TYKERB); PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib) that reduce cell proliferation ) and VEGF-A inhibitors, and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0183] In some embodiments, the additional therapeutic agent is an immune checkpoint inhibitor. In some embodiments, the additional therapeutic agent is selected from: anti-PD-1 antibody or antigen binding fragment thereof, anti-LAG3 antibody or antigen binding portion thereof, anti-VISTA antibody or antigen binding fragment thereof, anti-BTLA antibody or antigen binding fragment thereof, anti-TIM3 antibody or antigen binding fragment thereof, anti-CTLA4 antibody or antigen binding fragment thereof, anti-HVEM antibody or antigen binding fragment thereof, anti-CD27 antibody or antigen binding fragment thereof, anti-CD137 antibody or antigen binding fragment thereof, anti-OX40 antibody or antigen binding fragment thereof, anti-CD28 antibody or antigen binding fragment thereof, anti-PDL1 antibody or antigen binding fragment thereof, anti-PDL2 antibody or antigen binding fragment thereof, anti-GITR antibody or antigen binding fragment thereof, anti-ICOS antibody or antigen binding fragment thereof, anti-SIRPα antibody or antigen binding fragment thereof, anti-ILT2 antibody or antigen binding fragment thereof, anti-ILT3 antibody or antigen binding fragment thereof, anti-ILT4 antibody or antigen binding fragment thereof, anti-ILT5 antibody or antigen binding fragment thereof, and 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.

[0184] Co-administration includes administering the first and second amounts of the polypeptide and the chemotherapeutic agent in a substantially simultaneous manner, such as in a single pharmaceutical composition, e.g., a capsule having a fixed ratio of the first and second amounts, or each in a plurality of separate capsules. In addition, such co-administration also includes using each polypeptide in a sequential manner in either order.

[0185] When co-administration involves separate administration of a first amount of a polypeptide and a second amount of an additional therapeutic agent, the polypeptide is administered close enough in time to have the desired therapeutic effect. For example, the time period between each administration that can produce the desired therapeutic effect can range from several minutes to several hours, and can be determined based on the properties of each agent (such as efficacy, solubility, bioavailability, plasma half-life, and kinetic curves). For example, the polypeptide and the second therapeutic agent can be administered in any order within about 24 hours, within about 16 hours, within about 8 hours, within about 4 hours, within about 1 hour, or within about 30 minutes.

[0186] More specifically, the bispecific antibodies of the invention can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of the second anticancer agent to the subject.

[0187] It will be appreciated that methods of co-administering a first amount of a bispecific antibody and a second amount of an additional therapeutic agent may result in an enhanced or synergistic therapeutic effect, wherein the combined effect is greater than the additive effect resulting from separate administration of the first amount of the polypeptide and the second amount of the additional therapeutic agent.

[0188] The synergistic effect of a combination of therapies (e.g., a combination of prophylactic or therapeutic agents) can allow the use of one or more therapies at lower doses and / or the administration of the therapy to a subject at a lower frequency. The ability to utilize a lower dose of a therapy (e.g., a prophylactic or therapeutic agent) and / or the administration of the therapy at a lower frequency can reduce the toxicity associated with administering the therapy to a subject without reducing the effectiveness of the therapy in the treatment of cancer. In addition, the synergistic effect can result in improved effectiveness of the agent in preventing, managing, or treating a condition. Finally, the synergistic effect of a combination of therapies (e.g., a combination of prophylactic or therapeutic agents) can avoid or reduce the adverse or unwanted side effects associated with the use of any therapy alone.

[0189] When combination therapy using the bispecific antibodies of the invention is combined with another anti-cancer agent, both therapeutic agents can be administered so that the period of time between each administration can be longer (eg, days, weeks, or months).

[0190] The existence of synergistic effect can be determined using a method suitable for assessing drug interactions. Suitable methods include, for example, Sigmoid-Emax equation (Holford, NHG and Scheiner, LB, Clin.Pharmacokinet.6:429-453 (1981)), Loewe addition equation (Loewe, S. and Muischnek, H., Arch.Exp.PatholPharmacol.114:313-326 (1926)) and median effect equation (Chou, TC and Talalay, P., Adv.EnzymeRegul.22:27-55 (1984)). Each equation mentioned above can be applied together with experimental data to produce corresponding figures, to assist in assessing the effect of drug combination. The corresponding figures associated with the equation mentioned above are respectively concentration-effect curves, isobologram curves and combination index curves. Exemplary embodiments

[0191] The examples in this specification are not intended to and should not be used to limit the present invention; they are merely provided to illustrate the present invention. Example 1. Production of bispecific antibodies

[0192] The designed protein was generated by codon-optimized gene synthesis and inserted into pcDNA3.4 as an expression vector using Not I and Hind III restriction enzymes. Examples of 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, which are 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 comprising an NRP1 binding peptide. Examples of polynucleotide sequences encoding heavy chain polypeptides of bispecific antibodies are shown in SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 40 (encoding heavy chain polypeptides of SEQ ID NOs: 2-11 and 39, respectively). The polynucleotide sequence encoding the light chain polynucleotide combined with the heavy chain of the bispecific antibody is shown in SEQ ID NO: 25 (encoding the light chain polypeptide of SEQ ID NO: 12). The sequences shown in SEQ ID NO: 26-38 disclose polynucleotide sequences encoding GGGGS peptide linker subunits (the amino acid sequence of which is shown in SEQ ID NO: 13). The constructed expression vector includes a signal peptide and, in order to optimize transcription, a Kozak sequence may be included in the 5' untranslated region.

[0193] To obtain the amount of plasmid construct for transfection, transform the plasmid construct into One Shot TM Top10 E. coli competent cells and then cultured overnight. TM The construct plasmid was obtained using HiPure Expi plasmid large-scale preparation kit.

[0194] The fusion proteins were transiently expressed in a CHO-S system (Thermo Fisher Scientific Inc.). The proteins were expressed separately according to the manufacturer's instructions. TM SFM and ExpiFectamine TM A total of 0.8 μg of plasmid DNA with a 1:1 light chain to heavy chain ratio was prepared per ml of CHO-S culture. The mixture was plated at 6 × 10 6 CHO-S cells were added at a viable cell density of 10 cells / mL and greater than 98% viability. The cell culture was grown in a Nalgene shaker with a 19 mm orbit and shaking at 125 RPM. TM The culture was incubated overnight in a disposable PETG conical flask at 37°C, 80% humidity, 8% CO2. The next day, the culture was enhanced (ExpiCHO TMEnhancer; Thermo Fisher Scientific Inc.) and feed (ExpiCHO TM The culture was fed at 32°C, 80% humidity, 5% CO2 with shaking at 125 RPM with a 19 mm orbit. A second feeding was performed on day 5, and the culture was returned to 32°C until harvest on day 12. Harvesting was accomplished by centrifugation at 4000 × g for 20 minutes. The clarified supernatant was sterilized using an asymmetric polyethersulfone (PES) 0.22-μM filter assembly (Nalgene). The filtrate was stored at 4°C until purification the next day.

[0195] exist (GE Healthcare Life Sciences) using MabSelect prismA TM All antibody sterilized supernatants were purified by resin (GE Healthcare Life Sciences). The resin was balanced using 50mM sodium phosphate, 150mM NaCl, pH 7.0 buffer. The antibody supernatant was then loaded into the column. The resin was washed with 50mM sodium phosphate, 150mM NaCl, pH 7.0 buffer until the chromatographic baseline returned to the column equilibrium level. 100mM sodium acetate, 20% glycerol, pH 3.0 were then used for elution, and fractions were collected. Immediately, the fractions were neutralized with 1M Tris, pH 9. The fractions containing the main absorbance at a wavelength of 280nm were pooled in an Amicon 10-kDa ultrafiltration device for buffer exchange. Storage buffer (phosphate buffered saline) was used to remove the elution buffer by centrifugation seven times with half dilution in an Amicon concentrator. The material was subjected to SEC and subsequently stored at 4°C.

[0196] The antibody was purified using cation exchange chromatography. The cation exchange chromatography column (Capto S ImpAct) was disinfected with 1M NaOH and rinsed with MQ. It was balanced with 50mM NaAc pH 5.5 (starting buffer) and 50mM NaAc pH 5.5, 1M NaCl (elution buffer). The protein A purified antibody was loaded at a concentration of 1-2g antibody / mL resin. The column was then washed with 50mM NaAc pH 5.5. The antibody product was then eluted using a gradient of 5-60% elution buffer in 25 column volumes. Each peak in the CEX purification was collected separately and eluted by using Ultra-15 centrifugal filter units were concentrated by centrifugation at 4000 xg, followed by buffer exchange into PBS.

[0197] Size exclusion chromatography (SEC) analysis was performed on an Agilent Infinity 1260II Quaternary Pump High Performance Liquid Chromatography (HPLC) system with a diode array UV detector WR. Twenty (20) μg of antibody material was injected onto an aXBridgeProtein BEH SEC column. 2.5 μm, 4.6 mm×150 mm column. The mobile phase was 100 mM phosphate, 300 mM sodium chloride pH 7.0 at 50°C, and the flow rate was 0.3 mL / min. The antibody material was detected at wavelengths of 220, 280 and 330 nm at a sampling rate of 1 Hz during a 10 minute acquisition period. Example 2. Binding affinity of bispecific antibodies to EGFR and NRP1

[0198] BLI binding studies were performed using the Octet Red 96 system to evaluate the binding of the bispecific antibodies to recombinant huEGFR and huNRP1. Briefly, commercially sourced biotinylated huEGFR and huNRP1 were immobilized on streptavidin (Sa) biosensors and interrogated for binding and characterized with the generated constructs. Using BLI technology, the kinetics and binding affinity (equilibrium binding constant, K) of binding to the constructs were evaluated. D ) and were evaluated 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 assess their affinity for the targets. Constructs binding to huEGFR and huNRP1 and K D Determination.

[0199] huEGFR and huNRP1 (in their respective immobilization columns) were immobilized on Sa biosensors at a concentration of 0.2 μg / ml (loading signal range of 0.2-0.4 nanometers) in 2× kinetic buffer, with a loading time of 180 seconds per sensor. Baseline was run for 60 seconds after loading. Association of the constructs started at 100 nM (10 nM for high affinity binding), followed by 2 1:1 serial dilutions and a fourth well (blank) with buffer only. Dissociation of the constructs was then performed in the baseline wells. Reference sensors were generated by applying the constructs on the surface of a blank AMC biosensor or streptavidin biosensor. The association and dissociation steps were each 600 seconds. Data were analyzed using Octet analysis software, applying 1:1 and 2:1 model fits, which report the dissociation constants K D (M).

[0200] All constructs, including SEQ ID NO: 1 (control) and SEQ ID NO: 11, bound EGFR with double-digit picomolar affinities, see Table 1. For NRP-1 binding, the bsAb with heavy chain SEQ ID NO: 7 exhibited subnanomolar affinity, while SEQ ID NO: 1 bound NRP-1 with double-digit nanomolar affinity. Figure 1 A-1K shows the binding affinity curve of the bispecific antibody to immobilized huEGFR. Figure 2 A-2K shows the binding affinity curve of the bispecific antibody to immobilized huNRP1. Table 1. EGFR and NRP1 binding affinities of bispecific antibodies Example 3. Inhibition of VEGFR2 signaling in HUVEC cells

[0201] The bispecific antibody inhibition of VEGFR2 signaling in HUVEC cells was tested by probing electrophoresed cell lysates with anti-phosphorylated VEGFR2 on Western blot. HUVEC cells were grown overnight in EBM-2 medium supplemented with EGM-2 SingleQuots in 6-well plates. The next day, cells were incubated with 2ml of F-12K medium containing 0.1mg / ml heparin, endothelial cell growth supplements and 10% FBS for 4 hours. In order to observe the dose-dependent response to the antibody, cells were treated with bsAb for 30 minutes, and the bsAb was serially diluted 6 times from the highest concentration of 1μM to the lowest concentration of 0.005μM with serum-free medium, followed by 2.2ng / ml of VEGF165 or control for 10 minutes. Cell lysates were prepared by collecting cells with a cell scraper and incubating on ice with 100μL of NP-40 lysis buffer containing protease / phosphatase inhibitors for 20 minutes. The supernatant lysate was collected into a new tube after centrifugation at 13,000 rpm for 10 min, protein quantification was measured using BCA protein assay, and denatured at 70°C for 10 min after mixing with NuPAGE LDS sample buffer and LDS sample reducing buffer.

[0202] Cell lysate equal to 10 μg was loaded into a 4-12% Bis-Tris gel with 4 μl of PageRuler Plus prestained protein ladder and run at 200 V for 45 minutes. The gel was washed with distilled water and transferred to the membrane using the IBlot2 dry transfer system. The membrane was blocked with 5% milk in 1×TBST for 1 hour at room temperature and incubated overnight at 4°C in 5% milk in 1×TBST with anti-phosphorylated VEGFR2 (Y1175). The next day, the membrane was washed three times with TBST for 10 minutes and incubated with the secondary antibody in 5% milk at room temperature for 1 hour. The membrane was washed three times with 1×TBST for 10 minutes. Subsequently, the membrane was incubated with SuperSignal Femto chemiluminescent substrate for 1-2 minutes and imaged with Amersham ImageQuant 800. The blot was then stripped with Restore Plus Western Blot Stripping Buffer for 15 min at room temperature on a shaker, followed by three washes with 1× TBST, and the procedure was repeated starting with milk blocking, followed by incubation of anti-VEGFR2 primary antibody.

[0203] ImageJ software was used to analyze the intensity of the protein blotting bands of phosphorylated VEGFR2 (Y1175) and VEGFR2. The band density of the phosphorylated VEGFR2 (Y1175) obtained was normalized by VEGFR2, and the inhibition of VEGFR2 phosphorylation by bsAb was calculated by the ratio with VEGF165 control cell lysate (positive control). Prism9 software was used to determine the IC50 of bsAb to VEGFR2 phosphorylation. Figure 3 The Western blot in Figure 4 shows that incubation of HUVEC cells with the bispecific antibody of SEQ ID NO: 1 does not inhibit VEGF165-mediated VEGFR phosphorylation. Figures 4-10 show Western blots and IC50 graphs of inhibition of VEGFR2 phosphorylation after incubation with bispecific antibodies. The IC50 of the bispecific antibodies is shown in Table 2. Table 2. IC50 of bispecific antibodies for VEGFR signaling inhibition Example 4: Effect of bispecific antibodies on cell viability

[0204] Test the inhibition of cell viability of bispecific antibodies in H1975 lung cancer cell line. In brief, H1975 lung cancer cells were grown in RPMI1640 supplemented with 1% penicillin / streptomycin and 10% FBS. The cells were trypsinized and plated into 96-well 3D culture plates (3,000 cells / 120 μl / well) with round bottoms and incubated for 3-4 hours until they aggregated to form 3D spheroids. A reserve mother plate of bsAbs diluted 6 times from 5 μM to 0.6 μM was prepared. H1975 cells were treated with 30 μL of bsAb and incubated for 72 hours (in duplicate). Subsequently, CellTiter-Glo 3D reagent plus 25 μl of serum-free medium was added to each well, wrapped with foil, and incubated on a shaker for 7 minutes. The plate was read with a Varioskan Lux multi-mode plate reader and analyzed with Prism9 software.

[0205] Table 3 shows the IC50 of the bispecific antibodies. Fig. 11B ), the bispecific antibody having heavy chain SEQ ID NO: 1 (control) has Fig.11A The high IC50 (1.96 μM) shown in Figure 2. In the bispecific antibody with NRP1 short chain variable fragment (scFv) binding domain that improved cell growth inhibition, the IC50 of cell growth inhibition was much lower. Table 3. IC50 (μM) of cell growth inhibition Example 5: Tumor Growth Inhibition in the H1975 Xenograft Mouse Model Cell lines

[0206] The ability of bsAb to inhibit tumor growth in the H1975 cell line xenograft mouse model was tested. 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 2mmol / L glutamine (HyClone, Logan, UT, USA) at 37°C in a 5% CO2 incubator. Xenograft Models

[0207] Experiments and procedures involving mice were performed in accordance with the USD Department of Agriculture, Department of Health and Human Services and the NIH policy on the humane care and use of laboratory animals. Female athymic (nu / nu) mice aged 6 to 8 weeks from Charles River Laboratories (Wilmington, MA, USA) were housed in pathogen-free conditions with ad libitum access to laboratory food and water. All cell lines were screened for infectious agents (mycoplasma, etc.) prior to tumor cell line implantation. Xenografts were established by subcutaneous injection of 100 μL into the right flank, containing 1×10 per mouse mixed 1:1 in matrigel (Corning, Corning, NY, USA). 7 For efficacy studies, tumors were allowed to reach 150 to 300 mm before randomization and initiation of treatment. 3( 7 to 10 mice per group). Treatment and tumor measurements

[0208] Antibodies were diluted in sterile phosphate-buffered saline (Corning, Corning, NY, USA) and administered by ip injection at the designated dose level on the day of randomization with a total volume of 100 μL per mouse per treatment and twice weekly thereafter.

[0209] Tumor size was measured using digital calipers and the formula V = (W 2 × L) / 2, where W is the tumor width and L is the tumor length. Statistical analysis

[0210] Statistical analysis was performed using GraphPad Prism 7 software (La Jolla, CA, USA). Results are shown as mean values. Data between the control group and the experimental group at a single time point or endpoint were compared using Student's t-test. For growth curve analysis involving longitudinal data with repeated measures, type II ANOVA was used. Statistical differences of p < 0.05 were considered significant. TGI% was calculated according to the following formula: TGI (%) = (Vc1-Vt1) / (Vc0-Vt0) × 100, where V c1 and V t1 is the mean tumor volume in the control and treatment groups at the study endpoint, and V c0 and V t0 is the average tumor volume in the control and treatment groups at the beginning of the experiment.

[0211] Table 4 shows the bsAb tumor growth inhibition and percent tumor volume of treatment / control in xenograft mouse models. Figures 12A-12F The curve in shows the average tumor volume within days after implantation in mice treated with bsAbs. The bsAbs with a scFv NRP1 binding domain had the highest tumor growth inhibition, for example treatment with a bsAb with a heavy chain of SEQ ID NO: 11 resulted in 100% tumor growth inhibition. Table 4. Tumor growth inhibition and percentage of tumor volume for treatment / control Bispecific Antibody Peptides TGI% mean ± SEM T / C% SEQ ID NO: 1; 12 39.94 74.58％ SEQ ID NO:2;12 23.94 81.91％ SEQ ID NO:3; 12 71.13 37.15％ SEQ ID NO:7; 12 81.09％ 28.08％ SEQ ID NO:9; 12 94.96％ 18.86％ SEQ ID NO: 11; 12 102.56％ 12.54％ Example 6: Functional activities of additional bispecific antibodies

[0212] Another bispecific antibody was prepared, which was composed of a heavy chain comprising the amino acid sequence as shown 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 binding to NRP1 and EGFR are shown in Fig.13A and Fig. 13B The K of construct 12 binding to NRP1 D is 24.5 nM, and the K for binding to EGFR D is <0.1nM.

[0213] The anti-tumor activity of construct 12 was tested in the H1975 xenograft mouse model (described in Example 5) compared to construct 11 and isotype control. Mice were treated with bsAb (15 mg / kg, ip, QW) or isotype control (10.97 mg / kg, ip, QW) and the mean tumor volume was measured over time. The results are shown in Fig.14 , which demonstrated that construct 12 was as effective as construct 11 in reducing tumor growth in a xenograft model. Example 7: Receptor internalization and degradation activity of construct 11

[0214] In this example, construct 11 bsAb was tested for its ability to internalize NRP1 or EGFR and mediate receptor degradation compared to anti-NRP1 or anti-EGFR mAbs alone or both in combination.

[0215] For receptor internalization studies, H1975 or H1975-NRP1 OE cells (which overexpress NRP1) were treated with construct 11, anti-NRP1 mAb alone, anti-EGFR mAb alone, or anti-NRP1 + anti-EGFR in combination. For each sample, 200,000 cells were treated with 150 nM bsAb or mAb. Cell surface levels of NRP1 or EGFR (as % of control) were determined at 5 minutes, 1 hour, 5 hours, and 24 hours after treatment. Results for NRP1 expression are shown in Fig.16A and Fig. 16B The results of EGFR expression are shown in Fig. 16C and Fig.16D The results of H1975 cells are shown in Fig.16A and Fig. 16C The results of H1975-NRP1 OE cells are shown in Fig. 16B and Fig.16D For EGFR, the results showed that anti-NRP1 antibody alone did not internalize EGFR (as expected), while anti-EGFR mAb (alone or in combination with anti-NRP1) and construct 11 were both 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), NRP1 mAb alone or in combination with anti-EGFR mAb was able to partially internalize NRP1, while construct 11 was able to internalize NRP1 more effectively over time than anti-NRP1 mAb (alone or in combination with anti-EGFR).

[0216] For receptor degradation studies, cells were treated with construct 11, anti-NRP1 mAb alone, anti-EGFR mAb alone, or anti-NRP1 + anti-EGFR in combination, and the degradation of NRP1 and EGFR was determined. Briefly, H1975 cells (300,000 cells / sample) were cultured overnight in 6-well plates and subsequently treated with bsAb or mAb (150 nM) followed by incubation for 6 hours. Cell lysates were prepared and analyzed by standard Western blot to detect protein levels of EGFR, NRP1, and β-actin (used as a loading control). Results are shown in Fig.17 The results showed that construct 11 was more effective in degrading NRP1 and EGFR than single mAb treatment or combined treatment with anti-EGFR+anti-NRP1. Example 8: Production of anti-NRP1 monoclonal antibodies

[0217] The designed anti-NRP1 protein was produced by codon-optimized gene synthesis and inserted into pcDNA3.4 as an expression vector using Not I and Hind III restriction enzymes. Examples of 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.

[0218] The polynucleotide sequences encoding the heavy chain polypeptides of the monoclonal antibodies are shown in SEQ ID NOs: 55, 56, 57, 58, 59, 60 and 61 (which encode SEQ ID NOs: 41-47, respectively). The polynucleotide sequences encoding the light chain polypeptides combined with the heavy chain of the monoclonal antibodies are shown in SEQ ID NOs: 62, 63, 64, 65, 66, 67 and 68 (which encode SEQ ID NOs: 48-54, respectively). The constructed expression vector includes a signal peptide and, in order to optimize transcription, a Kozak sequence may be included in the 5' untranslated region.

[0219] To obtain the amount of plasmid construct for transfection, transform the plasmid construct into One Shot TM Top10 E. coli competent cells and then cultured overnight. TM The construct plasmid was obtained using HiPure Expi plasmid large-scale preparation kit.

[0220] The fusion proteins were transiently expressed in a CHO-S system (Thermo Fisher Scientific Inc.). The proteins were expressed separately according to the manufacturer's instructions. TM SFM and ExpiFectamine TM A total of 0.8 μg of plasmid DNA with a 1:1 light chain to heavy chain ratio was prepared per ml of CHO-S culture. The mixture was plated at 6 × 10 6 CHO-S cells were added at a viable cell density of 10 cells / mL and greater than 98% viability. The cell culture was grown in a Nalgene shaker with a 19 mm orbit and shaking at 125 RPM. TM The culture was incubated overnight in a disposable PETG conical flask at 37°C, 80% humidity, 8% CO2. The next day, the culture was enhanced (ExpiCHO TM Enhancer; Thermo Fisher Scientific Inc.) and feed (ExpiCHO TMThe culture was fed at 32°C, 80% humidity, 5% CO2 with shaking at 125 RPM with a 19 mm orbit. A second feeding was performed on day 5, and the culture was returned to 32°C until harvest on day 12. Harvesting was accomplished by centrifugation at 4000 × g for 20 minutes. The clarified supernatant was sterilized using an asymmetric polyethersulfone (PES) 0.22-μM filter assembly (Nalgene). The filtrate was stored at 4°C until purification the next day.

[0221] exist (GE Healthcare Life Sciences) using MabSelect prismA TM All antibody sterilized supernatants were purified by resin (GE Healthcare Life Sciences). The resin was balanced using 50mM sodium phosphate, 150mM NaCl, pH 7.0 buffer. The antibody supernatant was then loaded into the column. The resin was washed with 50mM sodium phosphate, 150mM NaCl, pH 7.0 buffer until the chromatographic baseline returned to the column equilibrium level. 100mM sodium acetate, 20% glycerol, pH 3.0 were then used for elution, and fractions were collected. Immediately, the fractions were neutralized with 1M Tris, pH 9. The fractions containing the main absorbance at a wavelength of 280nm were pooled in an Amicon 10-kDa ultrafiltration device for buffer exchange. Storage buffer (phosphate buffered saline) was used to remove the elution buffer by centrifugation seven times with half dilution in an Amicon concentrator. The material was subjected to SEC and subsequently stored at 4°C.

[0222] The antibody was purified using cation exchange chromatography. The cation exchange chromatography column (Capto S ImpAct) was disinfected with 1M NaOH and rinsed with MQ. It was balanced with 50mM NaAc pH 5.5 (starting buffer) and 50mM NaAc pH 5.5, 1M NaCl (elution buffer). The protein A purified antibody was loaded at a concentration of 1-2g antibody / mL resin. The column was then washed with 50mM NaAc pH 5.5. The antibody product was then eluted using a gradient of 5-60% elution buffer in 25 column volumes. Each peak in the CEX purification was collected separately and eluted by using The cells were concentrated by centrifugation at 4000 x g using an Ultra-15 centrifugal filter unit and resuspended in PBS.

[0223] Size exclusion chromatography (SEC) analysis was performed on an Agilent Infinity 1260II Quaternary Pump High Performance Liquid Chromatography (HPLC) system with a diode array UV detector WR. Twenty (20) μg of antibody material was injected onto an aXBridgeProtein BEH SEC column. 2.5 μm, 4.6 mm×150 mm column. The mobile phase was 100 mM phosphate, 300 mM sodium chloride pH 7.0 at 50°C, and the flow rate was 0.3 mL / min. The antibody material was detected at wavelengths of 220, 280 and 330 nm at a sampling rate of 1 Hz during a 10 minute acquisition period.

[0224] The activity of anti-NRP1 monoclonal antibodies can be evaluated by art-recognized methods. For example, binding affinity, inhibition of VEGFR2 signaling, and / or effects on in vitro cell viability or in vivo tumor growth can be evaluated as described in the Examples of U.S. Provisional Application No. 63 / 325,317 (the entire contents of which are expressly incorporated herein by reference). Sequence Listing Overview

Claims

1. A bispecific antibody comprising a first binding domain that binds to 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 shown in SEQ ID NO:79, HCDR2 consists of the sequence shown in SEQ ID NO:80, and HCDR3 consists of the sequence shown in any one of SEQ ID NOs:81-84; and (ii) an antibody light chain variable (VL) domain comprising CDR1, CDR2 and CDR3 regions (LCDR1, LCDR2 and LCDR3, respectively), wherein LCDR1 consists of the sequence shown in any one of SEQ ID NOs:85-87, LCDR2 consists of the sequence shown in SEQ ID NO:88, and LCDR3 consists of the sequence shown in SEQ ID NO:

89.

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

89.

3. A bispecific antibody comprising a first binding domain that binds to 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 comprises the sequence shown in SEQ ID NO:79, HCDR2 comprises the sequence shown in SEQ ID NO:80, and HCDR3 comprises the sequence shown in any one of SEQ ID NOs:81-84; and (ii) an antibody light chain variable (VL) domain comprising CDR1, CDR2 and CDR3 regions (LCDR1, LCDR2 and LCDR3, respectively), wherein LCDR1 comprises the sequence shown in any one of SEQ ID NOs:85-87, LCDR2 comprises the sequence shown in SEQ ID NO:88, and LCDR3 comprises the sequence shown in SEQ ID NO:

89.

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

5. The bispecific antibody of claim 4, 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 NRP1 binding single chain variable fragment (scFv), wherein the N-terminal variable heavy chain (VH) and the C-terminal scFv binding domain are located at opposite ends of a consecutive sequence.

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

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 comprises two polypeptides.

9. The bispecific antibody of claim 4, wherein 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), wherein HCDR1 consists of the sequence shown in SEQ ID NO:71, HCDR2 consists of the sequence shown in SEQ ID NO:72, and HCDR3 consists of the sequence shown 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 shown in SEQ ID NO:74, LCDR2 consists of the sequence shown in SEQ ID NO:75, and LCDR3 consists of the sequence shown in SEQ ID NO:

76.

10. The bispecific antibody of any one of claims 1-3, wherein the first binding domain comprises a VH domain comprising the sequence shown in SEQ ID NO: 77 and a VL domain comprising the sequence shown in SEQ ID NO:

78.

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

70.

12. 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 NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 39.

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 shown in SEQ ID NO:

12.

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

12.

15. The bispecific antibody of claim 4, wherein the binding affinity (K D ) is <0.1nM.

16. The bispecific antibody of claim 4, wherein the binding affinity (K D ) ranges from 0.1nM to 100nM.

17. The bispecific antibody of claim 4, wherein the EGFR binding domain comprises a K of the NRP1 binding domain to NRP1. D The K of the EGFR of 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 is greater than that of the EGFR of D .

18. 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).

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

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

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

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

23. A method for internalizing a neuropilin-1 (NRP1) receptor by a cell, the method comprising contacting the cell with the bispecific antibody of claim 1, such that NRP1 is internalized by the cell.

24. A method for internalizing epidermal growth factor receptor (EGFR) by a cell, the method comprising contacting the cell with the bispecific antibody of claim 4, such that EGFR is internalized by the cell.

25. A method of treating a cancer associated with aberrant NRPl expression in a subject, the method comprising administering to the subject the bispecific antibody of claim 1 or a pharmaceutical composition thereof.

26. A method of treating a cancer associated with aberrant EGFR expression in a subject, the method comprising administering to the subject the bispecific antibody of claim 4 or a pharmaceutical composition thereof.

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

28. The method of claim 26, further comprising administering to the subject at least one additional chemotherapeutic agent for combination therapy.

29. Use of the bispecific antibody according to claim 1 in the manufacture of a medicament for therapy.

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

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