Multifunctional recombinant antibodies and methods for their production and use

Abstract

The present invention discloses multifunctional recombinant antibodies and their preparation and use. The present invention provides not only monoclonal antibodies capable of specifically recognizing human CD276 protein, but also humanized recombinant antibodies thereof, which can be further engineered to recognize human CD276 and possess IL15 function, thereby effectively enhancing the tumor cell killing activity of the original antibody. At the same time, the present invention also introduces a mutant human IgG1 constant region to obtain a structurally engineered CD276-specific monoclonal antibody-IL15 bifunctional molecule, which effectively reduces antibody toxicity and improves safety. The antibodies of the present invention can recognize the CD276 protein in many tumor cells, thereby exerting their killing effect on many tumor cells. At the same time, the engineered IL15 recombinant antibodies targeting CD276 also have the advantages of a short metabolic cycle, low toxicity, and high safety, making them highly promising for future applications.

Description

[Technical Field]

[0001] The present invention belongs to the field of biotechnology and relates to an IL15 recombinant multifunctional antibody targeting Fc-mutated CD276, and its preparation and use. Specifically, the present invention relates to a multifunctional recombinant antibody that can recognize CD276, has IL15 function, and has reduced toxicity in the body, and its use. [Background technology]

[0002] CD276, or B7-H3, is a type I transmembrane protein that belongs to the B7 immune costimulatory / co-inhibitory family and has immunoregulatory functions. As a receptor protein involved in immunoregulation, its ligand has not yet been identified. While expressed at low levels in normal human tissues, CD276 is expressed on activated macrophages and monocytes, where it plays a suppressive role in T cells and prevents excessive immune activation.

[0003] CD276 is widely expressed in many malignant tumors, including lung cancer, liver cancer, intestinal cancer, gastric cancer, breast cancer, pancreatic cancer, and kidney cancer, etc. CD276 expression on tumor cells promotes tumor progression and leads to poor prognosis because CD276 can suppress antitumor immunity in the tumor microenvironment.

[0004] Therefore, CD276 can be used as a reliable target for tumor therapy.

[0005] First, as an immune checkpoint molecule, blocking its function can enhance the body's anti-tumor immunity.

[0006] Second, because CD276 is widely expressed on tumor cells, specific antibodies can kill tumor cells through mechanisms such as ADCC, CDC, and ADCP.

[0007] Furthermore, CD276 is specifically expressed on tumor cells, and specific killing of tumor cells can be achieved in the form of ADC.

[0008] IL15 is a cytokine expressed by many cell types, including monocytes, macrophages, epithelial cells, and fibroblasts, but not by T cells. Unlike many other cytokines, IL15 is generally secreted extracellularly and exerts no effect. Instead, it complexes with IL15Rα and localizes to specific cell membranes to stimulate nearby effector cells, primarily NK and CD8+ T cells. IL15 is closely related to IL2, and when it forms a complex with IL15Rα, it binds to the β / γ receptor shared with IL2 and mediates its biological activity. One advantage of IL15 over IL2 in terms of antitumor activity is that IL15 / Rα does not stimulate Treg proliferation.

[0009] Currently, many IL15-related molecules are under research and development for the treatment of malignant tumors. Among them, the most rapidly progressing is ALT-803, a complex consisting of IL15 and IL15Rαsushi-hFc1. Several clinical studies have shown that ALT-803 is effective against many tumors, including melanoma, but it also causes severe toxicities and side effects, primarily liver dysfunction, hypotension, and fever. Summary of the Invention

[0010] To address the above-mentioned problems, the present invention aims to provide a specific monoclonal antibody that can specifically recognize CD276 protein, a hybridoma secreting said monoclonal antibody, a humanized recombinant antibody derived from said monoclonal antibody, and a multifunctional recombinant antibody that can recognize human CD276 protein and also possesses human IL15 function, making it useful for treating malignant tumors. At the same time, we have also produced a structurally engineered specific monoclonal antibody-IL15 bifunctional molecule that maintains antitumor activity while significantly reducing toxicity and side effects.

[0011] To achieve the above objectives, the technical solutions used in the present invention are as follows:

[0012] The monoclonal antibody is capable of recognizing human CD276-ECD protein, and is characterized in that the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 5 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7.

[0013] In the present invention, a murine (Murin) monoclonal antibody capable of specifically recognizing human CD276 protein was produced by immunizing mice with recombinantly expressed human CD276-ECD protein and designated 147#. Biological analysis revealed the amino acid sequences of the heavy and light chain variable regions of this murine monoclonal antibody. This antibody can efficiently bind to CD276. Biological analysis revealed that the heavy chain variable region of this monoclonal antibody encodes 122 amino acid residues, and the light chain variable region encodes 107 amino acid residues.

[0014] Furthermore, the present invention claims protection of a recombinant antibody obtained by humanizing the monoclonal antibody 147#, in which the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 21 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 22.

[0015] In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain of the recombinant antibody is shown in SEQ ID NO: 23, and the amino acid sequence of the light chain is shown in SEQ ID NO: 24.

[0016] The inventors further analyzed the sequence of the obtained murin-derived monoclonal antibody 147# and replaced it with the CDR region of a human-derived template, further recombining the heavy chain variable region with a human IgG1 constant region and the light chain variable region with a human κ chain constant region. Based on the three-dimensional structure of the antibody, they also backmutated buried residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of the VL and VH of each antibody, ultimately obtaining a humanized antibody named hu147#. The amino acid sequences of the heavy chain variable region and light chain variable region, as well as the heavy chain and light chain amino acid sequences of the humanized recombinant antibody hu147#, are as described above.

[0017] Furthermore, the present invention claims to protect a multifunctional recombinant antibody, wherein the heavy chain comprises an antibody functional region that recognizes human CD276, a human IgG1 constant region functional domain, an IL15 functional region, and a non-functional amino acid fragment for linking each functional region, and the amino acid sequence of the antibody functional region that recognizes human CD276 comprises the amino acid sequence of the heavy chain variable region of the recombinant antibody.

[0018] The IL15 functional region can recognize the functional domain of the human IL2 / IL15β / γ receptor.

[0019] The present inventors further modified the humanized antibody obtained above by linking the heavy chain sequence of the antibody to a sequence having IL15 function, thereby obtaining a recombinant antibody that can also recognize human CD276 protein and has IL15 function.

[0020] In a preferred embodiment of the present invention, the human IgG1 constant region functional domain is a human IgG1 constant region, the amino acid sequence of which is shown in SEQ ID NO:8.

[0021] More preferably, the human IgG1 constant region functional domain is a mutated human IgG1 constant region, and the amino acid sequence of the mutated human IgG1 constant region is set forth in SEQ ID NO:25.

[0022] By using a human IgG1 constant region sequence containing mutations, the obtained IL15 recombinant antibody targeting CD276 has a shorter metabolic cycle, lower toxicity and side effects, and is safer.

[0023] In a preferred embodiment of the present invention, the amino acid sequence of the human IL15 functional domain is shown in SEQ ID NO:28.

[0024] More preferably, the amino acid sequence of the human IL15 functional region is set forth in SEQ ID NO:29.

[0025] In the preferred amino acid sequence of the functional region of human IL15 shown in SEQ ID NO: 29, human IL15Rsushi and human IL15 are linked via (GGGGS)6 to form a single-chain IL15, ie, IL15sc.

[0026] In a preferred embodiment of the present invention, the non-functional amino acid fragment connecting each functional domain of the CD276-targeting IL15 recombinant antibody is a GGGGS overlap, which is stable and plays an important role in constructing a biologically active fusion protein, ensuring correct protein folding, maintaining biological activity, and improving protein yield.

[0027] More preferably, the GGGGS duplication is (GGGGS)3.

[0028] In a preferred embodiment of the present invention, the amino acid sequence of the human IgG1 constant region functional domain of the multi-functional recombinant antibody is shown in SEQ ID NO: 8, the amino acid sequence of the heavy chain of the multi-functional recombinant antibody is shown in SEQ ID NO: 30, and the amino acid sequence of the light chain of the multi-functional recombinant antibody is shown in SEQ ID NO: 24.

[0029] In the present invention, a multifunctional antibody capable of recognizing CD276 and having IL15 function was produced and named SPGL007.

[0030] More preferably, the functional domain of the human IgG1 constant region of the multi-function recombinant antibody is a mutated human IgG1 constant region, the amino acid sequence of the mutated human IgG1 constant region is shown in SEQ ID NO: 25, the amino acid sequence of the heavy chain of the multi-function recombinant antibody is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain of the multi-function recombinant antibody is shown in SEQ ID NO: 24.

[0031] The multifunctional recombinant antibody prepared from the preferred multifunctional recombinant antibody sequence contains a mutated human IgG1 constant region sequence and is named SPGL008. The multifunctional recombinant antibody SPGL008 not only effectively recognizes CD276 in many tumor cells and effectively inhibits the growth of various cancer cells, but also has a shorter half-life, lower toxicity, and excellent safety.

[0032] Furthermore, the present invention claims protection of the nucleotide sequence encoding said monoclonal antibody, or said recombinant antibody, or said multifunctional recombinant antibody.

[0033] Based on the amino acid sequence of the provided monoclonal antibody, or the recombinant antibody, or the multifunctional recombinant antibody, the nucleotide sequence of the corresponding coding gene can be obtained.

[0034] In a preferred embodiment of the present invention, the nucleotide sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:4, and the nucleotide sequence of the light chain variable region is shown in SEQ ID NO:6.

[0035] Furthermore, the present invention claims protection for an expression vector containing said nucleotide sequence.

[0036] It can be obtained by constructing an expression vector containing the nucleotide sequence using molecular biology techniques.

[0037] Furthermore, the present invention claims the protection of host cells containing said expression vectors.

[0038] Using cell biology techniques, host cells capable of efficiently expressing the antibody protein can be constructed and obtained using the expression vector.

[0039] Furthermore, the present invention claims protection for the use of said monoclonal antibody, or said recombinant antibody, or said multifunctional recombinant antibody, or said nucleotide sequence, or said expression vector, or said host cell in the manufacture of a biologic for treating tumors.

[0040] Furthermore, the present invention claims protection for a biological product comprising at least one of said monoclonal antibody, or said recombinant antibody, or said multifunctional recombinant antibody, or said nucleotide sequence, or said expression vector, or said host cell.

[0041] Furthermore, the present invention provides a method for producing the monoclonal antibody, the recombinant antibody, or the multifunctional recombinant antibody, which comprises the following steps:

[0042] (1) obtaining an expression vector containing a gene fragment of the monoclonal antibody, recombinant antibody, or multifunctional recombinant antibody by artificial synthesis or molecular biology methods; (2) transfecting the expression vector into cells to express the protein; (3) Obtaining said monoclonal antibody or recombinant antibody or multifunctional recombinant antibody by protein purification.

[0043] The corresponding antibodies can be obtained by constructing expression vectors containing nucleotide sequences encoding the heavy or light chain sequences of the above antibodies, transfecting them into appropriate cells, and then expressing and purifying them.

[0044] In a preferred embodiment of the present invention, in step (1), producing an expression vector containing a gene fragment of the monoclonal antibody, recombinant antibody, or multifunctional recombinant antibody by molecular biology involves inserting the nucleotide sequence of the heavy chain or light chain of the antibody into a pcDNA3.4 expression vector.

[0045] In a preferred embodiment of the present invention, in the step (2), the cells for expressing the protein are Expi-293F cells.

[0046] In a preferred embodiment of the present invention, the protein is purified using Protein G in the step (3).

[0047] Compared with existing technologies, the present invention has the following technical advantages:

[0048] The present invention provides a monoclonal antibody capable of specifically recognizing human CD276 protein, as well as a humanized recombinant antibody thereof. Based on this, the inventors further utilized a CD276-specific recombinant antibody to prepare a multifunctional recombinant antibody that can recognize human CD276 and also possess IL15 function, thereby effectively enhancing the killing activity of the original antibody against tumor cells. At the same time, the present invention also introduces a mutant human IgG1 constant region to obtain a structurally engineered CD276-specific monoclonal antibody-IL15 bifunctional molecule. This bifunctional molecule efficiently shortens its metabolic cycle while maintaining in vivo antitumor activity, thereby reducing its toxicity and side effects in vivo and significantly improving safety. The multifunctional recombinant antibody of the present invention can recognize CD276 on various tumor cells, and has great potential for future applications. [Brief explanation of the drawings]

[0049] [Figure 1] FIG. 1 shows the results of measuring the binding activity of mouse-derived antibody 147# to human CD276-ECD protein by ELISA. [Figure 2] FIG. 2 shows the results of flow cytometry assay of binding of mouse-derived monoclonal antibodies to CD276. [Figure 3] FIG. 3 shows the results of Western blotting to confirm the binding of 147# to human CD276-ECD. [Figure 4] FIG. 4 shows the results of detecting the binding of 147# to a polypeptide in the human CD276 domain Ig-like C2 type 1 domain by ELISA. [Figure 5] FIG. 5 shows the results of detecting the binding of 147# to a polypeptide in the human CD276 domain Ig-like C2 type 1 domain by ELISA. [Figure 6] FIG. 6 shows the results of measuring the cross-reactivity of monoclonal antibody 147# with cynomolgus monkey CD276-ECD antigen. [Figure 7] FIG. 7 shows the results of measuring the binding activity of the humanized antibody hu147 and the mutant humanized antibody hu147mu to human CD276-ECD by ELISA. [Figure 8] FIG. 8 shows the results of measuring the binding activity of SPGL008 and SPGL007 to human CD276-ECD by ELISA. [Figure 9] FIG. 9 shows the results of SPGL008's simultaneous binding activity to human CD276 and human CD122 / 132. [Figure 10A] FIG. 10A shows the results of the cell proliferation-promoting activity of SPGL008. [Figure 10B] FIG. 10B shows the results of the cell proliferation-promoting activity of SPGL008. [Figure 11] FIG. 11 shows the results of SPGL008 inhibiting the growth of tumors transplanted with MC38 colorectal cancer cells in mice. [Figure 12] FIG. 12 shows the results of SPGL008 inhibiting the growth of tumors transplanted with human lung cancer cells NCI H1975. [Figure 13] Figure 13 shows the results of SPGL008, when used in combination with a HER2 monoclonal antibody, inhibiting the growth of JIMT-1 human breast cancer cell xenograft tumors in nude mice. [Figure 14] FIG. 14 shows the results of metabolism of SPGL008 in the bodies of huFcRn transgenic mice. [Figure 15] Figure 15 shows the results of detecting the binding of SPGL008 to various tumor cells by flow cytometry. [Figure 16] FIG. 16 shows the state of the protein after SPGL008 was left standing for 0 weeks. [Figure 17] FIG. 17 shows the state of the protein after SPGL008 was left standing at 4° C. for 5 weeks. [Figure 18] FIG. 18 shows the state of the protein after SPGL008 was left standing at 37° C. for 5 weeks. DETAILED DESCRIPTION OF THE INVENTION

[0050] Hereinafter, the present invention will be further described in conjunction with drawings and specific examples to better explain the objectives, technical solutions and advantages of the present invention.

[0051] Example 1 Immunization of Animals with Antigen and Production and Screening of Hybridomas Step 1: Immunization of mice with antigen Balb / c mice were immunized subcutaneously with recombinant human CD276-ECD protein (purchased from Beijing Acro BIOSYSTEMS; the amino acid sequence is set forth in SEQ ID NO: 1) using standard methods. On day 1, the human CD276-ECD protein was emulsified with Freund's complete adjuvant (CFA, Sigma) and thoroughly mixed to homogeneity, and then subcutaneously injected into the Balb / c mice (50 μg / mouse). On days 14 and 36, the human CD276-ECD protein was emulsified with Freund's incomplete adjuvant (IFA, Sigma) and thoroughly mixed to homogeneity, and then subcutaneously injected into the Balb / c mice (50 μg / mouse). On day 50, the human CD276-ECD protein was intraperitoneally injected at 50 μg / mouse for induction. Three to four days later, the spleens of the mice were harvested and used for fusion experiments.

[0052] Step 2: Hybridoma production and screening Three to four days after the last rush immunization, mouse spleen cells were fused with mouse myeloma cells SP2 / 0 using PEG (PEG1450, Sigma) using a standard hybridoma protocol. The fused cells were homogenously suspended in complete medium, which consisted of a 1:1 mixture of RPMI1640 and DMEM F12 medium, supplemented with 1% glutamine (Gibco), 1% sodium pyruvate (Gibco), 1% MEM-NEAA (Minimum Essential Medium-Non-Essential Amino Acid Solution, Gibco), 1% penicillin-streptomycin (Gibco), 50 μM β-mercaptoethanol (Gibco), and 20% fetal bovine serum (FBS, Gibco). 10 5 The cells were cultured overnight in a 96-well culture plate at 100 μl / well. The next day, 100 μl of complete medium containing 2× HAT (Sigma) was added to each well, bringing the culture volume to 200 μl / well (containing 1× HAT). After 7 to 12 days, the supernatant was collected and hybridoma wells positive for human CD276-ECD protein binding activity were screened by indirect enzyme-linked immunosorbent assay (ELISA).

[0053] Here, hybridoma wells positive for human CD276-ECD protein binding activity were screened by indirect enzyme-linked immunosorbent assay (ELISA) as follows: Recombinant human CD276-ECD protein was diluted to 1 μg / ml in coating solution (50 mM carbonate coating buffer, pH 9.6) and added to a microplate at 100 μl / well for overnight coating at 4°C. The plate was washed three times with PBST, and then 200 μl / well of blocking solution (2% BSA-PBST) was added and incubated at 37°C for 1 hour. The plate was then washed once with PBST and prepared for use. The collected hybridoma supernatants were then added to the blocked microplate at 100 μl / well and incubated at 37°C for 1 hour. The plate was then washed three times with PBST and then added with HRP-conjugated goat anti-mouse IgG secondary antibody (purchased from Abcam, catalog number ab6789) and incubated at 37°C for 30 minutes. After washing the plate five times with PBST, residual droplets were removed using absorbent paper. 100 μl of TMB (KPL) was added to each well and the plate was left to stand for 5 minutes at room temperature (20±5°C) away from light. 50 μl of 2M H2SO4 stop solution was added to each well to stop the substrate reaction. The OD value was read at 450 nm using a microplate reader, and the binding ability of the test antibody to the target antigen, CD276-ECD protein, was analyzed.

[0054] Ten hybridoma cell lines obtained by screening were amplified in serum-containing complete medium, centrifuged, and replaced with serum-free SFM medium until the cell density reached 1-2 × 10 7 The cells were cultured at a concentration of 1 / ml for 2 weeks at 37°C in 5% CO2. The culture supernatant was centrifuged and purified by Protein G affinity chromatography to obtain a mouse-derived anti-human CD276-ECD protein monoclonal antibody, designated 147#.

[0055] Example 2 Measurement of binding activity of mouse-derived antibody 147# to human CD276-ECD protein by ELISA The binding ability of mouse-derived antibodies to human CD276-ECD protein was measured by indirect enzyme-linked immunosorbent assay (ELISA). The specific method was as follows: human CD276-ECD protein was pre-coated and diluted to 2 μg / ml in coating solution (50 mM carbonate coating buffer, pH 9.6) and coated onto plates overnight at 4°C. The plates were then blocked with 5% nonfat dry milk at 37°C for 2 hours. The plates were washed three times with PBST, and 100 μl / well of the test antibodies, gradient-diluted in 1% BSA-PBST, was added to the blocked microplate and incubated at 37°C for 1 hour. The plates were then washed three times with PBST, and HRP-conjugated goat anti-mouse IgG secondary antibody (Millipore) was added and incubated at 37°C for 30 minutes. After washing the plate three times with PBST, residual droplets were removed using absorbent paper. 100 μl of TMB (KPL) was added to each well and the plate was left to stand for 5 minutes at room temperature (20±5°C) away from light. 50 μl of 2M H2SO4 stop solution was added to each well to stop the substrate reaction. The OD value was read at 450 nm using a microplate reader, and the binding ability of the test antibody to the target antigen, CD276-ECD protein, was analyzed.

[0056] As a result, as shown in Figure 1, antibody 147# had good binding activity and an EC 50 was 38.35 ng / ml, or 0.26 nM.

[0057] Example 3 Measurement of binding of mouse-derived monoclonal antibodies to CD276 by flow cytometry The binding affinity of 147# to human renal carcinoma cell line A498 was measured by fluorescence activated cell sorting (FACS).

[0058] In this experiment, human kidney cells A498 were used as the target cells, and 100 μl of 147#, diluted 6-fold from 10,000 ng / ml in four steps, was used as the primary antibody. 1 × 10 cells were suspended in 100 μl of RPMI-1640 serum-free medium (purchased from Gibco, catalog number 22400089). 5 The cells were incubated with 147# at 4°C for 1 hour (the highest concentration of 147# was 5 μg / ml, the lowest was 23 ng / ml). The cells were washed twice with PBS to remove unbound 147#. The cells were then incubated with 100 μl of 2 μg / ml Alexa Fluor 488-conjugated anti-mouse fluorescent secondary antibody (purchased from Thermo Invitrogen, catalog number A11001) at 4°C for 30 minutes. The cells were washed twice with PBS to remove unbound secondary antibody. Finally, the cells were resuspended in 100 μl of PBS and the binding affinity of 147# to the cells was measured using a flow cytometer. The data were fitted and analyzed using GraphPad Prism 6 software.

[0059] As shown in Figure 2, 147# specifically bound to human renal cancer cell line A498, which expresses high levels of CD276 on the cell surface, and its EC 50 was 59.15 ng / ml, i.e., 0.39 nM.

[0060] Example 4: Determination of antigen-binding epitope of 147# 4.1 Measurement of binding ability of reduced and denatured CD276-ECD to 147# The binding ability of 147# to reduced and denatured human CD276-ECD was measured by Western blot.

[0061] Reduced and denatured human CD276-ECD was subjected to SDS-PAGE electrophoresis (400 ng / lane), then electrophoretically transferred to a PVDF membrane. The membrane was blocked with 1% BSA-TBST (shaking at room temperature for 1 hour), and then incubated with 1 μg / mL (diluted in 1% BSA-PBST) mouse antibody 147# at room temperature for 2 hours with shaking. After washing three times with PBST, the membrane was incubated with HRP-conjugated goat anti-mouse IgG secondary antibody (purchased from Abcam, catalog number ab6789, diluted 10,000 times with 1% BSA-PBST according to the manufacturer's instructions) at room temperature for 1 hour with shaking. After washing three times with PBST, the membrane was transferred to an appropriate amount of Pierce TM An ECL Western blot substrate solution (purchased from Thermo, catalog number 32209) was added dropwise, and the sample was automatically imaged in a biomolecular imaging device (purchased from Thermo, model number CL1500) at room temperature, avoiding light.

[0062] As a result, as shown in Figure 3, specific immunoblots appeared between the target sites of CD276-ECD, indicating that 147# can specifically bind to reduced and denatured human CD276-ECD, suggesting that the epitope of antibody 147# that specifically binds to human CD276-ECD protein is a linear epitope.

[0063] 4.2 Identification of the target antigen-binding region of monoclonal antibody 147# The binding domain of 147# to human CD276-ECD was identified by conventional Western blot and ELISA methods.

[0064] To confirm the binding epitope of 147# on CD276, we searched the literature and NCBI to obtain the gene for the extracellular domain of human CD276 (CD276-ECD), which contains the functional domain Ig-like V type 1 (domain 1, amino acid sequence: SEQ ID NO: 16) and the functional domain Ig-like C2 type 1 (domain 2, amino acid sequence: SEQ ID NO: 17). The CD276 functional domains Ig-like V type 1 and Ig-like C2 type 1 were linked to a human Fc region (amino acid sequence: SEQ ID NO: 18) to form V type 1-Fc (amino acid sequence: SEQ ID NO: 19) and C2 type 1-Fc (amino acid sequence: SEQ ID NO: 20), respectively. These were then constructed in the pcDNA3.4 expression vector, transfected into Expi-293F cells, and purified using Protein A to obtain the respective Fc fusion proteins. Thereafter, the binding status of 147# to human CD276-ECD domain Ig-like V type 1 and Ig-like C2 type 1 was measured by conventional Western blotting, with reference to the method described in 4.1 above.

[0065] As a result, as shown in Figure 3, both Western blot detection results indicated that 147# binds to the second functional domain of human CD276-ECD, namely, Ig-like C2 type 1.

[0066] 4.3 Confirmation of binding polypeptide of monoclonal antibody 147# to the target antigen The binding ability of 147# to polypeptides in the human CD276 domain Ig-like C2 type 1 region of 147# was detected by ELISA, and the epitope of 147# binding to human Ig-like C2 type 1 was further confirmed.

[0067] The results of 4.1 and 4.2 confirmed that 147# can bind to denatured CD276-ECD and the Ig-like C2 type 1 domain, and determined that the binding epitope of 147# is a linear epitope. By synthesizing the polypeptide and detecting it by ELISA, the location of the binding epitope of 147# could be further clarified.

[0068] First, the human Ig-like C2 type 1 domain was divided into nine overlapping segments, and one N-terminal biotin-modified polypeptide was synthesized for each segment as follows: 1.bio-PSMTLEPNKD LRPGDTVTI(145~164); 2.bio-LRPGDTVTI TCSSYQGYPE(155~174); 3.bio-TCSSYQGYPE AEVFWQDGQG(165~184); 4.bio-AEVFWQDGQG VPLTGNVTTS(175~194); 5.bio-VPLTGNVTTS QMANEQGLFD(185~204); 6.bio-QMANEQGLFD VHSILRVVLG(195~214); 7.bio-VHSILRVVLG ANGTYSCLVR(205~224); 8.bio-ANGTYSCLVR NPVLQQDAHS(215~234); 9.bio-NPVLQQDAHS SVTIT(225~240).

[0069] The binding activity of 147# to the above nine polypeptides was measured by ELISA. As shown in Figure 4, 147# bound only to polypeptide 1, i.e., the polypeptide consisting of amino acids 145 to 154 at the N-terminus of human CD276-ECD protein, indicating that the binding epitope of 147# is located between P145 and I164 of the human CD276 protein.

[0070] Furthermore, by means of alanine scanning (single-site mutation of each non-alanine amino acid site to alanine), 20 polypeptides (underlined amino acid sites of mutation) with N-terminal biotin modifications were synthesized as follows: 1-1.bio- A SMTLEPNKD LRPGDTVTI(145~164); 1-2.bio-P AMTLEPNKD LRPGDTVTI(145~164); 1-3.bio-PS A TLEPNKD LRPGDTVTI(145~164); 1-4.bio-PSM A LEPNKD LRPGDTVTI(145~164); 1-5.bio-PSMT A EPNKD LRPGDTVTI(145~164); 1-6.bio-PSMTL A PNKD LRPGDTVTI(145~164); 1-7.bio-PSMTLE A NKD LRPGDTVTI(145~164); 1-8.bio-PSMTLEP A KD LRPGDTVTI(145~164); 1-9.bio-PSMTLEPN A D LRPGDTVTI(145~164); 1-10.bio-PSMTLEPNK A LRPGDTVTI(145~164); 1-11.bio-PSMTLEPNKD A RPGDTVTI(145~164); 1-12.bio-PSMTLEPNKD L A PGDTVTI(145~164); 1-13.bio-PSMTLEPNKD LR A GDTVTI(145~164); 1-14.bio-PSMTLEPNKD LRP A DTVTI(145~164); 1-15.bio-PSMTLEPNKD LRPG A TVTI(145~164); 1-16.bio-PSMTLEPNKD LRPGD A VTI(145~164); 1-17.bio-PSMTLEPNKD LRPGDT A TI(145~164); 1-18.bio-PSMTLEPNKD LRPGDTV A I(145-164); 1-19.bio-PSMTLEPNKD LRPGDTVT A (145-164); 1-20.bio-PSMTLEPNKD LRPGDTVTI(145~164).

[0071] As a result, as shown in Figure 5, 147# hardly bound to peptides 1-7, 1-10, 1-11, 1-12, 1-14, and 1-15, and its binding to peptides 1-9 and 1-17 was somewhat weaker. However, its binding to other polypeptides was consistent with that of the original polypeptide (PEP1). This indicates that the amino acids P / D / L / R / G / D in polypeptide PEP1 are the most important for the binding of 147#, followed by amino acids K / V. In other words, P151 / D154 / L155 / R156 / G157 / D158 in the human CD276 protein are the most important binding sites for monoclonal antibody 147#, and K153 / V161 are the next most important sites.

[0072] Example 5: Determination of cross-reactivity of monoclonal antibody 147# with cynomolgus monkey CD276-ECD antigen The binding affinity of 147# to cynomolgus monkey CD276-ECD (purchased from Beijing Acro BIOSYSTEMS, Inc., whose amino acid sequence is shown in SEQ ID NO: 2) and mouse CD276-ECD (purchased from Beijing Acro BIOSYSTEMS, Inc., whose amino acid sequence is shown in SEQ ID NO: 3) was measured by indirect enzyme-linked immunosorbent assay (ELISA). The binding affinity of mouse-derived antibodies to human CD276-ECD protein was measured by indirect enzyme-linked immunosorbent assay (ELISA). Cynomolgus monkey and mouse CD276-ECD proteins were pre-coated and diluted to 2 μg / ml in coating solution (50 mM carbonate coating buffer, pH 9.6) and coated on plates overnight at 4°C. The plates were then blocked with 5% nonfat dry milk at 37°C for 2 hours. The plate was washed three times with PBST, and 100 μl of the test antibody, gradient diluted with 1% BSA-PBST, was added to the blocked microplate in order and incubated at 37°C for 1 hour. The plate was washed three times with PBST, and HRP-conjugated goat anti-mouse IgG secondary antibody (Millipore) was added and incubated at 37°C for 30 minutes. After washing the plate three times with PBST, the plate was wiped clean with absorbent paper, and 100 μl of TMB (KPL) was added to each well. The plate was then incubated at room temperature (20±5°C) for 5 minutes, away from light. The substrate reaction was stopped by adding 50 μl of 2M H2SO4 stop solution to each well, and the OD reading at 450 nm was measured using a microplate reader to analyze the binding ability of the test antibody to its target antigen, cynomolgus monkey and mouse CD276-ECD protein.

[0073] As a result, as shown in Figure 6, the EC 50 The EC for binding to human CD276-ECD was 30.16 ng / ml, i.e., 0.2 nM. 50 The antibody 147# did not bind to mouse-derived CD276-ECD (38.35 ng / ml).

[0074] Example 6 Production of humanized antibody hu147 In this example, the heavy chain variable region and light chain variable region of hybridoma 147# were obtained by molecular biology-related methods, and a chimeric antibody was constructed.

[0075] RNA from 147# hybridoma cells was extracted using Trizol and mRNA was reverse transcribed to obtain cDNA. Then, using the cDNA as a template, PCR was performed using degenerate primers for the heavy and light chains of mouse-derived antibodies (the sequences of the combined primers were obtained from page 323 of "Antibody Engineering," Volume 1, Edited by Roland Kontermann and Stefan Dubel). The PCR products obtained were sequenced and analyzed using the Kabat database, confirming that the obtained sequences were the sequences of the variable regions of mouse-derived antibodies.

[0076] The relevant sequence information is as follows:

[0077] The gene sequences of the heavy chain variable regions of 147# are both 366 bp in length and encode 122 amino acid residues. The nucleotide sequence is shown in SEQ ID NO:4, and the amino acid sequence is shown in SEQ ID NO:5.

[0078] The gene sequences of the light chain variable regions of 147# are both 321 bp in length and encode 107 amino acid residues. The nucleotide sequence is shown in SEQ ID NO:6, and the amino acid sequence is shown in SEQ ID NO:7.

[0079] The amino acid sequences of the light and heavy chain variable regions of mouse antibody 147# were analyzed, and three antigen complementarity-determining regions (CDRs) and four framework regions (FRs) of mouse antibody 147# were determined using the Kabat rules. The amino acid sequences of the heavy chain complementarity-determining regions of 147# are HCDR1: KTSGYIFT (SEQ ID NO: 10), HCDR2: IGRIYP (SEQ ID NO: 11), and HCDR3: ARGGEVRRDFYALDY (SEQ ID NO: 12). The amino acid sequences of the light chain complementarity-determining regions of 147# are LCDR1: KASENVGTY (SEQ ID NO: 13), LCDR2: GASNRYT (SEQ ID NO: 14), and LCDR3: GQRYSYPFT (SEQ ID NO: 15).

[0080] Humanization templates that optimally matched the FR regions of each of the mouse-derived antibodies were selected from the Germline database. The CDR regions of the mouse-derived antibodies were then grafted onto the selected humanization templates in place of the CDR regions of the human-derived templates. The heavy chain variable region was then recombined with a human IgG1 constant region, and the light chain variable region with a human κ chain constant region. Based on the three-dimensional structure of the antibody, back mutations were performed on buried residues, residues that directly interact with the CDR regions, and residues that significantly affect the conformation of the VL and VH of each antibody, to form the humanized antibody heavy chain variable region (hu147VH, amino acid sequence SEQ ID NO: 21) and the humanized antibody light chain variable region (hu147VL, amino acid sequence SEQ ID NO: 22), respectively. The humanized antibody heavy chain (hu147H, amino acid sequence SEQ ID NO: 23) was formed by linking hu147VH to a human IgG1 constant region (amino acid sequence SEQ ID NO: 8). The hu147VL was linked to the human kappa chain constant region (amino acid sequence SEQ ID NO: 9) to form the humanized antibody light chain (hu147L, amino acid sequence SEQ ID NO: 24). The humanized antibody heavy and light chains were constructed in the pcDNA3.4 vector, transfected into Expi-293F cells, and purified with Protein A to obtain the humanized antibody hu147. SDS-PAGE electrophoresis and SEC-HPLC confirmed that the molecular weight of each antibody was accurate and that the purity was >95%.

[0081] Example 7: Preparation of a humanized antibody with Fc mutation (hu147mu) The heavy chain variable region of hu147 was linked to a mutant human IgG1 constant region (amino acid sequence shown in SEQ ID NO: 25) to form the heavy chain of hu147mu (amino acid sequence shown in SEQ ID NO: 26), and the light chain was the same as hu147 (amino acid sequence shown in SEQ ID NO: 24). These were then constructed into the pcDNA3.4 expression vector, transfected into Expi-293F cells, and purified with Protein G to obtain the antibody hu147mu. SDS-PAGE electrophoresis and SEC-HPLC confirmed that the molecular weight of each expressed antibody was approximately 150 kD and the purity of the antibody was >95%. The antibody was then quantified, aliquoted, and stored frozen at -80°C for future use.

[0082] Example 8 Measurement of the binding activity of humanized antibody hu147 and mutant humanized antibody hu147mu to human CD276-ECD by ELISA The binding affinity of the above humanized antibodies hu147 and hu147mu to human CD276-ECD was measured by ELISA. See Example 2 for the relevant experimental methods.

[0083] As a result of the experiment, as shown in Figure 7, the EC 50 The EC50 values ​​were 6.70 ng / ml and 6.80 ng / ml, i.e., 0.04 nM and 0.05 nM, respectively, both of which were significantly improved compared to 147# (EC50: 0.26 nM), indicating that hu147 and hu147mu have good affinity for human CD276-ECD and that the above mutations in the Fc do not affect antigen binding.

[0084] Example 9 Preparation of hu147-IL15 bifunctional molecules SPGL008 and SPGL007 The amino acid sequence of human IL15Rsusi is shown in SEQ ID NO:27, and the amino acid sequence of human IL15 is shown in SEQ ID NO:28. Human IL15Rsusi was linked to human IL15 via (GGGGS)6 to form single-chain IL15, i.e., IL15sc (shown in SEQ ID NO:29). The heavy chain sequence of hu147 (amino acid sequence shown in SEQ ID NO:23) was linked to the human IL15sc sequence via (GGGGS)3 to form the heavy chain of SPGL007 (amino acid sequence shown in SEQ ID NO:30). The heavy chain sequence of hu147mu (amino acid sequence shown in SEQ ID NO:26) was linked to the human IL15sc sequence via (GGGGS)3 to form the heavy chain of SPGL008 (amino acid sequence shown in SEQ ID NO:31). The light chain is the same as hu147 (amino acid sequence shown in SEQ ID NO:24). The heavy and light chains of SPGL007 were co-transfected into Expi-293F cells and purified with Protein G to yield the bifunctional antibody SPGL007. The heavy and light chains of SPGL008 were co-transfected into Expi-293F cells and purified with Protein G to yield the bifunctional antibody SPGL008. SDS-PAGE electrophoresis and SEC-HPLC confirmed that the molecular weight of each expressed antibody was approximately 190 kD and the purity of the antibody was >95%. The antibodies were quantified, aliquoted, and stored frozen at -80°C for further use.

[0085] Example 10 Measurement of binding activity of SPGL008 and SPGL007 to human CD276-ECD by ELISA The binding affinity of the above SPGL008 and SPGL007 to human CD276-ECD was measured by ELISA, and the relevant experimental method is described in Example 2.

[0086] As a result of the experiment, as shown in Figure 8, the EC 50 were 31.51 ng / ml and 32.75 ng / ml, i.e., 0.16 nM and 0.17 nM, respectively, both of which were slightly lower than those of hu147 (0.05 nM) or hu147mu (0.04 nM), but still maintained relatively high affinities.

[0087] Example 11 Simultaneous binding of SPGL008 to human CD276 and human CD122 / 132 The binding of SPGL008 and SPGL007, which bind to CD276-ECD, to human CD122 / 132 was measured using an ELISA assay, demonstrating that SPGL008 simultaneously binds to both CD276 and CD122 / 132. The method was as follows: human CD276-ECD protein was pre-coated and diluted to 2 μg / ml in coating solution (50 mM carbonate coating buffer, pH 9.6), and the plate was then incubated overnight at 4°C. The plate was then blocked with 5% nonfat dry milk for 2 hours at 37°C. The plate was washed three times with PBST, and the test antibody was diluted to 1 μg / ml in 1% BSA-PBST. 100 μl of the diluted antibody was added per well to the blocked microplate and incubated at 37°C for 1 hour. The plate was washed three times with PBST, gradient-diluted biotin-labeled CD122 / 132 (Beijing Acro BIOSYSTEMS) was added, and incubated at 37°C for 1 hour. After washing the plate three times with PBST, diluted HRP-labeled SA (Pierce) was added and incubated at 37°C for 30 minutes. After washing the plate three times with PBST, the plate was wiped clean with absorbent paper to remove residual droplets. 100 μl of TMB (KPL) was added to each well and incubated at room temperature (20±5°C) for 5 minutes, away from light. The substrate reaction was stopped by adding 50 μl of 2M H2SO4 stop solution to each well. The OD value was read at 450 nm using a microplate reader to analyze the binding ability of the test antibody to CD122 / 132.

[0088] As a result, as shown in Figure 9, both antibodies SPGL008 and SPGL007 have good binding activity, and under the conditions of this experiment, the EC 50 were 17.71 ng / ml and 15.08 ng / ml, i.e., 0.09 nM and 0.08 nM, respectively. As a control, hu147 was able to bind to CD276 (as shown in Example 8), but was unable to bind to CD122 / 132.

[0089] Example 12 Cell proliferation promoting activity of SPGL008 In this example, the biological activity of SPGL008 and SPGL007 was demonstrated through a CTLL2 cell proliferation experiment. The method was as follows: CTLL2 cells were cultured at 5 × 10 in 1640 medium containing 10% FBS. 4 IL2 was diluted to 30 ng / ml in 1640 medium containing 10% FBS, and then diluted three-fold to a total of eight gradients before being placed in the CTLL2 cell-containing culture plates. SPGL008 and SPGL007 were diluted to 5000 ng / ml in 1640 medium containing 10% FBS, and then further diluted three-fold to a total of eight gradients before being placed in the CTLL2 cell-containing culture plates. After 72 hours of culture in a CO2 cell culture incubator, the relative cell number in each well was measured using CCK8, and EC 50 was calculated to determine the activity of the sample.

[0090] As shown in Figure 10A, both SPGL008 and SPGL007 were able to stimulate the proliferation of CTLL2 cells and EC 50 The EC values ​​of SPGL008 and SPGL007 were 103.0 ng / ml and 91.0 ng / ml, i.e., 0.53 and 0.48 nM, respectively, indicating that SPGL008 had biological activity consistent with that of SPGL007. In contrast, the EC values ​​of IL2 were 103.0 ng / ml and 91.0 ng / ml, i.e., 0.53 and 0.48 nM, respectively, as shown in Figure 10B. 50 was 0.74 ng / ml, i.e., 0.048 nM.

[0091] Example 13: Clearly reduced toxicity of SPGL008 in mice compared to SPGL007 SPGL008 and SPGL007 were intraperitoneally injected twice into C57BL / 6 mice (Wetland Research Laboratories) on the first and third days, with an injection volume of 0.2 ml per injection. SPGL008 and SPGL007 were administered at doses of 0.5 mg / kg, 1 mg / kg, 2 mg / kg, and 4 mg / kg. The experimental mice were observed daily for mortality, and the control group was administered the same volume of PBS.

[0092] As shown in Table 1, on day 10 of the experiment, all animals survived at doses of 0.5 mg / kg, 1.0 mg / kg, and 2.0 mg / kg in the SPGL008 group (100% survival rate), while 70% of animals died at the 4.0 mg / kg dose (30% survival rate). In the SPGL007 group, 100% of animals died at doses of 4.0 mg / kg and 2.0 mg / kg (0% survival rate), 60% of animals died at the 1.0 mg / kg dose (40% survival rate), and no animals died at the 0.5 mg / kg dose (100% survival rate). These results indicate that after Fc mutation, SPGL008 is significantly less toxic to experimental mice than SPGL007.

[0093] Table 1. Survival rate of experimental animals in each dose group [Table 1]

[0094] Example 14 Inhibition of tumor growth by SPGL008 in MC38 mouse colon / rectal cancer cells In this example, the in vivo antitumor activity of SPGL008 and SPGL007 was evaluated using a mouse MC38 colorectal cancer cell transplant tumor model. The experimental method was as follows.

[0095] In vitro cultured mouse colon cancer MC38 cells were collected and the cell suspension was diluted to a concentration of 1 × 10 7 The cell suspension was adjusted to a concentration of 100 μl / ml. Under sterile conditions, 100 μl of the cell suspension was inoculated subcutaneously into the right flank of C57BL / 6 mice. The diameter of the subcutaneously implanted tumors in the mice was measured with a vernier caliper, and the average tumor volume was 100-200 mm. 3At the age of 18, the animals were randomly assigned to groups of 6 animals per group. SPGL008 and SPGL007 were administered at 0.5 mg / kg, while the control group received an equal volume of PBS. These were administered intraperitoneally twice a week in a volume of 0.2 ml per injection for two consecutive weeks. Throughout the experiment, the diameter of the transplanted tumors was measured three times a week, and the mice were weighed. The formula for calculating tumor volume (TV) was as follows: TV=1 / 2×a×b2

[0096] where a and b represent length and width, respectively. The relative tumor volume (RTV) was calculated from the measurement results using the formula RTV = Vt / V0. Here, V0 is the tumor volume measured at the time of administration (i.e., d0) and Vt is the tumor volume at each measurement. The evaluation index for antitumor activity is the relative tumor growth rate T / C (%), and the calculation formula is as follows: T / C(%)=(TRTV / CRTV)×100 Tumor inhibition rate TGI(%)=100-T / C(%)

[0097] TRTV is the RTV of the treatment group, and CRTV is the RTV of the negative control group.

[0098] As a result, as shown in Figure 11, both SPGL008 and SPGL007 exhibited strong antitumor activity, with TGI of 76.2% and 69.4%, respectively, which was not significantly different (p>0.05), indicating that the Fc mutation in SPGL008 did not affect its in vivo antitumor activity.

[0099] Example 15: Inhibition of growth of human lung cancer cell NCI H1975 transplanted tumors by SPGL008 In vitro cultured human lung cancer NCI-H1975 cells were collected and the cell suspension was diluted to a concentration of 8 × 10 7 The cell suspension was adjusted to a concentration of 100 μl / ml. Under sterile conditions, 100 μl of the cell suspension was inoculated subcutaneously into the right flank of nude mice. When the tumor cells formed solid tumors under the skin of the mice, the diameter of the transplanted tumor was measured with a vernier caliper, and the average tumor volume was 50-100 mm.3 The animals were randomly assigned to groups at the time of growth. The mice were administered 1.0 mg / kg, 0.3 mg / kg, and 0.1 mg / kg intraperitoneally, three times a week for a total of six doses. Throughout the experiment, the diameter of the transplanted tumors was measured twice a week, and the mice were weighed at the same time. The rest of the experiment was the same as in Example 14.

[0100] As a result, as shown in Figure 12, SPGL008 exhibited strong antitumor activity, with TGI of 70.0%, 60.8% and 62.5% at doses of 1.0 mg / kg, 0.3 mg / kg and 0.1 mg / kg, respectively.

[0101] Example 16: Inhibition of the growth of JIMT-1 human breast cancer cell xenograft tumors in nude mice by the combined use of SPGL008 and HER2 monoclonal antibody In vitro cultured human breast cancer JIMT-1 cells were collected and the cell suspension was diluted to a concentration of 8 × 10 7 The cell suspension was adjusted to a concentration of 100 μl / ml. Under sterile conditions, 100 μl of the cell suspension was inoculated subcutaneously into the right flank of nude mice. When the tumor cells formed solid tumors under the skin of the mice, the diameter of the transplanted tumor was measured with a vernier caliper, and the average tumor volume was 50-100 mm. 3 The animals were randomly assigned to groups at the time of growth. In the single-drug group, HER2 monoclonal antibody (trastuzumab) was administered at a dose of 20 mg / kg. In the combination group, trastuzumab was administered at a fixed dose of 20 mg / kg in combination with SPGL008 at 1.0 mg / kg and 0.3 mg / kg, respectively, twice a week via intraperitoneal injection for a total of six doses. Throughout the experiment, the diameter of the transplanted tumor was measured twice a week, and the mice were weighed at the same time. The rest of the procedure was the same as in Example 15.

[0102] As shown in Figure 13, the trastuzumab group showed weak antitumor activity, with a TGI of 55%, indicating that JIMT-1 is a tumor resistant to trastuzumab. When SPGL008 was administered in combination with trastuzumab at a dose of 1.0 mg / kg, the TGI reached 90%, and 50% (3 / 6) of the tumors completely disappeared (CR). When SPGD008 was administered in combination with trastuzumab at a dose of 0.3 mg / kg, the TGI reached 72%, demonstrating a significantly higher antitumor effect than trastuzumab alone.

[0103] Example 17 Metabolism of SPGL008 in huFcRn transgenic mice The pharmacokinetics of SPGL007 and SPGL008 were measured using human FcRn transgenic mice. The method was as follows: Eight mice were divided into two groups and intraperitoneally injected with SPGL007 and SPGL008 at a dose of 1 mg / kg. Blood was collected and serum obtained 2, 6, 24, 48, and 72 hours later. After appropriate dilution, serum drug concentrations were measured by ELISA, essentially as in Example 11.

[0104] As a result, as shown in Figure 14, the Fc mutant SPGL008 was clearly metabolized more quickly in the bodies of human FcRn genetically modified mice than the Fc wild-type SPGL007, with half-lives of 10 hours and 18 hours, respectively, which was a significant difference.

[0105] Example 18 Detection of CD276 expression in several types of tumor cells by SPGL008 The detection was carried out by flow cytometry, and the detection method was basically the same as in Example 3.

[0106] As a result, as shown in Figure 15, SPGL008 was able to bind to several types of human lung cancer cells (H1975, Calu-3, A549, H322, H292), human breast cancer cells (JIMT-1), human kidney cancer cells (A498), and human skin cancer cells (A431), suggesting that the above several types of tumors may be suitable indications for SPGL008.

[0107] Example 19: Excellent stability of SPGL008 The stability of SPGL008 was examined by size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) to detect changes in purity after leaving SPGL008 standing for different periods of time.

[0108] The analysis was performed using a TSKgel G3000SWXL chromatography column (TSK) on an HPLC Ultimate 3000 (Thermo) chromatograph. The detection mobile phase was PBS (pH 7.4), the constant flow rate was 0.8 ml / min, and the loading volume was 100 μg / 100 μl. The purity was expressed as the content (%) of the target protein in the total protein calculated using the integration method from the absorption peak at 280 nM.

[0109] As a result, as shown in Figures 16, 17, and 18, the purity of SPGL008 in PBS solution was 84.9% at 0 week, and after standing at 4°C for 5 weeks, the purity was 84.0%, with a change rate of <2%, and after standing at 37°C for 5 weeks, the purity was 83.9%, with a change rate of <2%, indicating that SPGL008 remained stable in PBS buffer solution under both 4°C and 37°C conditions.

[0110] It should be noted that the above examples are only for explaining the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art will understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the substance and scope of the technical solutions of the present invention.

Claims

1. A monoclonal antibody capable of recognizing human CD276-ECD protein, characterized in that the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:5 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

7.

2. A recombinant antibody obtained by humanizing the monoclonal antibody described in claim 1, characterized in that the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 21 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

22.

3. A multi-functional recombinant antibody, characterized in that the heavy chain comprises an antibody functional region that recognizes human CD276, a human IgG1 constant region functional domain, a functional region of human IL15, and a non-functional amino acid fragment for linking each functional region, and the amino acid sequence of the antibody functional region that recognizes human CD276 comprises the amino acid sequence of the heavy chain variable region of the recombinant antibody described in claim 2.

4. The multi-functional recombinant antibody described in claim 3, characterized in that the human IgG1 constant region functional domain is a mutated human IgG1 constant region, the amino acid sequence of the mutated human IgG1 constant region is shown in SEQ ID NO: 25, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain is shown in SEQ ID NO:

24.

5. A nucleotide sequence encoding the monoclonal antibody of claim 1, or the recombinant antibody of claim 2, or the IL15 recombinant antibody targeting CD276 of claim 3 or 4.

6. An expression vector comprising the nucleotide sequence of claim 5.

7. A host cell comprising the expression vector of claim 6.

8. Use of a monoclonal antibody described in claim 1, or a recombinant antibody described in claim 2, or an IL15 recombinant antibody targeting CD276 described in claim 3 or 4, or a nucleotide sequence described in claim 5, or an expression vector described in claim 6, or a host cell described in claim 7 in the manufacture of a biological drug for treating tumors.

9. A biological preparation characterized by comprising at least one of the monoclonal antibody described in claim 1, or the recombinant antibody described in claim 2, or the IL15 recombinant antibody targeting CD276 described in claim 3 or 4, or the nucleotide sequence described in claim 5, or the expression vector described in claim 6, or the host cell described in claim 7.

10. (1) obtaining an expression vector containing a gene fragment of the monoclonal antibody or recombinant antibody or IL15 recombinant antibody targeting CD276 by artificial synthesis or molecular biology methods; (2) transfecting the expression vector into cells to express the protein; (3) obtaining said monoclonal antibody or recombinant antibody or IL15 recombinant antibody targeting CD276 by protein purification; A method for producing the monoclonal antibody of claim 1, the recombinant antibody of claim 2, or the IL15 recombinant antibody targeting CD276 of claim 3 or claim 4, characterized in that it comprises:

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