Anti-human Delta-like4 antibody as well as preparation and application thereof

Abstract

The invention discloses an anti-human Delta-like4 (Delta-like4) antibody as well as preparation and application of the anti-human Delta-like4 antibody. The antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3. The invention also discloses nucleic acid for coding the antibody, a recombinant expression vector and a transformant containing the antibody, a pharmaceutical composition containing the antibody, a kit and a kit kit. The invention also discloses application of the antibody in preparation of drugs for promoting generation of non-functional angiogenesis and / or resisting tumors. The anti-human Delta-like4 antibody disclosed by the invention has an obvious inhibition effect on the growth and maturation of blood vessels, can promote the generation of non-functional blood vessels and has an anti-tumor drug effect, and the drug effect is equivalent to that of a positive control (an anti-DLL4 antibody from ABBVIE) in vitro.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an anti-human Delta-like 4 antibody and its preparation and application. Background Art

[0002] Koehler and Milsten developed the in vitro hybridoma technique to generate mouse monoclonal antibodies. Compared to polyclonal antibodies, monoclonal antibodies offer advantages such as high specificity, high potency, high purity, strong reproducibility, low cost, and the ability to be mass-produced. These excellent characteristics make monoclonal antibodies a hot topic in future therapeutic research.

[0003] Tumor angiogenesis plays a crucial role in tumor growth and migration. The DLL4-Notch signaling pathway plays a crucial role in regulating vascular development and formation. High expression of DLL4 (Delta-like 4) can be observed in many tumor vessels. Studies have shown that blocking DLL4 / Notch signaling can inhibit the growth of various solid tumors in mice. Within these tumor tissues, tumor blood vessel density increases, but these newly formed vessels are inactive and unable to provide blood flow to the tumor tissue, effectively inhibiting tumor growth. Therefore, DLL4 has been identified as an important target for effective anti-angiogenesis.

[0004] Currently, there are two main categories of clinical research drugs targeting the Notch signaling pathway: small molecule inhibitors (GSIs) that block γ-secretase and monoclonal antibodies that target Notch receptors and ligands. Most GSIs are not very specific and can also inhibit the cleavage of normal Notch receptors, causing gastrointestinal toxicity and side effects. Similarly, monoclonal antibodies targeting Notch1 and Notch2 receptors can also cause serious gastrointestinal toxicity and side effects. Studies have shown that anti-DLL4 monoclonal antibodies can kill tumors that are antagonistic to anti-VEGF-A antibodies.

[0005] Mouse monoclonal antibodies have the advantages of high affinity and strong specificity, but their application in humans still presents many challenges, such as different antibody subclasses, short serum half-life, and HAMA effect induction. The current approach to overcoming these difficulties is humanization of mouse monoclonal antibodies. Summary of the Invention

[0006] To address the current lack of an effective anti-DLL4-specific monoclonal antibody with both angiogenesis-promoting and anti-tumor efficacy, the present invention provides an anti-human Delta-like 4 antibody, its preparation, and use. The monoclonal antibody specifically binds to the extracellular domain of DLL4, blocking the DLL4-Notch signaling pathway.

[0007] To solve the above technical problems, one of the technical solutions provided by the present invention is: an antibody, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, and the light chain variable region comprising LCDR1, LCDR2 and LCDR3; wherein: the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 6.

[0008] In a specific embodiment of the present invention, the framework region of the heavy chain variable region and / or the light chain variable region is a murine framework region.

[0009] In a specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0010] In a specific embodiment of the present invention, the framework regions of the heavy chain variable region and / or light chain variable region are human framework regions.

[0011] In a specific embodiment of the present invention, the framework region of the heavy chain variable region is derived from the human germline heavy chain IGHV1-3*01; and / or, the framework region of the light chain variable region is derived from the human germline light chain IGKV3-11*01.

[0012] In a specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 12; and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 9, 10 or 11.

[0013] In a specific embodiment of the present invention, the antibody is a full-length antibody, Fab, Fab', F(ab')2 or Fv; the Fv is preferably a scFv.

[0014] In a specific embodiment of the present invention, the antibody is a full-length antibody, and the heavy chain constant region and / or light chain constant region thereof are derived from a human antibody.

[0015] In a specific embodiment of the present invention, the heavy chain constant region is derived from a human heavy chain IgG1 constant region; and / or the light chain constant region is derived from a human light chain κ chain constant region.

[0016] In a specific embodiment of the present invention, the amino acid sequence of the heavy chain constant region of the antibody is shown in SEQ ID NO: 13; and / or the amino acid sequence of the light chain constant region of the antibody is shown in SEQ ID NO: 14.

[0017] To solve the above technical problems, the present invention provides a second technical solution: an isolated nucleic acid encoding the antibody as described in one of the technical solutions of the present invention.

[0018] To solve the above technical problems, the third technical solution provided by the present invention is: a recombinant expression vector, which comprises the isolated nucleic acid as described in the second technical solution of the present invention.

[0019] In a specific embodiment of the present invention, the backbone of the recombinant expression vector is pCDNA3.1.

[0020] To solve the above technical problems, the fourth technical solution provided by the present invention is: a transformant, which comprises the nucleic acid as described in the second technical solution of the present invention or the recombinant expression vector as described in the third technical solution of the present invention, and the host cell of the transformant is a eukaryotic cell or a prokaryotic cell.

[0021] In a specific embodiment of the present invention, the eukaryotic cell is a mammalian cell, such as a 293F cell or an EXPI293 cell.

[0022] To solve the above technical problems, the fifth technical solution provided by the present invention is: a method for preparing an antibody, the method comprising culturing the transformant as described in the fourth technical solution of the present invention.

[0023] To solve the above technical problems, the sixth technical solution provided by the present invention is: a pharmaceutical composition, comprising the antibody as described in one of the technical solutions of the present invention, and a pharmaceutically acceptable carrier.

[0024] To solve the above technical problems, the present invention provides a seventh technical solution: a chimeric antigen receptor, which comprises the antibody as described in one of the technical solutions of the present invention.

[0025] To solve the above technical problems, the eighth technical solution provided by the present invention is: a genetically modified cell, wherein the genetically modified cell comprises the chimeric antigen receptor as described in the seventh technical solution of the present invention.

[0026] In a specific embodiment of the present invention, the genetically modified cells are eukaryotic cells, preferably isolated human cells; more preferably immune cells such as T cells or NK cells.

[0027] To solve the above technical problems, the present invention provides a ninth technical solution: an antibody-drug conjugate (ADC), wherein the antibody-drug conjugate comprises a cytotoxic agent or a label, and the antibody according to one of the technical solutions of the present invention.

[0028] To solve the above technical problems, the present invention provides a tenth technical solution: a kit, comprising the antibody according to one of the technical solutions of the present invention, the pharmaceutical composition according to the sixth technical solution of the present invention, the chimeric antigen receptor according to the seventh technical solution of the present invention, the genetically modified cell according to the eighth technical solution of the present invention, and / or the antibody-drug conjugate according to the ninth technical solution of the present invention.

[0029] To solve the above technical problems, the present invention provides an eleventh technical solution: a set of medicine boxes, comprising medicine box A and medicine box B, wherein:

[0030] The drug kit A contains the antibody according to one of the technical solutions of the present invention, the pharmaceutical composition according to the sixth technical solution of the present invention, the chimeric antigen receptor according to the seventh technical solution of the present invention, the genetically modified cell according to the eighth technical solution of the present invention, and / or the antibody-drug conjugate according to the ninth technical solution of the present invention;

[0031] The medicine kit B contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and / or other anti-tumor drugs.

[0032] To solve the above technical problems, the present invention provides a twelfth technical solution: a drug delivery device, comprising the antibody according to one of the technical solutions of the present invention, the pharmaceutical composition according to the sixth technical solution of the present invention, the chimeric antigen receptor according to the seventh technical solution of the present invention, the genetically modified cell according to the eighth technical solution of the present invention, and / or the antibody-drug conjugate according to the ninth technical solution of the present invention.

[0033] In a specific embodiment of the present invention, the drug delivery device further comprises a component for administering the antibody, the chimeric antigen receptor, the antibody drug conjugate, the genetically modified cell or the pharmaceutical composition to a subject, such as a syringe or an infusion device.

[0034] To solve the above technical problems, the present invention provides a thirteenth technical solution: a method for detecting a protein comprising the extracellular region of DLL4 for non-diagnostic purposes, the method comprising using the antibody described in one of the technical solutions of the present invention, the pharmaceutical composition described in the sixth technical solution of the present invention, the chimeric antigen receptor described in the seventh technical solution of the present invention, the genetically modified cell described in the eighth technical solution of the present invention and / or the antibody-drug conjugate described in the ninth technical solution of the present invention.

[0035] In a specific embodiment of the present invention, the DLL4 is human DLL4; and / or, the protein comprising the extracellular region of DLL4 is a full-length DLL4 protein.

[0036] To solve the above technical problems, the fourteenth technical solution provided by the present invention is: use of the antibody described in one of the technical solutions of the present invention, the pharmaceutical composition described in the sixth technical solution of the present invention, the chimeric antigen receptor described in the seventh technical solution of the present invention, the genetically modified cell described in the eighth technical solution of the present invention and / or the antibody-drug conjugate described in the ninth technical solution of the present invention in the preparation of drugs that promote non-functional angiogenesis and / or anti-tumor effects.

[0037] In a specific embodiment of the present invention, the tumor is a solid tumor.

[0038] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0039] The reagents and raw materials used in the present invention are commercially available.

[0040] The present invention provides a novel anti-Delta-like 4 antibody with a novel sequence that binds to DLL4 with high affinity, blocking the DLL4-Notch signaling pathway. This antibody significantly inhibits vascular growth and maturation, promotes the formation of non-functional angiogenesis, and exhibits anti-tumor efficacy comparable to that of a positive control (anti-DLL4 antibody from ABT-165 (ABBVIE)) in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The EC of Ab700 is the protein binding activity of DLL4 humanized antibody. 50 The EC value of antibody Ab710 was 204.1 ng / mL. 50 The EC value of antibody Ab720 was 43.83 ng / mL. 50 The EC value of antibody Ab7C11 was 91.15 ng / mL. 50 The value was 29.27 ng / mL, and the Isotype refers to the negative control without adding antibody.

[0042] Figure 2 The EC of Ab700 is the cell binding activity of humanized DLL4 antibody. 50 The EC value of antibody Ab710 was 80.98 ng / mL. 50 The EC value of antibody Ab720 was 10.26 ng / mL. 50The EC value of antibody Ab7C11 was 44.21 ng / mL. 50 The value was 22.52 ng / mL, and the Isotype refers to the negative control without adding antibody.

[0043] Figure 3 Microscopic photos of HUVEC angiogenesis induced by humanized DLL4 antibody in vitro.

[0044] Figure 4 Statistical graph of DLL4 humanized antibody-induced HUVEC angiogenesis in vitro. A is the negative control group, B is the anti-VEGF positive antibody group, C is the Ab700 antibody experimental group, D is the Ab710 antibody experimental group, E is the Ab720 antibody experimental group, and F is the anti-DLL4 positive antibody group. DETAILED DESCRIPTION

[0045] In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the procedures in molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA used herein are conventional procedures widely used in the corresponding fields. In addition, for a better understanding of the present invention, the following definitions and explanations of relevant terms are provided:

[0046] In the present invention, the letters in the amino acid sequence represent the single-letter abbreviations of amino acids known in the art, such as those described in J.Biol.Chem, 243, p3558 (1968): alanine: Ala-A, arginine: Arg-R, aspartic acid: Asp-D, cysteine: Cys-C, glutamine: Gln-Q, glutamic acid: Glu-E, histidine: His-H, glycine: Gly-G, asparagine: Asn-N, tyrosine: Tyr-Y, proline: Pro-P, serine: Ser-S, methionine: Met-M, lysine: Lys-K, valine: Val-V, isoleucine: Ile-I, phenylalanine: Phe-F, leucine: Leu-L, tryptophan: Trp-W, threonine: Thr-T.

[0047] In the present invention, the amino acid sequences of the listed CDRs are shown according to the definition of the Kabat numbering convention. However, it is well known to those skilled in the art that the CDRs of antibodies can be defined in the art by a variety of methods, such as Chothia (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), based on the three-dimensional structure of the antibody and the topology of the CDR loop, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT, World Wide Web imgt.cines.fr / ), and North based on affinity propagation clustering using a large number of crystal structures. CDR Definition. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions as defined by any of the above-mentioned known schemes described in the present invention.

[0048] Therefore, when referring to antibodies defined by specific CDR sequences defined herein, the scope of the antibodies also covers antibodies whose variable region sequences comprise the specific CDR sequences, but whose claimed CDR boundaries differ from the specific CDR boundaries defined herein due to the application of a different scheme (e.g., a different assignment system rule or combination). Although the scope of the present invention is based on the sequences shown in the definition according to the Kabat numbering convention, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of the present invention.

[0049] In the present invention, the term "full-length antibody" is used interchangeably to refer to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region is composed of one domain, CL. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulins IgA, IgD, IgE, IgG, and IgM. Complete antibodies form a "Y" shape. The stem of the Y is composed of the second and third constant regions of the two heavy chains (and the fourth constant region for IgE and IgM) bound together, and disulfide bonds (between chains) are formed in the hinge. The heavy chains γ, α, and δ have a constant region consisting of three tandem (in a row) Ig domains and a hinge region for increased flexibility; the heavy chains μ and ε have a constant region consisting of four immunoglobulin domains. The second and third constant regions are called "CH2 domains" and "CH3 domains," respectively. Each arm of the Y includes the variable region of a single heavy chain bound to the variable and constant regions of a single light chain and the first constant region. The variable regions of the light and heavy chains are responsible for antigen binding.

[0050] As used herein, a "Fab fragment" consists of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions within the CH3 domain. A "Fab' fragment" contains one light chain and a portion of one heavy chain comprising the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains. This allows for interchain disulfide bonds to form between the two heavy chains of the two Fab' fragments, forming a F(ab')2 molecule. A "F(ab')2 fragment" contains two light chains and two heavy chains comprising a portion of the constant region between the CH1 and CH2 domains. This allows for interchain disulfide bonds to form between the two heavy chains. Thus, a F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains. The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody, but lacking the constant region.

[0051] In the present invention, the scFv refers to a single chain antibody fragment, which includes a heavy chain variable region, a light chain variable region and a connecting peptide of 15 to 20 amino acids. The VL and VH domains are paired to form a monovalent molecule by a connecting peptide that enables them to be produced as a single polypeptide chain [see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules can have a general structure: NH2-VL-connecting peptide-VH-COOH or NH2-VH-connecting peptide-VL-COOH.

[0052] In the present invention, "nucleic acid" refers to a nucleotide chain of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerase.

[0053] In the present invention, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that permits expression of the mRNA, protein, polypeptide, or peptide by the host cell when the construct comprises a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and the vector is in contact with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell. The vectors of the present invention are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of the present invention may comprise any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthesized or partially obtained from natural sources, and which may contain natural, non-natural, or altered nucleotides. The recombinant expression vector may comprise naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. In exemplary aspects, the altered nucleotides or non-naturally occurring internucleotide linkages do not hinder transcription or replication of the vector.

[0054] The recombinant expression vector of the present invention can be any suitable recombinant expression vector that can be used to transform or transfect one or more genes or sequences of interest into any suitable host cell and preferably express the genes or sequences in the host cell. Suitable vectors include those designed for expansion and amplification or for expression or both, examples of vectors include but are not limited to viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0055] In the present invention, the term "host cell" refers to any type of cell that can contain the nucleic acid or vector described herein. In exemplary aspects, the host cell is a eukaryotic cell, such as a plant, animal, fungus or algae; or it can be a prokaryotic cell, such as a bacterium or protozoa.

[0056] In the present invention, pharmaceutical composition can comprise suitable pharmaceutically acceptable carrier such as pharmaceutical excipient, as pharmaceutical carrier as known in the art, pharmaceutical excipient, comprises buffer." pharmaceutically acceptable carrier " comprises any and all solvents, dispersion medium, isotonic agent and absorption delaying agent etc. that are compatible physiologically. When intravenously administering pharmaceutical composition, water is preferred carrier. Saline solution and aqueous dextrose and glycerol solution can also be used as liquid carrier, particularly for injectable solution. Can prepare comprising pharmaceutical composition of the present invention by mixing with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, the 16th edition, Osol, A. compiles (1980)) of the antibody of the present invention with required purity, preferably in the form of lyophilized preparation or aqueous solution.

[0057] The pharmaceutical composition of the present invention can also comprise more than one active ingredient, the active ingredient being required for the specific indication being treated, preferably having those active ingredients of complementary activity that do not adversely affect each other. For example, it is desirable to also provide other active ingredients, such as other antibodies, antiviral active agents, small molecule drugs or immunomodulators, etc. The active ingredients are suitably combined in an amount effective for the intended use. Sustained release formulations can be prepared, and suitable examples thereof include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody of the present invention, the matrix being a shaped article, such as a film or microcapsule form.

[0058] In the present invention, chimeric antigen receptor (CAR) is an engineered transmembrane protein that combines the specificity of antigen-specific antibodies with T cell receptor function. In general, CAR includes an extracellular domain, a transmembrane domain, and an intracellular domain. In exemplary aspects, the extracellular domain of CAR includes an antigen recognition region, which can be an scFV of an antigen-specific antibody.

[0059] In the present invention, the term "antibody-drug conjugate" generally refers to an antibody linked to a biologically active cytotoxic drug via a stable linker. The "cytotoxic drug" may include toxins, such as small molecule toxins or enzymatic toxins of bacterial, fungal, plant, or animal origin, radioactive isotopes, toxic drugs, chemotherapeutic drugs, antibiotics, or nucleolytic enzymes, or derivatives thereof.

[0060] In the present invention, application scenarios of "non-diagnostic purposes" include but are not limited to: for example, in vitro detection of the presence or absence of antigens (proteins containing the extracellular region of DLL4) in the laboratory; or as a positive antibody to screen other antibodies targeting Delta-like 4; or competing with other antibodies targeting Delta-like 4 for binding, detecting whether there is competition between antibodies, that is, whether the antigen epitopes are the same or similar, and other application scenarios.

[0061] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0062] Example 1: Screening of mouse monoclonal antibodies

[0063] Mouse monoclonal antibodies were prepared using hybridoma cell technology. For experimental protocols, please refer to the literature (Ed Harlow, David Lane. Antibody: A laboratory manual. 1988).

[0064] A full-length DLL4 gene plasmid was purchased (from Sino Biological Pharmaceuticals, Beijing). PCR was used to obtain the full-length and extracellular fragments of DLL4 and construct them into the plasmid pCDNA3.4 (common in the art), resulting in the full-length expression plasmid pCDNA3.4-DLL4-FL and the extracellular region expression plasmid pCDNA3.4-DLL4-ECD. The DLL4 extracellular region-HIS fragment (containing a HIS tag) was amplified by PCR and constructed into the pCDNA3.4 expression plasmid using HindIII and EcoRI, resulting in the expression plasmid pCDNA3.4-DLL4-ECD-HIS. The DLL4 extracellular region was amplified by PCR and constructed into the corresponding restriction site of pCDNA3.4-FC (containing an FC tag) using HindIII and BamHI, resulting in pCDNA3.4-DLL4-ECD-FC. The pCDNA3.4-DLL4-ECD-HIS and pCDNA3.4-DLL4-ECD-FC plasmids were transiently transformed into EXPI293 cells and cultured for 5 days. The supernatant was collected and the pCDNA3.4-DLL4-ECD-HIS protein was purified using a Ni affinity chromatography column, and the pCDNA3.4-DLL4-ECD-FC protein was purified using a Protein A affinity chromatography column.

[0065] BALB / c mice were initially immunized with a mixture of recombinant DLL4-ECD-FC protein and complete Freund's adjuvant (Sigma) by intraperitoneal injection. Subsequently, on days 14 and 35, mice were boosted with a mixture of recombinant protein DLL4-ECD-FC and incomplete Freund's adjuvant (Sigma) by intraperitoneal injection. On day 56, BALB / c mice were boosted with DLL4-ECD-HIS by intraperitoneal injection. Four days later, spleens were harvested for fusion.

[0066] Mouse spleen cells were fused with SP2 / 0 cells at a ratio of 4:1 and cultured in HAT (GBICO) medium in a 96-well plate (Corning). Hybridoma cell screening was then performed and the resulting cell clones were subjected to binding screening. The identification and screening process was divided into two steps:

[0067] ① Recombinant DLL4-ECD-HIS was immobilized on a 96-well enzyme-linked immunosorbent assay plate. The supernatant of clone expression was added and incubated for 1 hour. The plate was washed three times with PBST. The supernatant of cell clones with DLL4 binding activity was identified using goat anti-mouse IgG-HRP (Jackson Immuno). The plate was washed three times with PBST and developed with TMB to obtain positive clones that directly bind to DLL4.

[0068] ② Subsequently, the positive clones from step ① were transferred to 24-well plates (Corning) for culture to obtain more expression products. The cell supernatant was incubated with DLL4-overexpressing cells at 4°C for 1 hour, then washed twice with PBS. Goat anti-mouse IgG-HRP was added and incubated at 4°C for 0.5 hour, washed twice with PBS, and developed with TMB to identify positive clones that could bind to DLL4 on the cells.

[0069] Through the above screening process, the candidate mouse DLL4 monoclonal antibody D0808-7C11 was obtained.

[0070] Example 2: Antibody gene acquisition and chimeric antibody preparation

[0071] Hybridoma cell RNA expressing the positive antibody D0808-7C11 was extracted and reverse transcribed to obtain cDNA. Using this cDNA as a template, the light and heavy chain variable region nucleic acid sequences of the mouse IgG antibody were amplified by PCR. The heavy and light chain variable regions were analyzed. According to the Kabat numbering convention, the encoded heavy chain variable region comprises the heavy chain CDR1 (SYVMH) set forth in SEQ ID NO:1, the heavy chain CDR2 (YIIPYNDDTKYNEKFKG) set forth in SEQ ID NO:2, and the heavy chain CDR3 (GGDYEVFDY) set forth in SEQ ID NO:3. The light chain variable region comprises the light chain CDR1 (SVSSSVSYMH) set forth in SEQ ID NO:4, the light chain CDR2 (DTSKLTS) set forth in SEQ ID NO:5, and the light chain CDR3 (QQWSSNPFT) set forth in SEQ ID NO:6.

[0072] The heavy chain variable region was constructed into the pCDNA3.1-HC plasmid (purchased from Thermo Fisher Scientific Inc.) (containing a signal peptide and heavy chain IgG1 constant region), generating the chimeric antibody heavy chain plasmid pCDNA3.1-Ab7C11-HC. The light chain variable region was constructed into the pCDNA3.1-LC plasmid (containing a signal peptide and light chain kappa constant region), generating the chimeric antibody light chain plasmid pCDNA3.1-Ab7C11-LC. The light and heavy chain plasmids were co-transfected into 293F cells, cultured for 5 days, and the supernatant was collected and purified by protein A (GE) affinity chromatography to obtain recombinantly expressed anti-human DLL4 chimeric antibody Ab7C11.

[0073] The heavy chain variable region sequence of the anti-human DLL4 chimeric antibody Ab7C11 is shown in SEQ ID NO: 7, specifically:

[0074] QVKLEESGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYIIPYNDDTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGGDYEVFDYWGQGTTLTVSSAKTTPPSVYK

[0075] The light chain variable region sequence of the anti-human DLL4 chimeric antibody Ab7C11 is shown in SEQ ID NO: 8, specifically:

[0076] QIVLTQSPAIMSASPGEKVTMTCSVSSSVSYMHWYQQKSGTSPKRWIYDTSKLTSGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPFTFGPGTKVDIK

[0077] Example 3: Humanized design and expression

[0078] The heavy and light chain sequences of Ab7C11 were analyzed by Ig-BLAST, and IGKV3-11*01 was selected as the template for light chain humanization, and IGHV1-3*01 was selected as the template for heavy chain humanization. The mouse antibody and human FR were spliced ​​by CDR-grafting, and then multiple reverse mutation sequences were designed. The specific sequences are shown in Table 1. The variable regions in Table 1 were synthesized and directly constructed into the corresponding heavy and light chain constant region plasmids to obtain full-length heavy and light chain expression plasmids. The plasmids were extracted, and then different heavy and light chains were cross-paired and transfected into EXPI293. After 5 days of culture, the supernatant was collected and purified by protein A affinity chromatography. A total of 3 humanized antibodies were obtained, and the sequences of the 3 antibodies are shown in Table 2.

[0079] Table 1 Variable region sequences of Ab7C11 humanized antibody

[0080]

[0081] Table 2 Heavy and light chain variable region sequences of humanized antibodies

[0082] Antibody name Light chain variable region sequence Heavy chain variable region sequence Ab700 SEQ ID NO:9 SEQ ID NO:12 Ab710 SEQ ID NO: 10 SEQ ID NO:12 Ab720 SEQ ID NO:11 SEQ ID NO:12

[0083] The heavy chain constant region sequence of the humanized antibody is (SEQ ID NO: 13):

[0084] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0085] The light chain constant region sequence of the humanized antibody is (SEQ ID NO: 14):

[0086] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0087] Example 4: Humanized Antibody Protein Binding Activity

[0088] The protein binding activity of the prepared humanized antibody was assessed by coating the DLL4-ECD-HIS protein. After blocking the immunoplate, a gradient dilution of the antibody (starting at 1 μg / mL, 5-fold gradient dilution) was added and incubated for 1 hour. Wash twice, add a 1:10000 diluted secondary antibody and incubate for 0.5 hours. Finally, TMB was used for color development and 1M H2SO4 was used for termination. OD 450 Reading. The results show that ( Figure 1 ), humanized antibodies retain the protein binding activity of chimeric antibodies, but their EC values ​​are higher than those of chimeric antibodies. 50 value.

[0089] Example 5: Humanized Antibody Cell Binding Activity

[0090] DLL4-overexpressing cells were obtained by transiently transfecting 293F cells with the DLL4-FL expression plasmid pCDNA3.4-DLL4-FL. Cells were harvested, 200,000 cells were added to each well, and an equal volume of serially diluted humanized antibody (starting at 10 μg / mL, with 3-fold serial dilutions) was added and incubated at 4°C for 1 hour. The cells were washed once with PBS, and secondary antibody was added at a 1:10,000 dilution and incubated at 4°C for 0.5 hour. Finally, TMB was used for color development and 1M H2SO4 was used for quenching. OD 450 Reading. The results show that ( Figure 2 ), all humanized antibodies retained the cell binding activity of chimeric antibodies, among which Ab710 had a binding activity to EC 50 Superior to the chimeric antibody Ab7C11.

[0091] Example 6: Humanized Antibody Affinity Determination

[0092] Based on the protein- and cell-binding activities of the humanized antibodies, Ab700, Ab710, and Ab720 were selected for affinity testing. Affinity testing was performed using a gator Protein A probe. The antibodies were first loaded with different concentrations of antibody (three-fold dilutions starting from 500 nM) for 180 seconds, then equilibrated in PBS for 60 seconds, allowed to bind to the DLL4-HIS antigen for 240 seconds, and then dissociated in buffer for 600 seconds. Software was used to analyze the affinity of the different antibodies for the antigen. The results (Table 3) showed that the affinity KDs of Ab700, Ab710, and Ab720 were 1.24E-007, 5.74E-008, and 3.41E-007, respectively. Ab710 was comparable to the positive anti-DLL4 antibody (derived from the anti-DLL4 sequence of ABT-165 (ABBVIE)), all at the micromolar level.

[0093] Table 3 Affinity of humanized DLL4 antibodies

[0094] Antibody name KD Ab700 1.24E-007 Ab710 5.74E-008 Ab720 3.41E-007 anti-DLL4 positive 4.42E-008

[0095] Example 7: Effect of humanized antibodies on HUVEC angiogenesis in vitro

[0096] 100 μL of growth factor-reduced Matrigel was added to a 96-well plate and incubated at 37°C for polymerization and solidification. 2.5E4 HUVECs (human umbilical vein endothelial cells) mixed with 5 ng of VEGF (vascular endothelial growth factor) were suspended in 100 μL of ECM supplemented with 2% FBS and added to each well. A negative control group (no additional antibody) was set up, along with an anti-DLL4 positive control group (5 μg / mL anti-DLL4 positive, derived from the anti-DLL4 sequence of ABT-165 (ABBVIE)), experimental groups (5 μg / mL of homemade antibodies Ab700, Ab710, or Ab720), and an anti-VEGF positive control group (5 μg / mL anti-VEGF positive, Roche bevacizumab) were incubated at 37°C. After 4–16 hours, 50 μL of 6 μM Mcalcein AM dye was added to each well and incubated for 40 min in the dark. Calcein AM-labeled cultures were photographed at 40× magnification using an OLYMPUS inverted microscope, and the number of endothelial tubes was counted using the Image Pro Plus program.

[0097] The results show Figure 3 The experimental group showed the same vascular appearance as the anti-DLL4 positive control group, but with taller branches and thinner connections than the negative control group. The anterior end of the vessels was filled with buds and filopodia, and the vessels were more fragmented. The effect of Ab710 was particularly pronounced in the experimental group.

[0098] like Figure 4 As shown, compared with the negative control group, Ab700, Ab710, and Ab720 induced a significant increase in the number of HUVEC tubes formed, and the anti-VEGF antibody group inhibited capillary tube formation in the presence of VEGF. These data are consistent with established studies showing that blocking the DLL4-Notch1 signaling pathway leads to increased angiogenesis. The experimental groups displayed the same vascular appearance as the anti-DLL4 antibody group, with taller branches, thinner interconnections, and more pronounced scissor-like formation at the anterior end of the vessels compared to the negative control group. The effect of Ab710 was particularly pronounced in the experimental group.

Claims

1. An antibody, characterized in that The antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3; wherein: The amino acid sequence of the HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:

6.

2. The antibody according to claim 1, wherein The framework regions of the heavy chain variable region and / or light chain variable region are murine framework regions; Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

3. The antibody according to claim 1, wherein The framework regions of the heavy chain variable region and / or light chain variable region are human framework regions; Preferably, the framework region of the heavy chain variable region is derived from the human germline heavy chain IGHV1-3*01; and / or, the framework region of the light chain variable region is derived from the human germline light chain IGKV3-11*01; More preferably, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 12; and / or the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 9, 10 or 11.

4. The antibody according to any one of claims 1 to 3, wherein The antibody is a full-length antibody, Fab, Fab', F(ab')2 or Fv; the Fv is preferably scFv; Preferably, the antibody is a full-length antibody, and its heavy chain constant region and / or light chain constant region are derived from a human antibody; More preferably, the heavy chain constant region is derived from a human heavy chain IgG1 constant region; and / or the light chain constant region is derived from a human light chain κ chain constant region; Further more preferably, the amino acid sequence of the heavy chain constant region of the antibody is shown as SEQ ID NO: 13; and / or the amino acid sequence of the light chain constant region of the antibody is shown as SEQ ID NO:

14.

5. An isolated nucleic acid, characterized in that The nucleic acid encodes the antibody according to any one of claims 1 to 4.

6. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the isolated nucleic acid of claim 5; Preferably, the backbone of the recombinant expression vector is pCDNA3.

1.

7. A transformant, characterized in that: The transformant comprises the nucleic acid according to claim 5 or the recombinant expression vector according to claim 6, and the host cell of the transformant is a eukaryotic cell or a prokaryotic cell; Preferably, the eukaryotic cells are mammalian cells, such as 293F cells or EXPI293 cells.

8. A method for preparing an antibody, characterized in that: The method comprises culturing the transformant according to claim 7.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antibody according to any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

10. A kit, characterized in that The kit comprises the antibody according to any one of claims 1 to 4 and / or the pharmaceutical composition according to claim 9.

11. A medicine kit, characterized in that: The kit comprises kit A and kit B, wherein: The drug kit A contains the antibody according to any one of claims 1 to 4 and / or the pharmaceutical composition according to claim 9; The medicine kit B contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and / or other anti-tumor drugs.

12. A drug delivery device, characterized in that: The drug delivery device comprises the antibody according to any one of claims 1 to 4 and / or the pharmaceutical composition according to claim 9; Preferably, the drug delivery device further comprises a component for administering the antibody or the pharmaceutical composition to a subject, such as a syringe or an infusion device.

13. A method for detecting a protein comprising the extracellular region of DLL4 for non-diagnostic purposes, characterized in that: The method comprises using the antibody according to any one of claims 1 to 4 and / or the pharmaceutical composition according to claim 9; Preferably, the DLL4 is human DLL4; and / or, the protein comprising the extracellular region of DLL4 is a full-length DLL4 protein.

14. Use of the antibody according to any one of claims 1 to 4 and / or the pharmaceutical composition according to claim 9 in the preparation of a drug for promoting non-functional angiogenesis and / or anti-tumor effects; Preferably, the tumor is a solid tumor.

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