Anti-netrin-1 antibodies and uses thereof

Abstract

Disclosed are new anti-Netrin-1 antibodies that specifically bind to Netrin-1 and block the interaction of Netrin-1 with the receptor UNC5B / DCC, and induce cell death or apoptosis of tumor cells having a Netrin-1 receptor (such as an UNC5 receptor), which can be used for further development into humanized Netrin-1 antibodies, and provide a new treatment strategy for tumor treatment, particularly for treatment of tumors with high metastatic activity and reduced sensitivity to chemotherapy.

Description

Technical Field

[0001] This invention relates to the field of antibody drugs for tumor treatment, specifically to an anti-Netrin-1 antibody that targets and binds to neural inducing factor-1 (Netrin-1) and blocks the interaction between Netrin-1 and its receptor UNC5B / DCC, thereby inducing cell death or apoptosis in tumor cells with Netrin-1 receptors (such as UNC5 receptors), and its use in the preparation of drugs for treating cancer. Background Technology

[0002] According to the latest global cancer burden data released by the International Agency for Research on Cancer (WHO-IARC) of the World Health Organization, there were 19.29 million new cancer cases and 9.96 million new cancer deaths worldwide in 2020. Furthermore, with the increasing aging of the population, the cancer burden is expected to increase by 50% by 2040 compared to 2020, with the number of new cancer cases reaching nearly 30 million at that time.

[0003] Netrin-1 is a member of the axon guidance factor family, a secreted laminin factor. Other members in humans include Netrin-3 and Netrin-4. Netrin-1 was first discovered to play an important role in the development of the nervous system through axon guidance function (Serafini et al., 1994, Cell, 78(3):409-24). In addition, Netrin-1 has been found to play a key role in cellular immunity, cell migration, angiogenesis, and cell survival (Cirulli et al., 2007, Nature reviews. Molecular cell biology, 8(4):296-306). At the same time, a large number of studies have shown that Netrin-1 is closely related to a variety of diseases such as cancer, cardiovascular disease, and diabetes (Bongo et al., 2014, Journal of Cardiology, 63(2):95-8; Cassier et al., 2023, Nature, 620(7973):409-416). Netrin-1 is highly expressed in a large proportion of human tumors, particularly in inflammation-associated colorectal cancer, metastatic breast cancer, lung cancer, neuroblastoma, lymphoma, and melanoma, by cancer cells and the tumor microenvironment.

[0004] The main receptors for Netrin-1 are DCC and members of the UNC5 family (including UNC5A, UNC5B, UNC5C, and UNC5D in humans), which belong to the dependent receptor family (Masu et al., 1996, Cell, 87:175-185; Mehlen et al., 1998, Nature, 395:801-804; Hong et al., 1999, Cell, 97:927-941). In the absence of Netrin-1, they can actively induce apoptosis, and when they bind to Netrin-1, they can promote cell survival, proliferation, and migration. This property can be used as a protective mechanism against tumor progression and can also be used for anti-tumor therapy (Mehlen et al., 2011, Nature Reviews. Cancer, 11(3):188-97; Bourgeois et al., 2009, Journal of the National Cancer Institute, 101(4):237-47). Numerous studies have shown that the expression of DCC and the UNC5 family is downregulated in various tumors, thereby reducing receptor-dependent apoptosis in tumor cells (Kinzler et al., 1996, Proc Natl Acad Sci, 100:4173-4178; Shin et al., 2007, Gastroenterology, 133:1849-1857). Meanwhile, increased autocrine expression of the ligand Netrin-1 has been observed in many tumors, promoting tumor growth and metastasis, or reducing receptor-dependent apoptosis (Fitamant et al., 2008, Proc Natl Acad Sci, 105:4850-4855; Dudgeon et al., 2023, Cell Reports, 42(11):113369).

[0005] The interaction pattern between Netrin-1 and its receptor has been elucidated. The V domain (the repeating region of laminin-like epidermal growth factor) of Netrin-1 can bind to two DCC molecules simultaneously, while binding to UNC5B occurs only in the V-2 region (Finci et al., 2014, Neuron, 83(4):839-849; Grandin et al., 2016, Cancer Cell, 29(2):173-85). Previously, a monoclonal antibody that specifically binds to the V-2 region of Netrin-1 has been developed. NP137 can block the interaction between Netrin-1 and its receptor (especially UNC5B) and promote apoptosis in tumor cells (CN105979966B). Clinical data show that NP137 has significant therapeutic effects in both endometrial cancer and squamous cell carcinoma of the skin. In addition to inhibiting tumor cell proliferation and inducing apoptosis, it can also inhibit the EMT pathway of tumor cells (Cassier et al., 2023, Nature, 620(7973):409-416; Lengrand et al., 2023, Nature, 620(7973):402-408). Meanwhile, studies have reported that another family member, Netrin-3, and Netrin-1 are specifically and independently expressed in different types of tumor cells. For example, Netrin-3 is highly expressed in small cell lung cancer and neuroblastoma, where Netrin-1 expression is very low. In addition, due to its high homology with Netrin-1, Netrin-3 can also bind to the Netrin-1 receptor and perform the same function (Jiang et al., 2021, EMBO molecular medicine, 13(4):e12878). This suggests that Netrin-3 and Netrin-1 have similar functions in promoting tumor survival in different tumors, and that blocking the binding of both Netrin-1 and Netrin-3 to their receptors may have a better range and effect of tumor treatment than single-target blocking.

[0006] Therefore, there is a need in the art for novel Netrin-1 antibodies. This invention develops a novel monoclonal antibody that can bind to both Netrin-1 and Netrin-3 simultaneously, with properties comparable to or better than existing antibodies. Summary of the Invention

[0007] The purpose of this invention is to address the problems of tumor metastasis and drug resistance by providing an anti-Netrin1 antibody or its antigen-binding fragment that promotes tumor cell apoptosis and inhibits tumor cell metastasis by inhibiting the binding of Netrin-1 and its receptor, a pharmaceutical composition comprising the same, and its use in the preparation of a medicament for treating cancer.

[0008] In a first aspect, the present invention provides an anti-Netrin-1 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NO:1, 2 and 3, respectively, and the VL comprises VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NO:4, 5 and 6, respectively.

[0009] In some embodiments, the VH comprises an amino acid sequence as shown in SEQ ID NO:7, or an amino acid sequence having at least 85% sequence homology with the amino acid sequence shown in SEQ ID NO:7.

[0010] In some embodiments, the VL comprises an amino acid sequence as shown in SEQ ID NO:8, or an amino acid sequence having at least 85% sequence homology with the amino acid sequence shown in SEQ ID NO:8.

[0011] In some embodiments, the anti-Netrin-1 antibody comprises scFv antibody molecules, nanobodies, and antibody constant regions.

[0012] In some embodiments, the anti-Netrin-1 antibody is a complete antibody.

[0013] In some embodiments, the anti-Netrin-1 antibody comprises a heavy chain (HC) and a light chain (LC).

[0014] In some embodiments, the anti-Netrin-1 antibody is a chimeric anti-Netrin-1 antibody.

[0015] In some embodiments, the anti-Netrin-1 antibody is IgG1.

[0016] In some embodiments, the anti-Netrin-1 antibody contains a human κ light chain constant domain in LC and a human IgG1 heavy chain constant domain in HC.

[0017] In some embodiments, the HC comprises an amino acid sequence as shown in SEQ ID NO:9, or an amino acid sequence having at least 85% sequence homology with the amino acid sequence shown in SEQ ID NO:9.

[0018] In some embodiments, the LC comprises an amino acid sequence as shown in SEQ ID NO:10, or an amino acid sequence having at least 85% sequence homology with the amino acid sequence shown in SEQ ID NO:10.

[0019] The aforementioned anti-Netrin-1 antibody or its antigen-binding fragment has one or more of the following functions:

[0020] (1) Combining human and cynomolgus monkey Netrin-1;

[0021] (2) It binds to human Netrin-3 but not to human Netrin-4;

[0022] (3) Competing with UNC5B / DCC to combine with Netrin-1;

[0023] (4) Competing with UNC5B / DCC to combine with Netrin-3;

[0024] (5) Promotes apoptosis in Netrin-1-dependent tumor cells;

[0025] (6) It can induce apoptosis of tumor cells via caspase 3 / 7;

[0026] (7) Inhibits tumor epithelial-mesenchymal transition (EMT) and reduces tumor metastasis;

[0027] (8) Enhance the sensitivity of tumors to chemotherapy.

[0028] Secondly, the present invention provides a method for preparing the anti-Netrin-1 antibody or a fragment thereof.

[0029] Thirdly, the present invention provides host cells for preparing the anti-Netrin-1 antibody or fragments thereof of the present invention.

[0030] Fourthly, the present invention provides a pharmaceutical composition comprising an anti-Netrin-1 antibody or an antigen-binding fragment thereof as described in the first aspect and a pharmaceutically acceptable medium, carrier or diluent.

[0031] Fifthly, the present invention provides the use of the anti-Netrin-1 antibody or its antigen-binding fragment as described in the first aspect or the pharmaceutical composition as described in the fourth aspect in the preparation of a medicament for treating cancer, wherein the cancer has tumor cells expressing the Netrin-1 receptor, or tumor cells expressing the Netrin receptor in mesenchymal cells expressing Netrin-1, or tumor cells expressing or overexpressing the Netrin-1 receptor of UNC5 class and / or DCC.

[0032] In some embodiments, the UNC5 receptor is UNC5B and / or UNC5A and / or UNC5C and / or UNC5D.

[0033] In some embodiments, the tumor includes lung adenocarcinoma, breast cancer, endometriosis, and arthritis.

[0034] In a sixth aspect, the present invention also provides a method for blocking the netrin-1-mediated signaling pathway in tumor cells using the antibody of the present invention.

[0035] The beneficial effects of the anti-Netrin-1 antibody of the present invention:

[0036] The anti-Netrin-1 antibody of the present invention can specifically bind to human and cynomolgus monkey Netrin-1 and human Netrin-3, and can competitively bind to human Netrin-1 and human Netrin-3 with the ligand UNC5B / DCC. It can promote apoptosis of netrin-1-dependent tumor cells and induce apoptosis of tumor cells via caspase 3 / 7. It can be used for further development into a humanized Netrin-1 antibody, providing a new therapeutic strategy for tumor treatment, especially for tumors with high metastatic activity and reduced sensitivity to chemotherapy. Attached Figure Description

[0037] Figure 1 The ELISA binding assays of antibody 49B7-CHI and control antibody 4C11-HUMO3 to human Netrin-1 are shown.

[0038] Figure 2 The ELISA binding assays of antibody 49B7-CHI and control antibody 4C11-HUMO3 to mouse Netrin-1 are shown.

[0039] Figure 3 The ELISA binding assays of antibody 49B7-CHI and control antibody 4C11-HUMO3 to Netrin-1 in cynomolgus monkeys are shown.

[0040] Figure 4 The ELISA binding assays of antibody 49B7-CHI and control antibody 4C11-HUMO3 to human Netrin-3 are shown.

[0041] Figure 5 The ELISA binding assays of antibody 49B7-CHI, control antibody 4C11-HUMO3, and positive control antibody Anti-Netrin-4 to human Netrin-4 are shown.

[0042] Figure 6 The study demonstrated the inhibition of human Netrin-1 binding receptor UNC5B by antibody 49B7-CHI and control antibody 4C11-HUMO3.

[0043] Figure 7 The study demonstrated the inhibition of human Netrin-1 binding receptor DCC by antibody 49B7-CHI and control antibody 4C11-HUMO3.

[0044] Figure 8 The study demonstrated the inhibition of human Netrin-3 binding receptor UNC5B by antibody 49B7-CHI and control antibody 4C11-HUMO3.

[0045] Figure 9 The study demonstrated the inhibition of human Netrin-3 binding receptor DCC by antibody 49B7-CHI and control antibody 4C11-HUMO3.

[0046] Figure 10 The study demonstrated that antibody 49B7-CHI and control antibody 4C11-HUMO3 induced caspase 3 / 7 activity in human lung adenocarcinoma cells A549. Detailed Implementation

[0047] I. Netin-1 antigen and its antigen / receptor mechanism in cancer

[0048] Clinical studies have found that interfering with the interaction between Netrin-1 and its receptor UNC5B / DCC can induce tumor cell apoptosis and inhibit tumor cell growth (Broutier et al., EMBO Mol. Med, 2016, 8: 96-104; Boussouar et al., Cancer Res. 2020, 80: 747-756; Broutier et al., EMBO Mol. Med, 2016, 8: 96–104; Boussouar, A et al., Cancer Res, 2020(80): 747–756). Recent studies have shown that Netrins also participate in the regulation of tumor occurrence and development, playing an important regulatory role in various tumor tissues such as colorectal cancer and pancreatic ductal adenocarcinoma (Yang Jie et al., Chinese Journal of Biotechnology, 2018, 6: 876-887). Apoptosis is mainly mediated through exogenous apoptosis signaling pathways, endogenous apoptosis signaling pathways, and caspase-independent apoptosis signaling pathways. The caspase family includes caspase-2, caspase-8, caspase-9, and caspase-10 as initiators of apoptosis, and caspase-3, caspase-6, and caspase-7 as executors. Caspase-3-induced apoptosis is considered one of the important mechanisms for timely removal of abnormal cells and prevention of tumor formation (Wang Xuerong et al., *Journal of Ningxia Medical University*, 2004, 2: 149-151). Caspase-3 is one of the most important executors of apoptosis, capable of efficiently and irreversibly inducing apoptosis (Jia Lintao, Dissertation, Air Force Medical University of the Chinese People's Liberation Army, 2001).

[0049] Netrin-1 receptors DCC and UNC5B belong to a family of dependent receptors, which are functional receptors. Netrin-1 receptors are downregulated or absent in various tumors, including colorectal cancer (Lü Dan et al., *Chinese Journal of Cancer Biotherapy*, 2011, 1:92-96). DCC and UNC5C genes are associated with the development and progression of colorectal cancer, showing a negative correlation with its invasion and metastasis. Both play important roles in colorectal cancer invasion and metastasis. DCC and UNC5C regulate apoptosis, but neither DCC nor UNC5C alone can function (Liu Xudong, doctoral dissertation, Tianjin Medical University, 2006). Members of the DCC receptor family play important roles not only in neural guidance and cell migration but also in various stages of cancer formation and development (Sun Yueni et al., *Inner Mongolia Medical Journal*, 2018, 1:28-30). Studies have found that Netrin-1 interacts with its receptor family, thus possessing a unique dual-signal function. This interaction can control neuronal migration and axonal growth, and also participate in the regulation of tumorigenesis and development (Wan Qianlin et al., *Everyone's Health (Academic Edition)*, 2016, 9:73-74). It has been reported that netrin-1 and its receptor genes are downregulated in various tumors. DCC and UNC5H, which do not bind to the ligand netrin-1, can induce apoptosis, while binding the ligand inhibits apoptosis. Apoptosis pathways are usually suppressed in tumor cells. Therefore, netrin-1 and its receptor may play an important role in tumorigenesis (Qin Shutong et al., *Journal of Medical Molecular Biology*, 2006, 4:300-304). Netrin-1 receptors are tumor suppressor factors. As dependent receptors, they can induce apoptosis without binding to Netrin-1. This mechanism can serve as a protective mechanism to prevent tumor development (Mehlen P et al., *Nat Rev Cancer*, 2011; 11(3):188-197). Netrin-1 receptors are downregulated in various tumor tissues and exhibit "dependence receptor" properties, meaning they can induce apoptosis even in the absence of the Netrin-1 ligand, thus classifying them as belonging to the "dependence receptor" (DR) family (Cao Dong, *Tianjin Medical Journal*, 2019, 8: 869-873). Furthermore, in preclinical models simulating cancer, interference between Netrin-1 and its receptor has been found to induce cancer cell death and tumor growth inhibition (Cassier PA et al., *Nature*, 2023, 620(7973): 409-416).

[0050] II. Antibody and Antigen-Binding Fragments

[0051] On one hand, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to Netrin-1.

[0052] The specific Netrin-1 binding antibody of the present invention can be a polyclonal or monoclonal antibody that specifically binds to human Netrin-1 (anti-Netrin-1 antibody or antibody that binds to Netrin-1).

[0053] The anti-Netrin-1 polyclonal antibody of the present invention can be prepared, in particular, by immunizing animals such as rabbits and mice with a selected amino acid sequence and then collecting the serum of the immunized animals.

[0054] Anti-Netrin-1 monoclonal antibodies (mAbs) can be mouse, chimeric, humanized, or fully human monoclonal antibodies. The fragment can be any type of mAb fragment that essentially retains the ability of the entire antibody to bind to Netrin-1.

[0055] In some embodiments, the anti-human Netrin-1 monoclonal antibody of the present invention is a murine antibody. Murine anti-human Netrin-1 antibodies can be obtained according to conventional methods of lymphocyte fusion and hybridoma culture described by Kohler and Milstein (Nature, 1975, 256(5517):495-7). In some embodiments, the monoclonal antibody can be prepared by immunizing mammals (e.g., mice) and using lymphocyte fusion techniques to generate hybridomas.

[0056] In some implementations, alternative techniques to this conventional technique may be used, for example, monoclonal antibodies may be produced by expressing nucleic acids from a hybridoma clone.

[0057] The various mAbs proposed in this invention (e.g., the murine monoclonal antibody named 49B7 and its chimeric antibody 49B7-CHI, etc.) are antibodies or fragments thereof that specifically bind to the Netrin-1 peptide or its variants shown in SEQ ID NO:11.

[0058] Antibodies can be natural or conventional, in which two heavy chains are linked together by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda and kappa. The variable regions of the light chain (VL) and heavy chain (VH) determine the binding recognition and specificity to the antigen. The constant regions of the light chain (CL) and heavy chain (CH) confer important biological properties, such as antibody chain association, secretion, transplacental migration, complement fixation, and binding to the Fc receptor (FcR).

[0059] The specificity of antibodies lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is mainly composed of residues from the complementarity-determining region (CDR). Sometimes, residues in the framework region (FR) can affect the overall domain structure, thereby influencing the binding site.

[0060] The "complementarity-determining region" or "CDR" refers to the amino acid sequence that collectively defines the binding affinity and specificity of the native Fv region of the natural immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, named VLCDR1, VLCDR2, VLCDR3 and VHCDR1, VHCDR2, VHCDR3, respectively. Therefore, a typical antibody-antigen binding site comprises six CDRs, containing a set of CDRs from each of the variable regions of the heavy and light chains.

[0061] The "frame region" (FR) refers to the amino acid sequence inserted between CDRs, specifically those relatively conserved portions of the variable regions of the immunoglobulin light and heavy chains in different immunoglobulins within a single species. Each immunoglobulin light and heavy chain has four FRs, designated as FR1-VL, FR2-VL, FR3-VL, FR4-VL and FR1-VH, FR2-VH, FR3-VH, FR4-VH, respectively.

[0062] In some embodiments, the amino acid sequence of Netrin-1 is shown in SEQ ID NO:23, the nucleotide sequence of the nucleic acid encoding Netrin-1 is shown in SEQ ID NO:24, the amino acid sequence of the Netrin-1 peptide is shown in SEQ ID NO:11, and the nucleotide sequence of the nucleic acid encoding the Netrin-1 peptide is shown in SEQ ID NO:16.

[0063] Antibodies that can be used in this invention can be defined by their CDRs.

[0064] Preferably, the antibody is a monoclonal antibody or its antigen-binding fragment, which comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0065] The present invention therefore relates to an antibody or antigen-binding fragment that specifically binds to the Netrin-1 peptide of the amino acid sequence shown in SEQ ID NO:11, comprising one or more monoclonal antibodies having the amino acid sequence VHCDR3 as shown in SEQ ID NO:3 and / or VLCDR3 as shown in SEQ ID NO:6, and preferably having the complementarity-determining region (CDR) of both, wherein the monoclonal antibody has the property of binding Netrin-1 and inducing cell death or apoptosis in tumor cells via UNC5 class or DCC receptors.

[0066] More specifically, the monoclonal antibody may be further defined by the additional presence of CDR1, CDR2 of VH and / or CDR1 and CDR2 of VL. Therefore, the antibody may comprise one or more CDR1, CDR2 of VH and / or CDR1 and CDR2 of VL having the following amino acid sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:5.

[0067] In embodiments of the art, VHCDR1 contains amino acid substitutions that are no more than 95% sequence identical to the amino acid sequence shown in SEQ ID NO:1.

[0068] In embodiments of the art, VHCDR2 comprises amino acid substitutions that are no more than 95% sequence identical to the amino acid sequence shown in SEQ ID NO:2.

[0069] In embodiments of the art, VHCDR3 comprises amino acid substitutions that are no more than 95% sequence identical to the amino acid sequence shown in SEQ ID NO:3.

[0070] In embodiments of the art, VLCDR1 contains amino acid substitutions that are no more than 95% sequence identical to the amino acid sequence shown in SEQ ID NO:4.

[0071] In embodiments of the art, VLCDR2 contains amino acid substitutions that are no more than 95% sequence identical to the amino acid sequence shown in SEQ ID NO:5.

[0072] In embodiments of the art, VLCDR3 comprises amino acid substitutions that are no more than 95% sequence identical to the amino acid sequence shown in SEQ ID NO:6.

[0073] In embodiments of the art, the anti-Netrin-1 antibody is an antibody comprising VHCDR1 with the sequence SEQ ID NO:1, VHCDR2 with the sequence SEQ ID NO:2, and VHCDR3 with the sequence SEQ ID NO:3.

[0074] In some embodiments, the anti-Netrin-1 antibody is an antibody comprising VLCDR1 with the sequence SEQ ID NO:4, VLCDR2 with the sequence SEQ ID NO:5, and VLCDR3 with the sequence SEQ ID NO:6.

[0075] In some embodiments, the VH comprises VHCDR1, VHCDR2, and VHCDR3 with amino acid sequences as shown in SEQ ID NO:1, 2, and 3, respectively, and the VL comprises VLCDR1, VLCDR2, and VLCDR3 with amino acid sequences as shown in SEQ ID NO:4, 5, and 6, respectively.

[0076] In some embodiments, the amino acid sequences VHCDR1, VHCDR2 and VHCDR3 in the VH as shown in SEQ ID NO:1, 2 and 3 respectively, and the amino acid sequences VLCDR1, VLCDR2 and VLCDR3 in the VL as shown in SEQ ID NO:4, 5 and 6 respectively, are shown in Table 1.

[0077] In some embodiments, the Netrin-1 binding antibody has the property of binding Netrin-1 and inducing cell death or apoptosis in tumor cells via receptors containing UNC5 or DCC.

[0078] In some embodiments, the Netrin-1 binding antibody or anti-Netrin-1 antibody is preferably a monoclonal antibody. Various combinations of the anti-Netrin-1 antibody and antigen-binding fragments described herein will be described later.

[0079] In some embodiments, the anti-Netrin-1 monoclonal antibody (mAb) of the present invention may be a mouse, chimeric, humanized, or fully human monoclonal antibody. Specifically, these monoclonal antibodies are antibodies or fragments thereof that specifically bind to the Netrin1 polypeptide having the amino acid sequence shown in SEQ ID NO:11 or a variant thereof having at least 98% amino acid sequence difference.

[0080] In some embodiments, the anti-Netrin-1 antibody is a chimeric antibody.

[0081] A "chimeric antibody" is an antibody in which a constant region or a portion thereof is altered, replaced, or exchanged such that a non-human variable region is linked to a constant region of a different species or belonging to a different species. In some embodiments, a "chimeric antibody" also refers to an antibody in which a variable region or a portion thereof is altered, replaced, or exchanged such that a constant region is linked to a variable region of a different species or belonging to another antibody class or subclass.

[0082] In some embodiments, the anti-Netrin-1 antibody is a human-mouse chimeric antibody.

[0083] In some embodiments, the human-mouse chimeric anti-Netrin-1 antibody comprises the amino acid VH, such as sequence SEQ ID NO:7.

[0084] In some embodiments, the human-mouse chimeric anti-Netrin-1 antibody comprises the amino acid VL of sequence SEQ ID NO:8.

[0085] Preferably, the human-mouse chimeric antibody comprises amino acids such as VH of sequence SEQ ID NO:7 and VL of sequence SEQ ID NO:8.

[0086] In some exemplary embodiments, the human-mouse chimeric anti-Netrin-1 monoclonal antibody is an antibody named 49B7-CHI, which comprises the murine VH and VL of the murine monoclonal antibody 49B7. 49B7-CHI can be obtained by transplanting the CDR of the murine 49B7 antibody into human IgG1.

[0087] According to well-known techniques in the field of antibody technology, the sequence of the human-mouse chimeric anti-Netrin-1 monoclonal antibody 49B7-CHI can serve as a starting sequence for further constructing a humanized anti-Netrin-1 mAb. For example, based on the 49B7-CHI antibody, specific amino acid modifications can be made to the frame region (VH FRs) of VH and / or the FRs (VH FRs) of VL to further obtain a humanized anti-Netrin-1 antibody. The resulting humanized anti-Netrin-1 antibody has the same CDR as the parental antibody 49B7-CHI. The technique of making specific modifications to the frame region (VH FRs) of VH and / or the FRs (VH FRs) of VL to further obtain humanization is a common technique used by those skilled in the art of antibody research.

[0088] As used in this article, the “human frame region” is a frame region that is substantially identical (approximately 85% or more, particularly 90%, 95%, 97%, 99%, or 100%) to the frame region of a natural antibody.

[0089] In the context of this invention, the CDR / FR definition in the immunoglobulin light or heavy chain will be determined based on the Kabat or IMGT definition.

[0090] Preferably, the residues in the antibody variable domain are typically numbered according to a system designed by Kabat et al.

[0091] The term "humanized antibody" refers to an antibody that is originally wholly or partially non-human in origin and has been modified to replace certain amino acids, particularly in the framework regions of the heavy and light chains, to avoid or minimize human immune responses. The constant domains of humanized antibodies are most often human CH and CL domains. In one embodiment, the humanized antibody has human constant domains. The goal of humanization is to reduce the immunogenicity of xenobiotic antibodies (e.g., mouse antibodies) introduced into humans while maintaining the antibody's intact antigen-binding affinity and specificity.

[0092] In embodiments of the art, humanized antibodies can be produced using a variety of techniques, such as surface repair and CDR transplantation, based on the chimeric anti-Netrin-1 antibody of the present invention.

[0093] In embodiments described in this art, the chimeric or humanized monoclonal anti-Netrin-1 antibody further comprises a heavy chain constant region (CH) and a light chain constant region (CL).

[0094] In some embodiments, the anti-Netrin-1 antibody of the present invention may further comprise a human IgG1 constant heavy chain (CH) and / or a human IgG1 constant light chain (CL), particularly a human light chain constant domain.

[0095] In some embodiments, the anti-Netrin-1 antibody of the present invention may further comprise a human IgG1 constant heavy chain (CH) with the amino acid sequence shown in SEQ ID NO:12.

[0096] In some embodiments, the anti-Netrin-1 antibody of the present invention may further comprise a human κ constant light chain (CL) with the amino acid sequence shown in SEQ ID NO:13.

[0097] In some embodiments, the anti-Netrin-1 antibody of the present invention may further comprise a human IgG1 heavy chain (HC) with the amino acid sequence shown in SEQ ID NO:9.

[0098] In some embodiments, the anti-Netrin-1 antibody of the present invention may further comprise a human κ-type light chain (LC) with the amino acid sequence shown in SEQ ID NO:10.

[0099] In some implementations, an "antigen-binding fragment" (or "antibody-binding moiety") refers to one or more fragments of an antibody that retain the ability to specifically bind to Netrin-1. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, biantibodies, and bispecific and multispecific antibodies formed from antibody fragments. In one aspect, the fragment contains the VH and VL sequences of antibody 49B7-CHI.

[0100] Other allotypes can also be used. The specific binding of the mAb, Fab fragment, and F(ab')2 fragment of this chimeric antibody to Netrin-1 and its ability to inhibit the binding of Netrin-1 to its receptor UNC5B are demonstrated in the examples described herein (Example 3).

[0101] Netrin-1 is highly homologous across different species.

[0102] In some embodiments, the chimeric anti-Netrin-1 antibody of the present invention has a slightly weaker ability to specifically cross-bind mouse Netrin-1 than the control antibody 4C11-HUMO3.

[0103] In some embodiments, the chimeric anti-Netrin-1 antibody of the present invention specifically cross-binds to cynomolgus monkey Netrin-1 more strongly than the control antibody 4C11-HUMO3.

[0104] III. Homologous variants and alternatives of anti-Netrin-1 antibodies

[0105] Based on the CDR sequence of the aforementioned anti-human Netrin-1 antibody of the present invention, functionally conserved variants of the anti-human Netrin-1 antibody of the present invention, which can bind to the Netrin-1 peptide or its variants shown in SEQ ID NO:11, can be obtained by conventional techniques for preparing homologous antibodies by those skilled in the art of antibody preparation, through conserved amino acid modifications of no more than 1%, no more than 2%, or no more than 3%.

[0106] (1) In some embodiments, variants of the anti-Netrin-1 antibody of the present invention are variant antibodies containing VHCDR1 having at least 97, 98 or 99% identity with SEQ ID NO: 1.

[0107] (2) In some embodiments, variants of the anti-Netrin-1 antibody of the present invention are variant antibodies containing VHCDR2 having at least 97, 98 or 99% identity with SEQ ID NO: 2.

[0108] (3) In some embodiments, the variant of the anti-Netrin-1 antibody of the present invention is a variant antibody comprising VH=CDR3 having at least 97, 98 or 99% identity with SEQ ID NO:3.

[0109] (4) In some embodiments, variants of the anti-Netrin-1 antibody of the present invention are variant antibodies containing VLCDR1 having at least 97, 98 or 99% identity with SEQ ID NO: 4.

[0110] (5) In some embodiments, variants of the anti-Netrin-1 antibody of the present invention are variant antibodies comprising VL CDR2 having at least 97, 98 or 99% identity with SEQ ID NO: 5.

[0111] (6) In some embodiments, variants of the anti-Netrin-1 antibody of the present invention are variant antibodies comprising VLCDR3 having at least 97, 98 or 99% identity with SEQ ID NO: 6.

[0112] In some embodiments, the variants of the anti-Netrin-1 antibody include one or more CDR variants of (1)-(6).

[0113] Based on the amino acid sequence of the VH or VL of the aforementioned anti-human Netrin-1 antibody of the present invention, functionally conserved variants of the anti-human Netrin-1 antibody of the present invention that can bind to the Netrin-1 peptide or variants thereof shown in SEQ ID NO:11 can be obtained by conservative amino acid modification of no more than 3%, or no more than 5%, or no more than 6%, or no more than 8%, or no more than 10%, or no more than 15%. These variants are obtainable by conventional techniques for preparing homologous antibodies by those skilled in the art.

[0114] (7) In some embodiments, the VH of the antibody variant comprises an amino acid sequence having at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99.5%, or at least 99.9% sequence homology with the amino acid sequence shown in SEQ ID NO:7.

[0115] (8) In some embodiments, the VL of the antibody variant comprises an amino acid sequence having at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99.5%, or at least 99.9% sequence homology with the amino acid sequence shown in SEQ ID NO:8.

[0116] In some embodiments, the variant of the anti-Netrin-1 antibody is either (7) or (8).

[0117] Based on the HC or LC amino acid sequence of the aforementioned anti-human Netrin-1 antibody of the present invention, functionally conserved variants of the anti-human Netrin-1 antibody of the present invention that can bind to the Netrin-1 peptide or variants thereof shown in SEQ ID NO:11 can be obtained by conservative amino acid modification of no more than 3%, or no more than 5%, or no more than 6%, or no more than 8%, or no more than 10%, or no more than 15%. These variants are obtainable by conventional techniques for preparing homologous antibodies by those skilled in the art.

[0118] (9) In some embodiments, the HC of the antibody variant comprises an amino acid sequence having at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99.5%, or at least 99.9% sequence homology with the amino acid sequence shown in SEQ ID NO:9.

[0119] (10) In some embodiments, the LC of the antibody variant comprises an amino acid sequence having at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99.5%, or at least 99.9% sequence homology with the amino acid sequence shown in SEQ ID NO:10.

[0120] In some embodiments, the variant of the anti-Netrin-1 antibody is either (9) or (10).

[0121] In some embodiments of the invention, a sequence "at least 85% identical to a reference sequence" means a sequence having 85% or more, particularly 90%, 91%, 92%, 93%, or 94% of its full length. It also means having 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with the full length of the reference sequence.

[0122] The percentage of "sequence identity" can be determined by comparing two sequences that are best aligned within a comparison window. The portion of the polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence to achieve optimal alignment. The percentage of sequence identity is calculated by determining the number of positions in both sequences where the same amino acid residues appear, resulting in a number of matching positions. This number of matching positions is then divided by the total number of positions in the comparison window, and the result is divided by 100.

[0123] In some embodiments, the variants can be generated by substitution of conserved amino acids.

[0124] In the context of this invention, "conservative amino acid substitution" is a substitution in which one amino acid residue in a polypeptide fragment is replaced by another amino acid residue with a side chain group having similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitution does not significantly alter the functional properties of a protein. Example properties of amino acid groups with similar chemical side chains include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; 7) sulfur-containing side chains: cysteine ​​and methionine. Conservative amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine-tryptophan, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0125] Throughout this application, the term "comprising" should be interpreted to encompass all specifically mentioned features as well as optional, additional, or unspecified features. As used herein, the use of the term "comprising" also discloses embodiments in which no features other than those specifically mentioned are present (i.e., "consisting of", "contributing to").

[0126] IV. Functional Activity

[0127] The antibodies or their homologous variants of the present invention can specifically bind to Netrin-1 in humans and cynomolgus monkeys, inhibit the interaction between Netrin-1 and its receptors (specifically UNC5 or DCC, especially UNC5B and DCC), and induce apoptosis or cell death in tumor cells that express or overexpress Netrin-1 and its receptor.

[0128] In some embodiments, the aforementioned anti-Netrin-1 antibody or its antigen-binding fragment has one or more of the following functions:

[0129] (1) Combined with human, cynomolgus monkey and mouse Netrin-1;

[0130] (2) It binds to human Netrin-3 but not to human Netrin-4;

[0131] (3) Competing with UNC5B / DCC to combine with Netrin-1;

[0132] (4) Competing with UNC5B / DCC to combine with Netrin-3;

[0133] (5) Promotes apoptosis in Netrin-1-dependent tumor cells;

[0134] (6) It can induce apoptosis of tumor cells via caspase 3 / 7;

[0135] (7) Inhibits tumor epithelial-mesenchymal transition (EMT) and reduces tumor metastasis;

[0136] (8) Enhance the sensitivity of tumors to chemotherapy.

[0137] 1. Specific binding to Netrin-1

[0138] As used herein, the term "specificity" refers to the ability of an antibody to detectably bind to a specific peptide present on an antigen (e.g., Netrin-1) while having relatively low detectable reactivity with non-Netrin-1 proteins or structures (e.g., Netrin-1).

[0139] In some embodiments, the specificity of the antibody can be assessed by measuring the binding of the anti-Netrin-1 antibody of the present invention to human Netrin-1 homologous protein using an ELISA method.

[0140] In some embodiments, the ELISA method measures that the binding EC50 of the chimeric anti-Netrin-1 antibody of the present invention to human Netrin-1 (such as SEQ ID NO:23) is not higher than 100 ng / mL, preferably not higher than 80 ng / mL, more preferably not higher than 50 ng / mL, and most preferably not higher than 20 ng / mL, which is comparable to the specificity level of the control antibody 4C11-HUMO3.

[0141] In some embodiments, the ELISA-detected binding EC50 of the chimeric anti-Netrin-1 antibody to cynomolgus monkey Netrin-1 (e.g., SEQ ID NO:21) is no higher than 150 ng / mL, or preferably no higher than 120 ng / mL, or more preferably no higher than 110 ng / mL, or more preferably no higher than 80 ng / mL, more preferably no higher than 50 ng / mL, and most preferably no higher than 37 ng / mL, which is significantly stronger in specific binding ability than the control antibody 4C11-HUMO3.

[0142] In some embodiments, the EC50 of the chimeric anti-Netrin-1 antibody binding to mouse Netrin-1 (e.g., SEQ ID NO:22) as measured by ELISA is preferably no higher than 80 ng / mL, more preferably no higher than 50 ng / mL, and most preferably no higher than 25 ng / mL, which is significantly weaker in specific binding ability than the control antibody 4C11-HUMO3.

[0143] 2. Binding affinity with human Netrin-1

[0144] In some implementations, affinity for Netrin-1 can be determined using, for example, biolayer interferoc-metry (BLI) techniques to further assess specificity.

[0145] In some implementations, the term "affinity" refers to the strength of antibody-epitope binding. Antibody affinity is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant Ka is defined as 1 / Kd.

[0146] In some embodiments, during the BLI assay under the conditions described in this invention, the binding constant (ka) of the chimeric antibody of this invention to human Netrin-1 is 6.35 × 10⁻⁶.5 (1 / Ms), the dissociation constant (kd) is 2.66 × 10 -3 (1 / s), the dissociation constant (KD value) is not higher than 10.03 nM, or not higher than 9.52 nM, or not higher than 8.54 nM, or not higher than 7.18 nM, or not higher than 6.04 nM, or preferably not higher than 5.26 nM, or most preferably not higher than 4.18 nM.

[0147] 3. Binding level with human Netrin-1 homologous protein

[0148] In some embodiments, the homologous proteins of human Netrin-1 include human Netrin-3 (e.g., SEQ ID NO:17) and Netrin-4 (e.g., SEQ ID NO:18), which share 54% and 31% sequence similarity with human Netrin-1, respectively.

[0149] In some embodiments, the chimeric anti-Netrin-1 antibody described in this invention can bind strongly to the homologous protein human Netrin-3 (e.g., SEQ ID NO:17).

[0150] In some embodiments, the chimeric anti-Netrin-1 antibody described in this invention exhibits a dose-dependent interaction with human Netrin-3.

[0151] In some embodiments, the anti-Netrin-1 antibody of the present invention does not show significant binding to human Netrin-4 (e.g., SEQ ID NO:18).

[0152] 4. Inhibits the binding of human Netrin-1 to its receptor.

[0153] In some embodiments, the anti-Netrin-1 antibody of the present invention can block the interaction between human Netrin-1 and receptors UNC5B or DCC.

[0154] In some embodiments, in ELISA-type assays, the blocking effect of the anti-Netrin-1 antibody of the present invention on the interaction between human Netrin-1 and receptor UNC5B or DCC is expressed as an inhibition rate.

[0155] In some embodiments, under high antibody concentration conditions, the anti-Netrin-1 antibody of the present invention has a significantly stronger blocking effect on the interaction between human Netrin-1 and UNC5B than the control antibody 4C11-HUMO3.

[0156] In some embodiments, at an antibody concentration of 40 μg / mL, the anti-Netrin-1 antibody of the present invention exhibits a significantly stronger blocking effect on the interaction between human Netrin-1 and UNC5B than the control antibody 4C11-HUMO3.

[0157] In some embodiments, at an antibody concentration of 40 μg / mL, the anti-Netrin-1 antibody of the present invention inhibits the interaction between human Netrin-1 and UNC5B by more than 60%.

[0158] In some embodiments, under any antibody concentration conditions, the anti-Netrin-1 antibody of the present invention has a significantly stronger blocking effect on the interaction between human Netrin-1 and DCC receptor than the control antibody 4C11-HUMO3.

[0159] In some embodiments, under high antibody concentration conditions, the anti-Netrin-1 antibody of the present invention has a higher blocking effect on the interaction between human Netrin-1 and DCC receptor than under low antibody concentration conditions.

[0160] In some embodiments, the chimeric anti-Netrin-1 antibody of the present invention exhibits a dose-dependent blocking effect on the interaction between human Netrin-1 and DCC receptor.

[0161] In some embodiments, under high antibody concentration conditions of 40 μg / mL, the chimeric anti-Netrin-1 antibody of the present invention blocks the interaction between human Netrin-1 and DCC receptor to a higher level than under low antibody concentration conditions.

[0162] In some embodiments, at an antibody concentration of 20 μg / mL, the chimeric anti-Netrin-1 antibody of the present invention inhibits the interaction between human Netrin-1 and DCC receptor by approximately 25%.

[0163] In some embodiments, at an antibody concentration of 30 μg / mL, the chimeric anti-Netrin-1 antibody of the present invention inhibits the interaction between human Netrin-1 and DCC receptor by approximately 40%.

[0164] In some embodiments, at an antibody concentration of 40 μg / mL, the chimeric anti-Netrin-1 antibody of the present invention inhibits the interaction between human Netrin-1 and DCC receptor by nearly 60%.

[0165] 5. Inhibits the binding of human Netrin-1 homologs to their receptors.

[0166] In some embodiments, the anti-Netrin-1 antibody of the present invention can block the interaction between the human Netrin-1 homolog and the receptor UNC5B or DCC.

[0167] In some implementations, the homolog of human Netrin-1 is human Netrin-3.

[0168] In some embodiments, the anti-Netrin-1 antibody of the present invention can block the interaction between human Netrin-3 and receptors UNC5B or DCC.

[0169] In some embodiments, the anti-Netrin-1 antibody of the present invention has a significantly stronger blocking effect on the interaction between human Netrin-3 and UNC5B or DCC receptors than the control antibody 4C11-HUMO3.

[0170] In some embodiments, the anti-Netrin-1 inhibitor of the present invention has a stronger blocking effect on the interaction between human Netrin-3 and the UNC5B or DCC receptor than on the binding of human Netrin-1 to the receptor.

[0171] In some embodiments, at an antibody concentration of 20 μg / mL, the anti-Netrin-1 antibody of the present invention achieves a 100% inhibition rate in blocking the interaction between human Netrin-3 and receptors UNC5B or DCC.

[0172] 6. Induction of caspase 3 / 7 activity in human lung adenocarcinoma cells A549

[0173] In some embodiments, the chimeric anti-Netrin-1 antibody of the present invention promotes the activation of caspase 3 / 7 in cancer cells.

[0174] In some embodiments, the cancer cells are human lung adenocarcinoma cells.

[0175] Under the same concentration conditions, the chimeric anti-Netrin-1 antibody of the present invention exhibits a stronger pro-apoptotic ability than the control antibody 4C11-HUMO3.

[0176] In some embodiments, the fluorescence intensity of caspase 3 / 7 enzyme activity can be measured using fluorescence methods to evaluate the induction level of caspase 3 / 7 enzyme production induced by the chimeric anti-Netrin-1 antibody of the present invention.

[0177] In some embodiments, fluorescence intensity measurements showed that the caspase 3 / 7 enzyme induced by the chimeric anti-Netrin-1 antibody of the present invention was as high as 5 × 10⁻⁶. 5 .

[0178] V. Encoding Nucleic Acids

[0179] In another aspect, the present invention provides a composition comprising a nucleic acid encoding the heavy chain (VH) of an antibody that specifically binds to Netrin-1, and a nucleic acid encoding the light chain (VL), wherein,

[0180] (1) The encoded nucleic acid of the VH contains a polynucleotide sequence as shown in SEQ ID NO:19, or a polynucleotide sequence having at least 85% sequence homology with the polynucleotide sequence shown in SEQ ID NO:19.

[0181] (2) The nucleic acid encoding the VL contains a polynucleotide sequence as shown in SEQ ID NO:20, or a polynucleotide sequence having at least 85% sequence homology with the polynucleotide sequence shown in SEQ ID NO:20.

[0182] VI. Preparation Method

[0183] The anti-Netrin-1 antibody of the present invention can be prepared using various methods known in the art.

[0184] Anti-Netrin-1 antibodies or antibody fragments thereof can be prepared by any method known in the art.

[0185] In some embodiments, the anti-Netrin-1 antibody is a monoclonal antibody.

[0186] In some embodiments, the anti-Netrin-1 monoclonal antibody comprises a murine antibody or a chimeric antibody.

[0187] In some embodiments, the method includes hybridoma methods and genetic engineering methods.

[0188] In one exemplary embodiment, the antibody is a murine antibody.

[0189] In some embodiments, the anti-Netrin-1 antibody of the present invention is a murine anti-Netrin-1 antibody, which is prepared by the hybridoma method.

[0190] For example, immunogens that can be used for immune recipients may include: DNA encoding human Netrin-1 or a fragment thereof, a fusion protein containing the full-length extracellular domain of Netrin-1, or recombinant cells engineered to overexpress human Netrin-1.

[0191] In some embodiments, the present invention obtains high-titer, high-affinity, and high-specificity antiserum and specific immune cells by co-immunizing BALB / c mice with manganese adjuvant as an immune adjuvant and Netrin-1 peptide-KLH conjugate; then, mouse spleen lymphocytes are extracted and hybridomas are generated with mouse myeloma cells by electrofusion, and hybridoma parent clones that can bind human Netrin-1 are screened; further, mouse monoclonal clones with Netrin-1 binding activity are isolated by limiting dilution, and the heavy chain variable region sequence and light chain variable region sequence of the mouse clones are obtained by sequencing.

[0192] In some embodiments, murine chimeric anti-human Netrin-1 antibodies can also be obtained through genetic engineering recombination technology. For example, DNA molecules encoding the heavy and light chain genes of the antibody of the present invention can be obtained through chemical synthesis or PCR amplification. The obtained DNA molecules are inserted into an expression vector, then transfected into host cells, and the transfected host cells are cultured under specific conditions to express the antibody of the present invention.

[0193] In one exemplary embodiment, the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of a murine monoclonal antibody named 49B7 obtained by hybridoma method can be obtained by sequencing the antibody. These sequences can be used as the VH and VL of the further constructed 49B7 murine chimeric antibody 49B7-CHI.

[0194] Generating coding nucleic acid sequences from known protein amino acid sequences is a skill that ordinary technicians in the antibody field are proficient in, such as PCR.

[0195] In some embodiments, the chimeric anti-Netrin-1 antibody of the present invention comprises VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 of the aforementioned murine antibody 49B7, respectively, as shown in SEQ ID NO:1-6.

[0196] In some embodiments, the anti-human Netrin-1 antibody of the present invention is a chimeric antibody prepared using a genetic engineering recombinant expression method. To recombinantly express the anti-human Netrin-1 chimeric antibody, host cells can be transfected with a heavy chain recombinant expression vector and a light chain recombinant expression vector, the recombinant expression vector comprising DNA fragments encoding the immunoglobulin light chain and heavy chain, respectively, such that the light chain and heavy chain are expressed in the host cells and optionally secreted into the culture medium culturing the host cells, from which the chimeric anti-Netrin-1 antibody of the present invention can be recovered. The standard recombinant DNA method is used to obtain the antibody heavy chain and light chain genes, incorporate these genes into the recombinant expression vector, and introduce the vector into the host cells. The heavy chain recombinant expression vector is obtained by linking the heavy chain variable region encoding nucleic acid (SEQ DI NO:19) of the murine anti-human Netrin-1 antibody of the present invention with an expression vector containing the human IgG1 heavy chain constant region (CH) encoding nucleic acid using genetic engineering technology. The light chain recombinant expression vector is obtained by linking the light chain variable region encoding nucleic acid (SEQ DI NO:20) of the murine anti-human Netrin-1 antibody of the present invention with an expression vector containing the human κ-type light chain constant region (CL) encoding nucleic acid using genetic engineering technology.

[0197] In some exemplary embodiments, the chimeric antibody against human Netrin-1 of the present invention is named 49B7-CHI.

[0198] Preparing humanized antibodies based on chimeric antibodies is a common technique mastered by skilled technicians in the field of antibody technology.

[0199] To reduce the immunogenicity of the antibody, a humanized anti-human Netrin-1 antibody can be further prepared based on the chimeric anti-human Netrin-1 antibody technique of the present invention, for example, by replacing its constant region with a constant region derived from human immunoglobulins. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing antigens while exhibiting reduced immunogenicity in humans, as compared to murine antibodies.

[0200] "Humanized antibody" refers to a chimeric antibody containing amino acid residues from a non-human CDR and amino acid residues from a human FR. In some embodiments, all or substantially all of the CDRs of a humanized antibody correspond to the CDRs of a non-human antibody, such as a mouse antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. The humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. The "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been designed and humanized.

[0201] Based on the aforementioned chimeric anti-Netrin-1 antibody of this invention, those skilled in the art of antibody technology can generate humanized Netrin-1 specific binding antibodies using genetic engineering methods.

[0202] For example, the nucleic acid encoding the heavy chain variable region of a humanized anti-Netrin-1 antibody can be designed based on the structure of VHFR1-VHCDR1-VHFR2-VHCDR2-VHFR3-VHCDR3-VHFR4, according to...

[0203] The design of the structure VLFR1-VLCDR1-VLFR2-VLCDR2-VLFR3-VLCDR3-VLFR4 encodes the nucleic acid of the light chain variable region of the humanized anti-Netrin-1 antibody.

[0204] For example, the chimeric anti-Netrin-1 antibody according to the present invention can obtain a humanized anti-Netrin-1 antibody whose heavy chain and / or light chain variable domains, in addition to containing a CDR sequence from a Netin-1 antibody such as a mouse chimeric antibody 49B7-CHI, will also contain a specific sequence human antibody frame region (FR) fragment that is highly homologous to the FR region of the chimeric antibody. Humanized anti-Netrin-1 antibodies can be achieved, for example, by humanizing the heavy chain variable region (VH) and light chain variable region (VL) encoded nucleic acids of a chimeric antibody that specifically binds to human Netrin-1 at the nucleic acid level through CDR transplantation, resulting in a CDR containing a murine antibody. However, the VH and VL domains contain the human antibody's shared frame region (human shared FR). Furthermore, referring to the frame region (FR) of the murine antibody, multiple amino acid substitutions are performed on the human antibody's shared FR to perform reversion mutations, thereby designing and generating the heavy chain variable region encoded nucleic acid sequence and light chain variable region encoded nucleic acid sequence of the humanized anti-human Netrin-1 antibody of this invention. Further, a recombinant expression plasmid is constructed and recombinantly expressed in host cells, containing the polynucleotide encoding the heavy and light chains of the humanized anti-Netrin-1 antibody.

[0205] The terms "shared human antibody frame region" and "human shared frame" are used interchangeably and represent the frame containing the most common amino acid residues selected for the humanized heavy chain variable region and humanized light chain variable region frame sequences. Typically, the selection of humanized heavy chain variable region and humanized light chain variable region sequences in human immunoglobulins is derived from a subgroup of variable domain sequences.

[0206] Once the DNA fragments encoding VH and VL containing the chimeric or humanized anti-Netrin-1 antibody are obtained, these fragments can be further manipulated using standard recombinant DNA techniques, such as converting the variable region gene into a full-length antibody chain gene, a Fab fragment gene, or an scFv gene. In these operations, the DNA fragment encoding VL or VH is operatively ligated to another DNA fragment encoding another protein, such as the antibody constant region or a flexible linker. For example, the isolated DNA encoding the VH region of the anti-Netrin-1 antibody of the present invention is converted into a full-length heavy chain gene (and a Fab heavy chain gene) by operatively ligating the DNA encoding VH to another DNA molecule encoding the heavy chain constant region domain (CH1, CH2, CH3, or optionally CH4). The sequence of the wild-type human heavy chain constant region gene is known in the antibody field. DNA fragments covering these regions can be obtained by standard PCR amplification. In some embodiments, the heavy chain constant region may be the human IgG1 constant region. For example, isolated DNA encoding the VL region of an anti-human Netrin-1 antibody can be converted into a full-length light chain gene (and a Fab light chain gene) by operably linking the DNA encoding VL to another DNA molecule encoding the light chain constant region CL. The sequence of the wild-type human light chain constant region gene is known in the art.

[0207] For example, to express the antibody or antigen-binding fragment of the present invention that specifically binds to human Netrin-1, those skilled in the art of antibodies can, through genetic engineering, insert the polynucleotides encoding the amino acid sequence VH shown in SEQ ID NO:7 and the amino acid sequence VL shown in SEQ ID NO:8 into a eukaryotic expression vector containing the nucleic acid encoding the human IgG1 constant region and a eukaryotic expression vector containing the nucleic acid encoding the human κ light chain constant region, respectively, to obtain a recombinant humanized heavy chain gene and a humanized light chain encoding nucleic acid operably linked to the transcription and translation control sequences of the humanized heavy chain recombinant expression vector and the humanized light chain recombinant expression vector, respectively, wherein the selected eukaryotic expression vector and expression control sequence are compatible with the expression host cell used.

[0208] According to the aforementioned method, those skilled in the art of antibodies can design, through CDR transplantation and reverse mutation, nucleic acids encoding amino acid sequences of heavy chain variable regions and light chain variable regions, such as those containing humanized frame regions (FRs), to construct expression plasmids containing the amino acid sequences shown in SEQ ID NO:12 and SEQ ID NO:13, respectively. Further, through genetic engineering, recombinant vectors expressing nucleic acids encoding the heavy chain (encoding the amino acid sequence shown in SEQ ID NO:9) and the light chain (encoding the amino acid sequence shown in SEQ ID NO:10) are obtained, respectively. These are then expressed in mammalian host cells to produce heavy and light chains containing humanized FRs. Optionally, in the humanized antibody, the CDRs of the murine anti-Netrin-1 antibody can be separated by spacer regions, such as frame regions (FRs) (e.g., the frame sequences described herein or the frame regions of germline common sequences of human antibodies).

[0209] For example, after co-transfecting a recombinant expression vector containing a full-length heavy chain encoding nucleic acid encoding a chimeric antibody or humanized antibody that specifically binds to Netrin-1 according to the present invention and a recombinant expression vector containing a full-length light chain encoding nucleic acid into mammalian host cells, the host cells are cultured sufficiently to allow antibody secretion to produce the anti-Netrin-1 chimeric antibody of the present invention, or the humanized anti-Netrin-1 antibody obtained by further humanizing the sequence of the chimeric anti-Netrin-1 antibody according to the present invention.

[0210] A variety of techniques can be used to achieve this goal, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques.

[0211] In some preferred embodiments, a eukaryotic expression plasmid vector encoding a nucleic acid for an anti-human Netrin-1 antibody is transfected into mammalian host cells via electroporation. The methods and conditions used for culturing the resulting transfected cells and for recovering the resulting antibody molecules are known to those skilled in the art and can be varied or optimized based on methods known in this specification and the prior art, depending on the specific expression vector and mammalian host cells used.

[0212] In some embodiments, the host cell is an expiCHOS cell.

[0213] Once the anti-Netrin-1 antibody or its antigen-binding fragment of the present invention has been generated through recombinant expression, it can be purified by any method known in the art (such as methods suitable for purifying immunoglobulin molecules), for example by chromatography.

[0214] VII. Composition

[0215] In another aspect, the present invention provides a pharmaceutical composition comprising at least one anti-Netrin-1 antibody according to the present invention and a pharmaceutically acceptable carrier or excipient. In some embodiments, the antibody is murine chimeric.

[0216] "Pharmaceutical-acceptable" or "pharmaceutical-admissible" means a molecular entity and composition that, when administered appropriately to mammals, particularly humans, does not produce adverse, allergic, or other adverse reactions. In this invention, a pharmaceutically acceptable carrier (or excipient) means any type of non-toxic or liquid filler, diluent, encapsulating material, or formulation aid.

[0217] The form and route of administration of the pharmaceutical composition containing the antibodies of the present invention naturally depend on the condition to be treated, the severity of the disease, the patient's age, weight, and sex, etc.

[0218] The antibodies of this invention can be formulated for topical, parenteral, intravenous, intramuscular, subcutaneous, or intraocular administration.

[0219] In some embodiments, the antibody of the present invention may be administered intravenously.

[0220] In particular, pharmaceutical compositions containing the antibodies of the present invention may contain a carrier that is pharmaceutically acceptable for injectable formulations. These may be, in particular, isotonic, sterile saline solutions (monophosphate or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or mixtures thereof), or dry, especially lyophilized compositions, which, when added, can be formulated into injectable solutions if sterile water or physiological saline is used.

[0221] Suitable drug forms for injection include sterile aqueous solutions or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid enough to be easily injected. It must be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi.

[0222] The excipient or carrier can be a solvent or dispersion medium, comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants, stabilizers, cryoprotectants, or antioxidants.

[0223] Sterile injectable solutions are prepared by incorporating the desired amount of the active compound with several other ingredients listed above (as needed) into a suitable solvent, followed by filtration and sterilization. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile carrier containing a basic dispersion medium and the desired other ingredients from those listed above. For sterile powders used to prepare sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying techniques to produce a powder containing the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

[0224] After preparation, the solution will be administered in a manner compatible with the dosage form and at a therapeutically effective amount. This formulation is readily available in various dosage forms, such as the injectable solution type described above.

[0225] For example, for parenteral administration in aqueous solution form, the solution should be appropriately buffered if necessary, and the liquid diluent should first be isotonic with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.

[0226] In embodiments described in this art, the pharmaceutical compositions of the present invention are used in combination with anticancer drugs and chemotherapy drugs in patients.

[0227] In embodiments of the art, the pharmaceutical composition and the chemotherapy drug are configured as a kit of parts, wherein the chemotherapy drug is used as an anticancer drug in patients treated with peptides or antibodies as disclosed herein.

[0228] In embodiments of the art, the chemotherapy drugs in the kit and the composition containing the aforementioned anti-Netrin-1 antibody are used to be administered to the patient simultaneously, separately, or sequentially.

[0229] VIII. Use in the preparation of medicines for treating diseases

[0230] In another aspect, the present invention provides the use of the aforementioned anti-Netrin-1 antibody in the preparation of a medicament for treating cancer.

[0231] In some well-known embodiments in the field of antibody pharmaceuticals, the anti-Netrin-1 antibody is combined with a chemotherapy drug to prepare a drug for treating cancer.

[0232] In some embodiments of the art, the tumor cells express or overexpress the Netrin-1 receptor (specifically, UNC5 class, particularly UNC5B and / or UNC5A and / or DCC). Typically, the tumor cells evade Netrin-1 receptor-associated apoptosis because Netrin-1 binds to the receptor, specifically UNC5 class, particularly UNC5B and / or UNC5A, and / or DCC, in the presence of Netrin-1.

[0233] In some embodiments of the field, the tumor cells express or overexpress Netrin-1.

[0234] In some embodiments of the field, the cancers expressing or overexpressing Netrin-1 include metastatic breast cancer, non-small cell lung cancer, aggressive neuroblastoma, pancreatic cancer, primary melanoma, metastatic melanoma, ovarian cancer, glioblastoma, acute myeloid leukemia, chronic lymphocytic leukemia, aggressive B-cell lymphoma, sarcoma, renal adenocarcinoma, head and neck cancer, testicular cancer (e.g., embryonal carcinoma, teratoma, yolk sac tumor), kidney cancer, gastric cancer, and uterine cancer.

[0235] In some embodiments of the art, the present invention provides the use of the aforementioned anti-Netrin-1 antibody and chemotherapy drugs in combination to prepare combined anticancer drug compositions or kits for simultaneous, separate or sequential administration to patients.

[0236] For example, the chemotherapy drugs mentioned may be cytotoxic drugs.

[0237] According to some embodiments in the art, the chemotherapeutic agent can be an alkylating agent. The alkylating agent can be a platinum derivative, such as cisplatin, carboplatin, oxaliplatin, or other alkylating agents such as cyclophosphamide, ifosfamide, melphalan, thiotepa. Other classes include epipodophylotoxine, such as etoposide; topoisomerase inhibitors (camptotecines), such as irinotecan, topotecan; and DNA minor groove alkylating agents, such as trabected, methotrexate, pemetrexed, raltitrexed.

[0238] In some embodiments of the art, the cytotoxic agent may be a cytotoxic antibiotic. Cytotoxic antibiotics may be actinomycins, anthracyclines, bleomycin, prikamycin, or mitomycin. Anthracyclines may be doxorubicin, daunorubicin, pentorubicin, idarubicin, or epirubicin.

[0239] In some embodiments of the art, the cytotoxic drug may be taxane or other tubulin-targeting agents. Taxane may be paclitaxel, docetaxel, or eribuline.

[0240] In some embodiments of the art, cytotoxic drugs may be antitumor agents.

[0241] In some embodiments of the art, the antitumor agent is a monoclonal antibody: for example, cetuximab, panitumumab, bevacizumab;

[0242] In some exemplary embodiments in the art, the antitumor agent may be a kinase inhibitor, such as imatinib, nilotinib, dasatinib, erlotinib, gefitinib, afatinib, sunitinib, sorafenib, pazopanib, crizotinib, and axitinib.

[0243] IX. Treatment methods

[0244] In another aspect, the present invention provides a method for treating cancer, wherein a therapeutically effective amount of a pharmaceutical composition comprising at least one anti-Netrin-1 antibody or antigen-binding fragment according to the present invention and a pharmaceutically acceptable carrier or excipient is administered to a subject in need. Therefore, the composition and method may comprise any or a combination of features disclosed in the antibodies disclosed herein.

[0245] In one embodiment, the cancer is one in which tumor cells express or overexpress the Netrin-1 receptor (specifically, UNC5 class, particularly UNC5B and / or UNC5A and / or DCC). Typically, the tumor cells evade Netrin-1 receptor-related apoptosis because Netrin-1 binds to the receptor, specifically, UNC5 class, particularly UNC5B and / or UNC5A, and / or DCC, in the presence of Netrin-1. According to one characteristic, the cancer is one in which tumor cells express or overexpress Netrin-1.

[0246] In one embodiment, some implementations of the cancers expressing or overexpressing Netrin-1 include metastatic breast cancer, non-small cell lung cancer, aggressive neuroblastoma, pancreatic cancer, primary melanoma, metastatic melanoma, ovarian cancer, glioblastoma, acute myeloid leukemia, chronic lymphocytic leukemia, aggressive B-cell lymphoma, sarcoma, renal adenocarcinoma, head and neck cancer, testicular cancer (e.g., embryonal carcinoma, teratoma, yolk sac tumor), kidney cancer, gastric cancer, and uterine cancer.

[0247] The term "treating cancer" refers to the inhibition of the growth of malignant cells in a tumor and / or the metastatic progression from said tumor. Such treatment can also lead to the regression of tumor growth, i.e., a measurable reduction in tumor size.

[0248] In this invention, the term "patient" or "patient in need" is intended for use in humans or non-human mammals that are affected by or may be affected by malignant tumors.

[0249] The "therapeutic effective dose" of the polypeptide or antibody of the present invention refers to an adequate amount that treats the cancerous disease with a reasonable benefit / risk ratio suitable for any medical treatment. However, it is understood that the total daily dosage of the polypeptide or antibody of the present invention will be determined by the attending physician within a reasonable medical judgment. The specific therapeutic effective dose level for any particular patient will depend on a variety of factors, including the condition being treated and its severity; the activity of the specific antibody used; the specific composition used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the specific antibody used; the duration of treatment; drugs used in combination with or concurrently with the specific antibody used; and similar factors well known in the medical field. In a specific embodiment, the therapeutic effective dose of the polypeptide or antibody administered to the patient is a dose range of 5 mg / m² to 500 mg / m², more specifically 150 mg / m² to 450 mg / m², per body surface area.

[0250] In a further embodiment, the polypeptide or antibody of the present invention is repeatedly administered according to an operating protocol dependent on the patient to be treated (age, weight, treatment history, etc.), which can be determined by a skilled physician.

[0251] Table 1. Sequence List

[0252]

[0253]

[0254]

[0255]

[0256] Example

[0257] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0258] Example 1: Antibody generation and screening

[0259] The protein used for animal immunization is a peptide-hemocyanin (KLH) conjugate. The peptide sequence (SEQ ID NO: 11) is selected from the V-2 domain of human Netrin-1 (SEQ ID NO: 23), which contains the major interaction site between Netrin-1 and UNC5B that has been publicly reported (Grandin et al., 2016, Cancer Cell, 29: 173-85). The peptide is conjugated to KLH via 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC). The synthesis of the peptide and its conjugation to KLH were both performed by Jier Biochemical (Shanghai) Co., Ltd.

[0260] Each BALB / c mouse received four multi-site intramuscular / subcutaneous injections of 60 μg peptide-KLH conjugate and 300 μg manganese adjuvant (Qimeng Biotechnology, MS0001) (once a week, with the adjuvant concentration adjusted to 1 mg / mL with physiological saline). Hybridoma fusion was performed four weeks after the first immunization. Three days prior to fusion, mice were given an intraperitoneal immunoshock with 60 μg peptide-KLH conjugate. Hybridoma supernatants were analyzed using ELISA to screen for monoclonal antibodies that simultaneously bind to human netrin-1 (Beijing Baipusaisi, NE1-H52H3), cynomolgus monkey netrin-1 (expressed by Baiying Biotechnology), and mouse netrin-1 (Wuhan Huamei Biotechnology, CSB-MP016127MO). ELISA was used to select monoclonal antibodies that could block the interaction between netrin-1 and UNC5B or DCC. Through these experiments, the present invention screened out a suitable mouse monoclonal antibody, clone number 49B7, whose heavy chain and light chain variable regions are composed of sequences SEQ ID NO: 7 and 8, respectively.

[0261] Example 2: Antibody Expression and Purification

[0262] This study used methods known to those skilled in the art for generating monoclonal antibodies based on encoding heavy and light chain nucleic acid sequences. The specific implementation method is as follows: sequences encoding the variable regions of the 49B7 heavy and light chains were cloned into heavy chain expression vectors and light chain expression vectors, respectively. The constant regions of both heavy and light chain expression vectors were derived from human IgG1 (heavy chain constant region SEQ ID NO: 12, light chain constant region SEQ ID NO: 13). Then, following the manufacturer's instructions, the plasmid DNA encoding 49B7-CHI was transiently transfected into expiCHO S cells (Thermo Fisher) using the ExpiCHO transfection kit (Gibco). The cells were cultured in ExpiCHO-S expression medium for 10 days, and the cell supernatant was collected, filtered (using a 0.22 μm filter), and then purified by affinity chromatography. Affinity chromatography capture was performed using Mab Select Sure LX (Cytiva) packing material with a retention time of 6 minutes. The fermentation supernatant was loaded onto the column at a loading rate of 40 mg protein per mL of packing material, with the target protein in elution buffer at pH 3.8. The resulting antibody solution was subjected to buffer replacement (PBS) and concentration via a 30 kDa ultrafiltration tube. The protein concentration was determined using Nanodrop (Thermo Fisher), and purity was analyzed by HPLC-SEC. The results showed a purity between 98% and 100%, yielding the chimeric monoclonal antibody 49B7-CHI (heavy chain SEQ ID NO: 9, light chain SEQ ID NO: 10). The control antibody 4C11-HUMO3 (heavy chain SEQ ID NO: 14, light chain SEQ ID NO: 15, from patent CN105979966B) was generated using the same method.

[0263] Example 3: ELISA assay of antibody binding to netrin-1

[0264] Incubate 50 μL of 1 μg / mL human, cynomolgus monkey, or mouse netrin-1 solution (prepared using PBS phosphate buffer) overnight at 4°C in a flat-bottomed 96-well plate. After washing three times with 300 μL of PBS solution containing 0.05% Tween-20 (PBS-T), add 200 μL of PBS-T solution containing 5% skim milk powder to each well and incubate at 37°C for 2 hours. After washing three times with 300 μL of PBS-T, add different concentrations of anti-netrin-1 antibody 49B7-CHI or 4C11-HUMO3 (control antibody) and incubate at 37°C for 1 hour. Dilute the antibody into a series of concentration gradients with PBS solution containing 1% BSA, starting at 20 μg / mL, 5-fold dilutions, for a total of 11 gradients. After washing three times with 300 μL of PBS-T, add 50 μL of horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody (Beijing Yiqiao Shenzhou, SSA005) dilution buffer (1:10000 diluted in 1% BSA-PBST solution) and incubate at 37°C for 1 hour. After washing three times with 300 μL of PBS-T, add 50 μL of single-component TMB chromogenic solution (Beijing Solarbio, PR1200) to each well. After color development, add 50 μL of 2M H2SO4 solution to terminate the reaction. Read the photometric value at 450 nm using a Thermofisher, Multiskan FC microplate reader.

[0265] Figure 1 The dose-dependent interaction of the 49B7-CHI chimeric antibody with human netrin-1 in an ELISA assay was demonstrated. The results showed that the 49B7-CHI antibody had a slightly higher 50% (EC50) human netrin-1 binding concentration than the control antibody 4C11-HUMO3, indicating that the 49B7-CHI antibody's ability to bind human netrin-1 in an ELISA assay was slightly weaker than that of the control antibody. Figure 2 The dose-dependent interaction of the 49B7-CHI chimeric antibody with mouse netrin-1 was demonstrated in an ELISA assay. Results showed that the 49B7-CHI antibody had a slightly weaker binding capacity to mouse netrin-1 than the control antibody. Figure 3 The study demonstrated the dose-dependent interaction between the 49B7-CHI chimeric antibody and cynomolgus netrin-1 in an ELISA assay. Results showed that the EC50 value of 49B7-CHI binding to cynomolgus netrin-1 was significantly lower than that of 4C11-HUMO3, and the maximum binding signal was also much greater than that of 4C11-HUMO3, indicating that the binding ability of 49B7-CHI to cynomolgus netrin-1 was significantly stronger than that of the control antibody 4C11-HUMO3.

[0266] Example 4: Detection of the binding affinity between antibody and human netrin-1 using biomembrane interferometry.

[0267] The affinity of the antibody for human netrin-1 was detected using biolayer interferometry (BLI). Human netrin-1-His was serially diluted from 100 nM to seven concentration points using PBST, with a zero-concentration reference well included. Antibody 49B7-CHI was diluted to 5 μg / mL. The molecular interaction instrument (ForteBio, Sartorius, Octet R8) was set to the following operating conditions: temperature 30°C, shake speed 1000 rpm. The antibody was captured using a pre-coated AHC2 probe (Sartorius, catalog number 18-5142) for 180 s; binding to the serially diluted netrin-1-His sample took 120 s; dissociation took 300 s; and regeneration was performed three times with regeneration buffer (10 mM glycine, pH 1.7), 30 s each time. Detection was performed using ForteBio's Octet System. After obtaining the sensor data, the binding constant (ka) and dissociation constant (kd) were analyzed using Octet BLIAnalysis software. An ideal binding-dissociation curve was fitted, and the equilibrium dissociation constant KD (kd / ka) between the antibody and antigen was calculated. The results showed that the binding constant (ka) of the chimeric antibody 49B7-CHI was 6.35 × 10⁻⁶. 5 (1 / Ms), the dissociation constant (kd) is 2.66 × 10 -3 (1 / s), with a dissociation constant (KD value) of 4.18 nM.

[0268] Example 5: ELISA assay of antibody binding to human netrin-1 homologous protein

[0269] The goal of these studies was to assess the binding specificity of 49B7-CHI to human netrin-1 homologs, including human netrin-3 and netrin-4, which share 54% and 31% sequence similarity with human netrin-1, respectively.

[0270] Incubate 50 μL of 1 μg / mL human netrin-3 (Wuhan Huamei Biotechnology, CSB-EP016128HU) solution (prepared with PBS) in a flat-bottomed 96-well plate overnight at 4°C. After washing three times with 300 μL of PBS-T, add 200 μL of PBS-T solution containing 5% skim milk powder to each well and incubate at 37°C for 2 hours. After washing three times with 300 μL of PBS-T, add different concentrations of 49B7-CHI or 4C11-HUMO3 and incubate at 37°C for 1 hour. Dilute the antibody into a series of concentration gradients with PBS solution containing 1% BSA, starting at 20 μg / mL, 5-fold dilution, for a total of 11 gradients. After washing three times with 300 μL of PBS-T, add 50 μL of HRP-conjugated goat anti-human IgG antibody dilution buffer (1:10000 diluted in 1% BSA-PBST solution) and incubate at 37°C for 1 hour. After washing three times with 300 μL of PBS-T, add 50 μL of single-component TMB chromogenic solution to each well. After color development, add 50 μL of 2MH2SO4 solution to terminate the reaction. Read the photometric value at 450 nm using a microplate reader. Figure 4 The results show the binding of the 49B7-CHI chimeric antibody to human netrin-3 in an ELISA assay. The results indicate a dose-dependent interaction between 49B7-CHI and human netrin-3, suggesting that 49B7-CHI can bind strongly to its homolog, human netrin-3, in addition to human netrin-1. In contrast, the control antibody 4C11-HUMO3 showed no significant binding to human netrin-3 in the ELISA assay.

[0271] Incubate 50 μL of 1 μg / mL human netrin-4 (Wuhan Huamei Biotechnology, CSB-MP881014HU) solution (prepared with PBS) in a flat-bottomed 96-well plate overnight at 4°C. After washing three times with 300 μL of PBS-T, add 200 μL of PBS-T solution containing 5% skim milk powder to each well and incubate at 37°C for 2 hours. After washing three times with 300 μL of PBS-T, add different concentrations of 49B7-CHI, 4C11-HUMO3, or Anti-netrin-4 antibody (Beijing Yiqiao Shenzhou, 203393-T08) and incubate at 37°C for 1 hour. Dilute the antibody into a series of concentration gradients with PBS solution containing 1% BSA, starting at 80 μg / mL, 2-fold dilution, for a total of 12 gradients. After washing three times with 300 μL of PBS-T, add 50 μL of HRP-conjugated goat anti-human IgG antibody dilution buffer (1:10000 diluted in 1% BSA-PBST solution) and incubate at 37°C for 1 hour. After washing three times with 300 μL of PBS-T, add 50 μL of single-component TMB chromogenic solution to each well. After color development, add 50 μL of 2M H2SO4 solution to terminate the reaction. Read the photometric value at 450 nm using a microplate reader. Figure 5 The results show the binding of the 49B7-CHI chimeric antibody to human netrin-4 in an ELISA assay. The results indicate that neither 49B7-CHI nor 4C11-HUMO3 significantly binds to human netrin-4, while the positive control antibody Anti-netrin-4 exhibits dose-dependent binding.

[0272] Example 6: Antibody inhibits the binding of human netrin-1 to its receptor

[0273] Incubate 50 μL of 1 μg / mL human UNC5B (Beijing Yiqiao Shenzhou, 13606-H02H) or human DCC (Beijing Baipusaisi, NEC-H5254) solution (prepared with PBS) in a flat-bottomed 96-well plate at 4°C overnight. After washing three times with 300 μL of PBS-T, add 200 μL of PBS-T solution containing 5% skim milk powder to each well and incubate at 37°C for 2 hours. Alternatively, mix human netrin-1-his (Beijing Baipusaisi, NE1-H52H3) with different concentrations of 49B7-CHI or 4C11-HUMO3 antibody in a flat-bottomed 96-well plate (prepared with PBS solution containing 1% BSA) and incubate at 37°C for half an hour. After sealing, the 96-well coated plates were washed three times with 300 μL of PBS-T. Then, a pre-mixed antibody and human netrin-1-his mixture was added. The final concentrations of 49B7-CHI or 4C11-HUMO3 antibodies were 0, 20, 30, and 40 μg / mL, with three replicates per group. The final concentration of human netrin-1-his was 1 μg / mL. The plates were incubated at 37°C for 1 hour. After washing three times with 300 μL of PBS-T, 50 μL of HRP-conjugated mouse anti-his antibody (Beijing Yiqiao Shenzhou, 105327-MM02T-H) dilution buffer (1:5000 diluted in 1% BSA-PBST solution) was added, and the plates were incubated at 37°C for 1 hour. After washing three times with 300 μL of PBS-T, 50 μL of single-component TMB chromogenic solution was added to each well. After chromogenic development, 50 μL of 2M H2SO4 solution was added to terminate the reaction. The photometric value at 450 nm was read using an ELISA reader. Figure 6 and Figure 7 The results of blocking the interaction between human netrin-1 and its receptors UNC5B or DCC using the 49B7-CHI chimeric antibody in ELISA assays are shown. The blocking effect is presented using the inhibition rate. Groups with an antibody concentration of 0 were designated as the control group, and groups containing neither antibody nor human netrin-1 protein were designated as the blank control group. The inhibition rate was calculated using the formula below.

[0274] Inhibition rate (%) = 100 * (Signal value of control group - Signal value of antibody group) / (Signal value of control group - Signal value of blank control group)

[0275] Experimental results showed that at antibody concentrations of 20 and 30 μg / mL, the blocking effect of 49B7-CHI on the interaction between human netrin-1 and UNC5B was slightly weaker than that of 4C11-HUMO3. However, at an antibody concentration of 40 μg / mL, the blocking effect of 49B7-CHI on the interaction between human netrin-1 and UNC5B was significantly stronger than that of the control antibody 4C11-HUMO3. Under all antibody concentration conditions, the blocking effect of 49B7-CHI on the interaction between human netrin-1 and the DCC receptor was stronger than that of the control antibody 4C11-HUMO3, especially at the high antibody concentration of 40 μg / mL, where the blocking effect of 49B7-CHI was significantly higher.

[0276] Example 7: Antibody inhibits the binding of human netrin-3 to its receptor

[0277] Incubate 50 μL of 1 μg / mL human UNC5B (Beijing Yiqiao Shenzhou, 13606-H02H) or human DCC (Beijing Baipusaisi, NEC-H5254) solution (prepared with PBS) in a flat-bottomed 96-well plate at 4°C overnight. After washing three times with 300 μL of PBS-T, add 200 μL of PBS-T solution containing 5% skim milk powder to each well and incubate at 37°C for 2 hours. Alternatively, mix human netrin-3-his (Wuhan Huamei Biotechnology, CSB-EP016128HU) with different concentrations of 49B7-CHI or 4C11-HUMO3 antibody in a flat-bottomed 96-well plate (prepared with PBS solution containing 1% BSA) and incubate at 37°C for half an hour. After sealing, the 96-well coated plate was washed three times with 300 μL of PBS-T. Then, a pre-mixed antibody and human netrin-3-his solution was added. The final concentrations of 49B7-CHI or 4C11-HUMO3 antibody were 0, 20, 30, and 40 μg / mL, with three replicates per group. The final concentration of human netrin-3-his was 2 μg / mL. The plate was incubated at 37°C for 1 hour. After washing three times with 300 μL of PBS-T, 50 μL of HRP-conjugated mouse anti-his antibody (Beijing Yiqiao Shenzhou, 105327-MM02T-H) dilution buffer (1:5000 diluted in 1% BSA-PBST solution) was added, and the plate was incubated at 37°C for 1 hour. After washing three times with 300 μL of PBS-T, 50 μL of single-component TMB chromogenic solution was added to each well. After chromogenic development, 50 μL of 2M H2SO4 solution was added to terminate the reaction. The photometric value at 450 nm was read using an ELISA reader. Figure 8 and Figure 9The results of blocking the interaction between human netrin-3 and its receptors UNC5B or DCC using the 49B7-CHI chimeric antibody in ELISA assays are shown. The blocking effect is presented using the inhibition rate. Groups with an antibody concentration of 0 were designated as the control group, and groups containing neither antibody nor human netrin-3 protein were designated as the blank control group. The inhibition rate was calculated using the formula below.

[0278] Inhibition rate (%) = 100 * (Signal value of control group - Signal value of antibody group) / (Signal value of control group - Signal value of blank control group)

[0279] Experimental results showed that, under all antibody concentration conditions, 49B7-CHI significantly enhanced the blocking effect of human netrin-3 on the interaction between UNC5B or DCC receptors compared to the control antibody 4C11-HUMO3. Furthermore, 49B7-CHI exhibited a greater blocking effect on the binding of human netrin-3 to its receptor than on the binding of human netrin-1 to its receptor. These findings indicate that 49B7-CHI demonstrates a clear advantage in blocking the interaction between human netrin-3 and UNC5B or DCC receptors.

[0280] Example 8: Antibody-induced activity of caspase 3 / 7 in human lung adenocarcinoma cells A549

[0281] On day 1, A549 cells resuspended in complete culture medium (F12K medium containing 10% fetal bovine serum) were seeded into 96-well cell culture plates at a density of 1 × 10⁶ cells per well. 5 Cells were added to each well. Simultaneously, 49B7-CHI or 4C11-HUMO3 antibody was added to a final concentration of 10 μg / mL, with a final volume of 100 μL per well. A control group consisted of wells without antibody. Each group had three replicates. The next day, the supernatant was discarded, and Caspase- The 3 / 7 Assay System reagent (Promega, G8091) was used to detect the activity of caspase 3 / 7 enzyme.

[0282] Figure 10 The study showed that 49B7-CHI promotes the activation of caspase 3 / 7 in A549 human lung adenocarcinoma cells. 49B7-CHI promotes tumor cell apoptosis by neutralizing cell-secreted netrin-1 in the culture medium, thereby inhibiting downstream pathways. At the same concentration, 49B7-CHI exhibited a stronger pro-apoptotic effect than the control antibody 4C11-HUMO3.

Claims

1. An anti-netrin-1 antibody or its antigen-binding fragment, comprising a heavy chain variable region (VH) and a light chain variable region (VL), characterized in that, The VH contains VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NO:1, 2 and 3 respectively, and the VL contains VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NO:4, 5 and 6 respectively.

2. The anti-netrin-1 antibody or its antigen-binding fragment according to claim 1, characterized in that, The VH contains an amino acid sequence as shown in SEQ ID NO:7, or an amino acid sequence having at least 85% sequence homology with the amino acid sequence shown in SEQ ID NO:

7.

3. The anti-netrin-1 antibody or its antigen-binding fragment according to claim 1, characterized in that, The VL contains an amino acid sequence as shown in SEQ ID NO:8, or an amino acid sequence having at least 85% sequence homology with the amino acid sequence shown in SEQ ID NO:

8.

4. The anti-netrin-1 antibody or its antigen-binding fragment according to any one of claims 1-3, characterized in that, The anti-netrin-1 antibody may include scFv antibody molecules, nanobodies, antibody constant regions, or complete antibodies.

5. The anti-netrin-1 antibody or its antigen-binding fragment according to any one of claims 1-4, characterized in that... The anti-netrin-1 antibody is a complete antibody, and / or the anti-netrin-1 antibody is IgG1, which further includes a human κ constant domain in LC and a human IgG1 constant domain in HC.

6. The anti-netrin-1 antibody or its antigen-binding fragment according to any one of claims 1-5, characterized in that, The anti-netrin-1 antibody comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence as shown in SEQ ID NO:9, or an amino acid sequence having at least 85% sequence homology with the amino acid sequence shown in SEQ ID NO:9, and / or the LC comprises an amino acid sequence as shown in SEQ ID NO:10, or an amino acid sequence having at least 85% sequence homology with the amino acid sequence shown in SEQ ID NO:

10.

7. The anti-netrin-1 antibody or its antigen-binding fragment according to any one of claims 1-5, characterized in that, The anti-netrin-1 antibody or its antigen-binding fragment has one or more of the following functions: (1) Combined with human, cynomolgus monkey and mouse netrin-1; (2) It binds to human netrin-3 but not to human netrin-4; (3) mimics the receptor UNC5B / DCC and / or competes with UNC5B / DCC for binding to netrin-1; (4) mimics the receptor UNC5B / DCC and / or competes with UNC5B / DCC for binding to netrin-3; (5) Promotes apoptosis in netrin-1-dependent tumor cells; (6) It can induce apoptosis of tumor cells via caspase 3 / 7; (7) Inhibits tumor epithelial-mesenchymal transition (EMT) and reduces tumor metastasis; (8) Enhance the sensitivity of tumors to chemotherapy.

8. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-7 and a pharmaceutically acceptable medium, carrier or diluent.

9. Use of the anti-netrin-1 antibody of any one of claims 1-7 or its antigen-binding fragment, or the pharmaceutical composition of claim 8, in the preparation of a medicament for treating cancer, characterized in that, The cancer is defined as: tumor cells expressing the Netrin-1 receptor, or tumor cells expressing the neuroinducible factor-1 receptor in mesenchymal cells expressing Netrin-1, or tumor cells expressing or overexpressing the neuroinducible factor-1 receptor of UNC5 class receptors and / or DCC receptors.

10. The use according to claim 9, wherein the UNC class 5 receptor is UNC5B and / or UNC5A.

11. The pharmaceutical composition according to claim 8, characterized in that, The composition further comprises a chemotherapy drug.

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