Multifunctional recombinant antibodies and methods of making and using the same

Abstract

The application discloses a multifunctional recombinant antibody and a preparation method and application thereof, and provides a monoclonal antibody capable of specifically recognizing human CD276 protein, and also provides a humanized recombinant antibody, and a multifunctional antibody capable of recognizing human CD276 and having IL15 function is further restructured on the basis, and the multifunctional antibody can effectively enhance the killing effect of the original antibody on tumor cells. Meanwhile, the application also introduces a mutant human IgG1 constant region to obtain a restructured specific CD276 monoclonal antibody-IL15 bifunctional molecule, and the restructured specific CD276 monoclonal antibody-IL15 bifunctional molecule can effectively reduce the toxicity of the antibody and improve the safety. The antibody of the application can recognize CD276 protein in various tumor cells, and then has a killing effect on various tumor cells, and the restructured CD276 targeted IL15 recombinant antibody has the advantages of short drug metabolism cycle, low toxicity and high safety, and has a great application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to an Fc mutant CD276-targeted IL15 recombinant multifunctional antibody, a preparation method and application thereof, in particular to a multifunctional recombinant antibody capable of recognizing CD276 and having IL15 function, and having reduced toxicity in vivo, and application thereof. BACKGROUND

[0002] CD276, also known as B7-H3, is a type I transmembrane protein, belongs to the B7 immune co-stimulation and co-inhibition family, and has immune regulation function. As an immune regulation related receptor protein, its ligand is still inconclusive. The expression level in normal human tissues is low, CD276 is expressed in activated macrophages and monocytes, and can inhibit T cells to avoid excessive activation of immunity.

[0003] CD276 is widely expressed in various malignant tumors, including lung cancer, liver cancer, intestinal cancer, gastric cancer, breast cancer, pancreatic cancer and renal cancer, etc. The expression of CD276 in tumor cells can promote the progression of tumors and lead to poor prognosis, because CD276 can inhibit anti-tumor immunity in the tumor microenvironment.

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

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

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

[0007] Thirdly, the specific expression of CD276 in tumor cells can achieve specific killing of tumor cells through ADC.

[0008] IL15 is a cytokine expressed by various cells, including monocytes, macrophages, epidermal cells and fibroblasts, but not T lymphocytes. Unlike other cytokines, IL15 is generally not secreted by cells to play a role, but is positioned on the specific cell membrane after combining with IL15Rα, so as to stimulate the nearby effector cells, mainly NK and CD8+T cells. IL15 is closely related to IL2, and the complex of IL15 and IL15Rα can bind to the shared β / γ receptor of IL2 to mediate biological activity. In terms of anti-tumor effect, one advantage of IL15 over IL2 is that IL15 / Rα does not stimulate the proliferation of Tregs.

[0009] Currently, there are a variety of IL15 related molecules in the development stage for the treatment of malignant tumors. The most advanced is ALT-803, which is a complex formed by IL15 and IL15R alpha sushi-hFcl. Multiple clinical studies show that ALT-803 is effective for a variety of tumors including melanoma, but at the same time, serious side effects occur, mainly including liver damage, hypotension and fever, etc. SUMMARY

[0010] To solve the above problems, the present application aims to provide a specific monoclonal antibody capable of specifically recognizing CD276 protein, a hybridoma cell secreting the monoclonal antibody; and a recombinant antibody obtained by humanization of the monoclonal antibody; and a multifunctional recombinant antibody capable of recognizing human CD276 protein and having human IL15 function, which can be used for malignant tumors; at the same time, we prepared a modified specific monoclonal antibody-IL15 bifunctional molecule, which greatly reduces the side effects while maintaining the anti-tumor activity.

[0011] To achieve the above-mentioned object, the technical scheme adopted by the present application is:

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

[0013] The present application prepared a murine monoclonal antibody capable of specifically recognizing human CD276 protein by immunizing mice with recombinant expressed human CD276-ECD protein, named 147#. Through biological analysis, the amino acid sequences of the heavy chain variable region and the light chain variable region of the murine monoclonal antibody were obtained. The antibody can effectively bind to CD276. Through biological analysis, the heavy chain variable region of the monoclonal antibody encodes 122 amino acid residues, and the light chain variable region encodes 107 amino acid residues.

[0014] Further, the present application also claims a recombinant antibody obtained by humanization of the monoclonal antibody 147#, the amino acid sequence of the heavy chain variable region of the recombinant antibody is shown in SEQ ID NO: 21, and the amino acid sequence of the light chain variable region of the recombinant antibody is shown in SEQ ID NO: 22.

[0015] As a preferred embodiment of the present application, 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 of the recombinant antibody is shown in SEQ ID NO: 24.

[0016] The inventors of the present application further analyze the sequence of the obtained mouse-derived monoclonal antibody 147#, replace the CDR region of the human template, recombine the heavy chain variable region with the human IgG1 constant region, recombine the light chain variable region with the human kappa chain constant region, and based on the three-dimensional structure of the antibody, perform back-mutation on the buried residues, residues that have direct interaction with the CDR region, and residues that have important influence on the conformation of the VL and VH of each antibody, to finally obtain a humanized recombinant antibody, named hu147#. The amino acid sequences of the heavy chain variable region and the light chain variable region of the humanized recombinant antibody hu147#, the heavy chain amino acid sequence, and the light chain amino acid sequence are shown above.

[0017] Further, the present application also protects a multifunctional recombinant antibody, the heavy chain of the multifunctional recombinant antibody comprising an antibody functional region recognizing human CD276, a human IgG1 constant region functional domain, an IL15 functional region, and a non-functional amino acid fragment for connecting the functional regions; the amino acid sequence of the antibody functional region recognizing human CD276 comprising the heavy chain variable region amino acid sequence of the recombinant antibody.

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

[0019] The inventors of the present application further modify the obtained humanized antibody above, connect the antibody heavy chain sequence with the sequence having the IL15 function, and further obtain a recombinant antibody that can recognize human CD276 protein and has IL15 function.

[0020] As a preferred embodiment of the present application, the human IgG1 constant region functional domain is a human IgG1 constant region, and the amino acid sequence 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 shown in SEQ ID NO: 25.

[0022] The inventors of the present application obtain a CD276-targeted IL15 recombinant antibody by using the sequence containing the mutated human IgG1 constant region, and the metabolic cycle of the recombinant antibody is shorter, the toxic side effects are lower, and the safety is better.

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

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

[0025] The amino acid sequence of the functional region of the preferred human IL15 shown in SEQ ID NO: 29 is that the human IL15 R sushi is connected with the human IL15 through (GGGGS) 6 to form a single-chain IL15, namely IL15sc.

[0026] As a preferred embodiment of the present application, the non-functional amino acid fragment for connecting each functional region in the CD276-targeted IL15 recombinant antibody is a GGGGS repeat. The linker plays an important role in constructing a stable, biologically active fusion protein, which can ensure the correct folding of the protein, maintain the biological activity, and improve the protein yield, etc.

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

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

[0029] The present application prepares a multifunctional antibody capable of recognizing CD276 and having IL15 function, named SPGL007.

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

[0031] The multifunctional recombinant antibody prepared by using the preferred multifunctional recombinant antibody sequence contains the sequence of the mutated human IgG1 constant region, named SPGL008; the multifunctional recombinant antibody SPGL008 not only can effectively recognize CD276 in a variety of tumor cells, but also can effectively inhibit the growth of a variety of cancer cells, and has a shorter half-life, lower toxicity, and good safety.

[0032] Further, the present application also claims the nucleotide sequence encoding the monoclonal antibody, or the recombinant antibody, or the multifunctional recombinant antibody.

[0033] According to the amino acid sequence of the monoclonal antibody, or the recombinant antibody, or the multifunctional recombinant antibody, the corresponding encoding gene nucleotide sequence can be obtained.

[0034] As a preferred embodiment of the present application, 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] Further, the present application also claims an expression vector comprising the nucleotide sequence.

[0036] By means of molecular biology, an expression vector comprising the nucleotide sequence can be constructed.

[0037] Further, the present application also claims a host cell comprising the expression vector.

[0038] By means of cell biology, a host cell capable of effectively expressing the antibody protein can be constructed by using the expression vector.

[0039] Further, the present application also claims the use of the monoclonal antibody, or the recombinant antibody, or the multifunctional recombinant antibody; or the nucleotide sequence, or the expression vector, or the host cell in the preparation of a biological agent for treating tumors.

[0040] Further, the present application also claims a biological agent comprising at least one of the monoclonal antibody, or the recombinant antibody, or the multifunctional recombinant antibody; or the nucleotide sequence, or the expression vector, or the host cell.

[0041] Further, the present application also provides a preparation method of the monoclonal antibody or the recombinant antibody, or the multifunctional recombinant antibody, comprising the following steps:

[0042] (1) obtaining an expression vector comprising a gene fragment of the monoclonal antibody or the recombinant antibody or the multifunctional recombinant antibody by artificial synthesis or molecular biology;

[0043] (2) transfecting cells with the expression vector for protein expression;

[0044] (3) obtaining the monoclonal antibody or the recombinant antibody or the multifunctional recombinant antibody by protein purification.

[0045] By constructing an expression vector comprising the heavy chain or light chain sequence of the above antibody, transfecting suitable cells for expression and purification, the corresponding antibody can be obtained.

[0046] As a preferred embodiment of the present application, in step (1), the expression vector comprising the gene fragment of the monoclonal antibody or the recombinant antibody or the multifunctional recombinant antibody is prepared by molecular biology, i.e. inserting the heavy chain or light chain nucleotide sequence of the antibody into a pcDNA3.4 expression vector.

[0047] As a preferred embodiment of the present application, in the step (2), the cell for protein expression is Expi-293F cell.

[0048] As a preferred embodiment of the present application, the protein purification method in the step (3) is Protein G purification.

[0049] Compared with the prior art, the present application has the following technical effects:

[0050] The present application provides a monoclonal antibody capable of specifically recognizing human CD276 protein, and also provides a humanized recombinant antibody thereof. On this basis, the inventors further prepared a multifunctional recombinant antibody capable of recognizing human CD276 and having IL15 function by using CD276 specific recombinant antibody, which can effectively enhance the killing effect of the original antibody on tumor cells. At the same time, the present application introduces a mutant human IgG1 constant region to obtain a modified specific CD276 monoclonal antibody-IL15 bifunctional molecule. The bifunctional molecule can effectively shorten its metabolic cycle while maintaining its anti-tumor activity in vivo, thereby reducing its toxic side effects in vivo and greatly improving its safety. The multifunctional recombinant antibody of the present application can recognize CD276 on a variety of tumor cells, and has great application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 Results of ELISA method for determining the binding activity of mouse-derived antibody 147# to human CD276-ECD protein

[0052] Figure 2 Results of flow cytometry for determining the binding of mouse-derived monoclonal antibody to CD276.

[0053] Figure 3 Results of Western blot method for determining the binding of 147# to human CD276-ECD.

[0054] Figure 4 、 Figure 5 Results of ELISA method for detecting the binding of 147# to polypeptide in the human CD276 domain Ig-like C2-type 1 region.

[0055] Figure 6 Results of cross-reactivity determination of monoclonal antibody 147# with cynomolgus monkey CD276-ECD antigen.

[0056] Figure 7 Results of ELISA method for determining the binding activity of humanized antibody hu147 and mutant humanized antibody hu147mu to human CD276-ECD.

[0057] Figure 8ELISA assay results of SPGL008 and SPGL007 binding activity to human CD276-ECD.

[0058] Figure 9 SPGL008 binding activity to human CD276 and human CD122 / 132.

[0059] Figure 10A 、 10B SPGL008 cell proliferation promoting activity results.

[0060] Figure 11 SPGL008 inhibiting mouse colorectal cancer cell MC38 tumor growth results.

[0061] Figure 12 SPGL008 inhibiting human lung cancer cell NCI H1975 tumor growth results.

[0062] Figure 13 SPGL008 combined with HER2 monoclonal antibody inhibiting human breast cancer cell JIMT-1 tumor growth in nude mice results.

[0063] Figure 14 SPGL008 metabolic results in huFcRn transgenic mice.

[0064] Figure 15 Flow cytometry results of SPGL008 binding to various tumor cells.

[0065] Figure 16 SPGL008 protein after 0 weeks of storage.

[0066] Figure 17 SPGL008 protein after 5 weeks of storage at 4℃.

[0067] Figure 18 SPGL008 protein after 5 weeks of storage at 37℃. DETAILED DESCRIPTION

[0068] For the purpose of better illustrating the object, technical scheme and advantages of the present application, the present application will be further described below in conjunction with the drawings and specific examples.

[0069] Example 1 Preparation and screening of antigen immunized animals and hybridomas

[0070] Step 1: Immunize mice with antigen

[0071] Balb / c mice were routinely immunized subcutaneously with recombinant human CD276-ECD protein (purchased from Beijing Baipusaisi Co., Ltd., Acro BIOSYSTEMS, amino acid sequence as shown in SEQ ID NO: 1). On day 1, human CD276-ECD protein was emulsified with Freund's complete adjuvant (CFA, Sigma) and thoroughly mixed, and then subcutaneously injected into Balb / c mice (50 μg / mouse). On days 14 and 36, human CD276-ECD protein was emulsified with Freund's incomplete adjuvant (IFA, Sigma) and thoroughly mixed, and then subcutaneously boosted into Balb / c mice (50 μg / mouse). On day 50, 50 μg / mouse of human CD276-ECD protein was injected intraperitoneally to challenge the mice. Three to four days later, the spleens of the mice were harvested for fusion experiments.

[0072] Step 2: Preparation and screening of hybridomas

[0073] Three to four days after the final immunization in mice, mouse spleen cells and mouse myeloma cells SP2 / 0 were fused using a standard hybridoma technique via PEG (PEG1450, Sigma). The fused cells were then suspended and homogenized in complete culture medium, which consisted of a 1:1 mixture of RPMI 1640 and DMEM F12, followed by the addition of 1% Glutamine (Gibco), 1% Sodium pyruvate (Gibco), 1% MEM-NEAA (minimal basal medium-nonessential amino acid solution, Gibco), 1% Penicillin-streptomycin (Gibco), 50 μM β-mercaptoethanol (Gibco), and 20% FBS (fetal bovine serum, Gibco). The cells were then fused at a 10:1 ratio. 5 100 μl of cells per well was aliquoted into 96-well plates and cultured overnight. The next day, 100 μl of complete culture medium containing 2×HAT (Sigma) was added to each well, bringing the total culture medium in the 96-well plate to 200 μl (containing 1×HAT). After 7–12 days, the supernatant was harvested, and hybridoma wells showing positive human CD276-ECD protein binding activity were screened using an indirect enzyme-linked immunosorbent assay (ELISA).

[0074] The method for screening the positive hybridoma wells of human CD276-ECD protein activity by indirect enzyme-linked immunosorbent assay is as follows: the recombinant human CD276-ECD protein is diluted to 1 μg / ml with coating solution (50 mM carbonate coating buffer, pH 9.6), 100 μl / well is added to the enzyme-labeled plate, and 4°C is coated overnight. Wash the plate with PBST for 3 times, add 200 μl / well of blocking solution (2% BSA-PBST), and place at 37°C for 1 h, then wash the plate with PBST once and stand by. The collected hybridoma supernatant is sequentially added to the blocked enzyme-labeled plate, 100 μl / well, 37°C for 1 h. Wash the plate with PBST for 3 times, add HRP-labeled goat anti-mouse IgG secondary antibody (purchased from Abeam, product number ab6789), and place at 37°C for 30 min; wash the plate with PBST for 5 times, then try to pat dry the residual droplets on the blotting paper, add 100 μl of TMB (KPL company) to each well, and place at room temperature (20±5°C) in the dark for 5 min; add 50 μl of 2M H2SO4 stop solution to each well to terminate the substrate reaction, and read the OD value at 450 nm by enzyme-labeled instrument, to analyze the binding ability of the antibody to be tested to the target antigen human CD276-ECD protein.

[0075] The 10 hybridoma cell strains obtained by screening are expanded in serum-containing complete culture medium, centrifuged to replace the serum-free culture medium SFM, and cultured at a cell density of 1-2×10 7 / ml, 5% CO2, 37°C for 2 weeks, centrifuged to obtain culture supernatant, purified by Protein G affinity chromatography, and obtained mouse anti-human CD276-ECD protein monoclonal antibody, named 147#.

[0076] Example 2: ELISA method for determining the binding activity of mouse antibody 147# to human CD276-ECD protein

[0077] The indirect enzyme-linked immunosorbent assay was used to determine the binding ability of the mouse-derived antibody to the human CD276-ECD protein. The specific method is as follows: the human CD276-ECD protein was coated in advance, diluted to 2 μg / ml with coating solution (50 mM carbonate coating buffer, pH 9.6), 4°C, overnight; then blocked with 5% skimmed milk powder, 37°C, 2 hours; washed the plate 3 times with PBST, and then the antibody to be tested was added to the coated enzyme-labeled plate in gradient dilution with 1% BSA-PBST, 100 μl / well, 37°C for 1 hour. The plate was washed 3 times with PBST, and then HRP-labeled goat anti-mouse IgG secondary antibody (Millipore Corporation) was added, 37°C for 30 minutes; after washing the plate 3 times with PBST, the residual droplets were dried as much as possible on the blotting paper, 100 μl of TMB (KPL Corporation) was added to each well, and the plate was placed at room temperature (20±5°C) in the dark for 5 minutes; 50 μl of 2M H2SO4 stopping solution was added to each well to terminate the substrate reaction, and the OD value was read at 450 nm by an enzyme-labeled instrument, to analyze the binding ability of the antibody to be tested to the target antigen human CD276-ECD protein.

[0078] The results, as shown in Figure 1 Table 1, showed that the antibody 147# had good binding activity, with an EC 50 of 38.35 ng / ml, i.e. 0.26 nM.

[0079] Example 3 Flow cytometry method for determining the binding of mouse-derived monoclonal antibody to CD276

[0080] The binding affinity of 147# to human renal cancer cells A498 was determined by the method of fluorescence activated cell sorting (FACS).

[0081] In this experiment, human renal cancer cells A498 were used as target cells, and 100 μl of 147# was used as a primary antibody, which was diluted at a gradient of 6 times from 10000 ng / ml to 4 gradients, and was added to 1×10 5A498 cells were incubated with 147# at 4°C for 1 h (the highest working concentration of 147# was 5 ug / ml, and the lowest was 23 ng / ml), and the cells were washed twice with PBS to remove unbound 147#, and then the cells were incubated with 100 μl, 2 μg / ml, Alexa Fluor 488 labeled anti-mouse secondary antibody (purchased from Thermo, invitrogen company, item number A11001) at 4°C for 30 min, and the cells were washed twice with PBS to remove unbound secondary antibody, and finally the cells were resuspended in 100 μl PBS, and the binding affinity of 147# to the cells was determined by flow cytometry, and the data were analyzed by GraphPad Prism 6 software.

[0082] The results are shown in Figure 2 The results show that 147# can specifically bind to human renal cancer cell A498 with high expression of cell surface CD276, and the EC50 is 59.15 ng / ml, i.e. 0.39 nM.

[0083] Example 4 Determination of 147# binding to antigen epitope

[0084] 4.1. Determination of the binding ability of 147# to reduced denatured CD276-ECD

[0085] The binding ability of 147# to reduced denatured human CD276-ECD was determined by Western Blot.

[0086] After SDS-PAGE electrophoresis of reduced denatured human CD276-ECD (400 ng / lane), the PVDF membrane was transferred by electrotransfer method, blocked in 1% BSA-TBST (room temperature shaking for 1 h), 1 μg / mL (1% BSA-PBST diluted) mouse antibody 147# was added, and incubated at room temperature for 2 h, and then washed with PBST for 3 times, HRP labeled goat anti-mouse IgG secondary antibody (purchased from Abeam company, item number ab6789, diluted 10000 times with 1% BSA-PBST according to the instructions) was added, and incubated at room temperature for 1 h, and then washed with PBST for 3 times, and then a suitable amount of Pierce ECL Western Blot Substrate Solution (purchased from Thermo company, item number 32209) was added on the PVDF membrane, and then the automatic imaging was performed on the biomolecular imager (purchased from Thermo company, model CL1500) at room temperature in the dark. TM ECL Western Blot Substrate Solution (purchased from Thermo company, item number 32209), and then the automatic imaging was performed on the biomolecular imager (purchased from Thermo company, model CL1500) at room temperature in the dark.

[0087] The results are shown in Figure 3As shown, specific immunoblotting appeared between the CD276-ECD target positions, indicating that 147# can specifically bind to reduced and denatured human CD276-ECD, suggesting that the epitope of the antibody 147# specifically binding to human CD276-ECD protein is a linear epitope.

[0088] 4.2. Determination of the binding region of the target antigen of the monoclonal antibody 147#

[0089] The binding region of 147# to human CD276-ECD was determined by conventional Western blot and ELISA methods.

[0090] To confirm the binding epitope of 147# to CD276, the extracellular domain gene (CD276-ECD) of human CD276 was obtained by literature review and NCBI, which contains functional domain Ig-like V-type 1 (Domain 1, amino acid sequence as shown in SEQ ID NO: 16), functional domain Ig-like C2-type 1 (Domain 2, amino acid sequence as shown in SEQ ID NO: 17). The CD276 functional domains Ig-like V-type 1 and Ig-like C2-type 1 were spliced with the human Fc region (amino acid sequence as shown in SEQ ID NO: 18), respectively, to form V-type 1-Fc (amino acid sequence as shown in SEQ ID NO: 19) and C2-type 1-Fc (amino acid sequence as shown in SEQ ID NO: 20), respectively, which were constructed into pcDNA3.4 expression vectors, transfected into Expi-293F cells, and each Fc fusion protein was obtained by Protein A purification. Subsequently, the binding of 147# to human CD276-ECD domains Ig-like V-type 1 and Ig-like C2-type 1 was determined by the conventional Western blot method as described in 5.1 above.

[0091] The results are shown in Figure 3 As shown, the Western blot detection results showed that 147# only binds to the second functional domain of human CD276-ECD, i.e., Ig-like C2-type 1.

[0092] 4.3. Confirmation of the polypeptide bound by the monoclonal antibody 147# to the target antigen

[0093] The binding ability of 147# to polypeptides in the Ig-like C2-type 1 domain of human CD276 was detected by ELISA method, and the epitope of 147# binding to human Ig-like C2-type 1 was further confirmed.

[0094] From the results of 4.1 and 4.2, it has been determined that 147# can bind to denatured CD276-ECD and Ig-like C2-type 1 domain, and it is judged that the epitope bound by 147# is a linear epitope, which can be detected by using synthetic polypeptides by ELISA method to further determine the position of the epitope bound by 147#.

[0095] First, the human Ig-like C2-type 1 domain is divided into 9 parts with overlaps, and each part is synthesized into an N-terminal biotin-modified polypeptide, i.e.:

[0096] 1. bio-PSMTLEPNKD LRPGDTVTI (145-164);

[0097] 2. bio-LRPGDTVTI TCSSYQGYPE (155-174);

[0098] 3. bio-TCSSYQGYPE AEVFWQDGQG (165-184);

[0099] 4. bio-AEVFWQDGQG VPLTGNVTTS (175-194);

[0100] 5. bio-VPLTGNVTTS QMANEQGLFD (185-204);

[0101] 6. bio-QMANEQGLFD VHSILRVVLG (195-214);

[0102] 7. bio-VHSILRVVLG ANGTYSCLVR (205-224);

[0103] 8. bio-ANGTYSCLVR NPVLQQDAHS (215-234);

[0104] 9. bio-NPVLQQDAHS SVTIT (225-240).

[0105] The binding activity of 147# to the above 9 polypeptides was determined by ELISA method, and the results are shown in Table 1. Figure 4 As shown in Table 1, 147# only binds to polypeptide 1, i.e. the polypeptide of amino acids 145-154 at the N terminus of human CD276-ECD protein, indicating that the binding epitope of 147# is located between P145 and I164 of human CD276 protein.

[0106] Further, 20 N-terminal biotin-modified polypeptides (underlined part is the mutated amino acid site) were synthesized by alanine scanning (mutating the non-alanine amino acid site to alanine one by one), i.e.

[0107] 1-1.bio- A SMTLEPNKD LRPGDTVTI(145-164);

[0108] 1-2.bio-P A MTLEPNKD LRPGDTVTI(145-164);

[0109] 1-3.bio-PS A TLEPNKD LRPGDTVTI(145-164);

[0110] 1-4.bio-PSM A LEPNKD LRPGDTVTI(145-164);

[0111] 1-5.bio-PSMT A EPNKD LRPGDTVTI(145-164);

[0112] 1-6.bio-PSMTL A PNKD LRPGDTVTI(145-164);

[0113] 1-7.bio-PSMTLE A NKD LRPGDTVTI(145-164);

[0114] 1-8.bio-PSMTLEP A KD LRPGDTVTI(145-164);

[0115] 1-9.bio-PSMTLEPN A D LRPGDTVTI(145-164);

[0116] 1-10.bio-PSMTLEPNK A LRPGDTVTI(145-164);

[0117] 1-11.bio-PSMTLEPNKD A RPGDTVTI(145-164);

[0118] 1-12.bio-PSMTLEPNKD L A PGDTVTI(145-164);

[0119] 1-13.bio-PSMTLEPNKD LR A GDTVTI(145-164);

[0120] 1-14.bio-PSMTLEPNKD LRP A DTVTI (145-164);

[0121] 1-15.bio-PSMTLEPNKD LRPG A TVTI (145-164);

[0122] 1-16.bio-PSMTLEPNKD LRPGD A VTI (145-164);

[0123] 1-17.bio-PSMTLEPNKD LRPGDT A TI (145-164);

[0124] 1-18.bio-PSMTLEPNKD LRPGDTV A I(145-164);

[0125] 1-19.bio-PSMTLEPNKD LRPGDTVT A (145-164);

[0126] 1-20.bio-PSMTLEPNKD LRPGDTVTI(145-164);

[0127] The results are as follows Figure 5 As shown, SPGA03-147 hardly binds 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 is slightly weakened. However, its binding to other peptides is consistent with that of the original peptide (PEP1). This indicates that amino acids P / D / L / R / G / D in peptide PEP1 are the most critical for binding to 147#, followed by amino acids K / V. In other words, P151 / D154 / L155 / R156 / G157 / D158 in human CD276 protein are the most critical sites for binding to monoclonal antibody 147#, while K153 / V161 are secondary critical sites.

[0128] Example 5: Cross-activity assay of monoclonal antibody 147# with cynomolgus monkey CD276-ECD antigen

[0129] The indirect enzyme-linked immunosorbent assay was used to determine the binding ability of 147# to cynomolgus monkey CD276-ECD (purchased from Beijing Bupsails Company, Acro BIOSYSTEMS, the amino acid sequence is shown in SEQ ID NO: 2) and mouse CD276-ECD (purchased from Beijing Bupsails Company, Acro BIOSYSTEMS, the amino acid sequence is shown in SEQ ID NO: 3). The specific method is as follows: the indirect enzyme-linked immunosorbent assay was used to determine the binding ability of the mouse-derived antibody to human CD276-ECD protein. The specific method is as follows: the cynomolgus monkey and mouse CD276-ECD proteins were coated in advance, diluted to 2 μg / ml with coating solution (50 mM carbonate coating buffer, pH 9.6), 4°C, overnight; then blocked with 5% skimmed milk powder, 37°C, 2 hours; PBST was used to wash the plate 3 times, and the antibody to be tested was added to the blocked enzyme-labeled plate in gradient dilution with 1% BSA-PBST, 100 μl / well, 37°C for 1 hour. PBST was used to wash the plate 3 times, and HRP-labeled goat anti-mouse IgG secondary antibody (Millipore Company) was added, 37°C for 30 min; after PBST was used to wash the plate 3 times, the residual droplets were dried as much as possible on the blotting paper, 100 μl of TMB (KPL Company) was added to each well, and it was placed at room temperature (20±5°C) for 5 min; 50 μl of 2M H2SO4 termination solution was added to each well to terminate the substrate reaction, and the OD value was read at 450 nm by an enzyme-labeled instrument, and the binding ability of the antibody to be tested to the target antigen cynomolgus monkey and mouse CD276-ECD protein was analyzed.

[0130] The results are shown in Figure 6 The EC50 of 147# antibody binding to monkey CD276-ECD protein is 30.16 ng / ml, i.e. 0.2 nM, which is consistent with the EC50 of binding to human CD276-ECD (38.35 ng / ml); 147# antibody does not bind to mouse-derived CD276-ECD. 50

[0131] Example 6 Preparation of humanized antibody hu147

[0132] In this embodiment, the heavy chain variable region and the light chain variable region of the hybridoma 147# were obtained by related methods of molecular biology, and a chimeric antibody was further constructed.

[0133] ​The RNA of 147# hybridoma cells was extracted by Trizol and mRNA reverse transcription was performed to obtain cDNA, and then cDNA was used as a template, and the heavy chain and light chain degenerate primers of the mouse antibody (Antibody Engineering Volume 1, Edited by Roland Kontermann and Stefan Dübel, the sequence of the combined primer is from page 323) were used for PCR, the obtained PCR products were sequenced and analyzed by kabat database to determine that the obtained sequence is the variable region sequence of the mouse antibody.

[0134] The relevant sequence information is as follows:

[0135] The heavy chain variable region gene sequence of 147# is 366 bp in full length, encoding 122 amino acid residues, the nucleotide sequence is shown as SEQ ID NO: 4, and the amino acid sequence is shown as SEQ ID NO: 5;

[0136] The light chain variable region gene sequence of 147# is 321 bp in full length, encoding 107 amino acid residues, the nucleotide sequence is shown as SEQ ID NO: 6, and the amino acid sequence is shown as SEQ ID NO: 7;

[0137] The amino acid sequences of the light chain variable region and the heavy chain variable region of the mouse antibody 147# were analyzed, and the 3 antigen complementarity determining regions (CDRs) and 4 framework regions (FRs) of the mouse antibody 147# were determined according to the Kabat rule. Among them, 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); and the amino acid sequences of the light chain complementarity determining regions are LCDR1: KASENVGTY (SEQ ID NO: 13), LCDR2: GASNRYT (SEQ ID NO: 14), and LCDR3: GQRYSYPFT (SEQ ID NO: 15).

[0138] The best matching humanized template was selected in the Germline database according to the above mouse antibody FR region. Then the CDR region of the mouse antibody was transplanted to the selected humanized template to replace the CDR region of the human template. The heavy chain variable region was recombined with the human IgG1 constant region, and the light chain variable region was recombined with the human kappa chain constant region. Based on the three-dimensional structure of the antibody, the buried residues, the residues directly interacting with the CDR region, and the residues having important influence on the conformation of VL and VH of each antibody were subjected to back mutation to form the humanized antibody heavy chain variable region (hu147 VH, the amino acid sequence is shown in SEQ ID NO: 21) and the humanized antibody light chain variable region (hu147 VL, the amino acid sequence is shown in SEQ ID NO: 22). The hu147 VH was spliced with the human IgG1 constant region (the amino acid sequence is shown in SEQ ID NO: 8) to form the humanized antibody heavy chain (hu147 H, the amino acid sequence is shown in SEQ ID NO: 23). The hu147 VL was spliced with the human kappa chain constant region (the amino acid sequence is shown in SEQ ID NO: 9) to form the humanized antibody light chain (hu147 L, the amino acid sequence is shown in SEQ ID NO: 24). The heavy chain and the light chain of the humanized antibody were respectively constructed into the pcDNA3.4 expression vector, transfected into Expi-293F cells, and the humanized antibody hu147 was obtained by Protein A purification. The molecular weight of each antibody was determined by SDS-PAGE electrophoresis and SEC-HPLC to be correct and the purity was more than 95%.

[0139] Example 7 Preparation of Fc mutant humanized antibody (hu147mu)

[0140] The hu147 heavy chain variable region sequence was spliced with the mutant human IgG1 constant region (the amino acid sequence is shown in SEQ ID NO: 25) to form the heavy chain of hu147mu (the amino acid sequence is shown in SEQ ID NO: 26), and the light chain was the same as hu147 (the amino acid sequence is shown in SEQ ID NO: 24). They were respectively constructed into the pcDNA3.4 expression vector, transfected into Expi-293F cells, and the antibody hu147mu was obtained by Protein G purification. The molecular weight of each antibody was determined by SDS-PAGE electrophoresis and SEC-HPLC to be about 150 kD, the purity of the antibody was more than 95%, and the antibody was quantified, aliquoted, and stored at -80°C for standby.

[0141] Example 8 Determination of the binding activity of humanized antibody hu147 and mutant humanized antibody hu147mu to human CD276-ECD by ELISA method

[0142] The binding affinity of the above humanized antibodies hu147 and hu147mu to human CD276-ECD was determined by ELISA method, and the relevant experimental method was referred to Example 2.

[0143] The experimental results are shown in Figure 7 As shown in the table, the EC50 of the humanized antibodies hu147 and hu147mu binding to human CD276-ECD were 6.70 ng / ml and 6.80 ng / ml, i.e. 0.04 nM and 0.05 nM, which were significantly improved compared with 147# (EC50 was 0.26 nM), indicating that hu147 and hu147mu had good affinity to human CD276-ECD, and the above mutation of Fc did not affect the binding to the antigen.

[0144] Example 9 Preparation of hu147-IL15 bifunctional molecules SPGL008 and SPGL007

[0145] The amino acid sequence of human IL15Rsushi is shown in SEQ ID NO: 27; the amino acid sequence of human IL15 is shown in SEQ ID NO: 28; human IL15Rsusi and human IL15 are connected by (GGGGS)6 to form a 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) is spliced with the sequence of human IL15sc by (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) is spliced with the sequence of human IL15sc by (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 chain and light chain of SPGL007 were co-transfected into Expi-293F cells, and the bifunctional antibody SPGL007 was obtained by Protein G purification; the heavy chain and light chain of SPGL008 were co-transfected into Expi-293F cells, and the bifunctional antibody SPGL008 was obtained by Protein G purification; the molecular weight of each antibody expressed was about 190 kD, the purity of the antibody was > 95%, the quantity was determined, the antibody was aliquoted and stored at -80°C for standby.

[0146] Example 10 ELISA method for determining the binding activity of SPGL008 and SPGL007 to human CD276-ECD

[0147] The binding affinity of SPGL008 and SPGL007 to human CD276-ECD was determined by ELISA method, and the relevant experimental method was referred to Example 2.

[0148] The experimental results are shown in Table 1. Figure 8 The EC50 of SPGL008 and SPGL007 binding to human CD276-ECD was 31.51 ng / ml and 32.75 ng / ml, i.e. 0.16 nM and 0.17 nM, respectively, which was lower than that of hu147 (0.05 nM) or hu147mu (0.04 nM), but still maintained a high affinity. 50

[0149] Example 11 SPGL008 simultaneously binds to human CD276 and human CD122 / 132

[0150] The binding of SPGL008 and SPGL007 to human CD122 / 132 was determined by ELSA method, which showed that SPGL008 had the function of simultaneously binding to CD276 and CD122 / 132. The method is as follows: human CD276-ECD protein was coated in advance, diluted to 2 μg / ml with coating solution (50 mM carbonate coating buffer, pH 9.6), 4°C, overnight; then blocked with 5% skimmed milk, 37°C, 2 hours; PBST wash plate 3 times, dilute the test antibody to 1 ug / ml with 1% BSA-PBST gradient, add to the blocked enzyme-labeled plate, 100 μl / well, 37°C for 1 hour. PBST wash plate 3 times, add gradient-diluted biotin-labeled CD122 / 132 (purchased from Beijing Bipsy Company), 37°C, 1 hour, PBST wash plate 3 times, then add diluted HRP-labeled SA (Pierce Company), 37°C for 30 min; PBST wash plate 3 times, try to pat dry the residual droplets on the blotting paper, add 100 μl of TMB (KPL Company) to each well, and place it at room temperature (20±5°C) for 5 min; add 50 μl of 2M H2SO4 termination solution to each well to terminate the substrate reaction, and read the OD value at 450 nm with an enzyme-labeled instrument to analyze the binding ability of the test antibody to human CD122 / 132.

[0151] The results are shown in Table 2. Figure 9 The EC50 of SPGL008 and SPGL007 binding to human CD122 / 132 protein was 17.71 ng / ml and 15.08 ng / ml, i.e. 0.09 nM and 0.08 nM, respectively; as a control, hu147 could bind to CD276 (as shown in Example 8), but could not bind to CD122 / 132. 50 ​​

[0152] Promoting cell proliferation activity of SPGL008

[0153] This example illustrates the biological activity of SPGL008 and SPGL007 with CTLL2 cell proliferation experiment. The method is as follows: CTLL2 cells are diluted to 5 x 105 / ml with 1640 culture solution containing 10% FBS, 100ul / well is added to the cell culture plate. IL2 is diluted to 30ng / ml with 1640 culture solution containing 10% FBS, and then 3-fold dilution is performed for a total of 8 gradients, which are then added to the above-mentioned CTLL2 cell-containing culture plates; SPGL008 and SPGL007 are diluted to 5000ng / ml with 1640 culture solution containing 10% FBS, and then 3-fold dilution is performed for a total of 8 gradients, which are then added to the above-mentioned CTLL2 cell-containing culture plates; after 72 hours of incubation in a CO2 cell incubator, the relative cell number of each well is determined by CCK8, and the EC50 is calculated to determine the activity of the sample. 4

[0154] The results are shown in Figure 10A SPGL008 and SPGL007 can both stimulate the proliferation of CTLL2 cells, with EC50 of 103.0ng / ml and 91.0ng / ml, i.e. 0.53 and 0.48nM, respectively, indicating that SPGL008 has consistent biological activity with SPGL007. As a control, the EC50 of IL2 is 0.74ng / ml, i.e. 0.048nM, as shown in Figure 10B

[0155] Example 13: SPGL008 has significantly reduced toxicity in vivo compared to SPGL007

[0156] SPGL008 and SPGL007 were intraperitoneally injected into C57BL / 6 mice (Vantianhua Company) on day 1 and day 3, with a volume of 0.2ml / injection, and SPGL008 and SPGL007 were administered at doses of 0.5mg / kg, 1mg / kg, 2mg / kg and 4mg / kg. The death of the experimental mice was observed every day, and the control group was administered the same volume of PBS.

[0157] ​​The results are shown in Table 1. On the 10th day of the experiment, all experimental animals survived (survival rate 100%) at doses of 0.5 mg / kg, 1.0 mg / kg and 2.0 mg / kg in the SPGL008 group, and 70% of experimental animals died (survival rate 30%) at a dose of 4.0 mg / kg; but 100% of experimental animals died (survival rate 0%) at doses of 4.0 mg / kg and 2.0 mg / kg in the SPGL007 group, 60% of experimental animals died (survival rate 40%) at a dose of 1.0 mg / kg, and no experimental animals died (survival rate 100%) at a dose of 0.5 mg / kg. These results show that the toxicity of SPGL008 to experimental mice is significantly reduced compared to SPGL007 after Fc mutation.

[0158] Table 1 Survival rate of experimental animals in each dose group

[0159] Antibody 0.5 mg / kg 1.0 mg / kg 2.0 mg / kg 4.0 mg / kg SPGL007 100％ 40％ 0％ 0％ SPGL008 100％ 100％ 100％ 30％

[0160] Example 14 Inhibition of growth of mouse colorectal cancer cell MC38 transplanted tumor by SPGL008

[0161] This example uses a mouse colorectal cancer cell MC38 transplanted tumor model to evaluate the in vivo anti-tumor activity of SPGL008 and SPGL007. The experimental method is as follows:

[0162] Mouse colorectal cancer MC38 cells cultured in vitro were collected, and the cell suspension concentration was adjusted to 1 x 10 7 / ml. Under sterile conditions, 100 μl of cell suspension was inoculated subcutaneously on the right flank of C57BL / 6 mice. The diameter of the transplanted tumor was measured with a vernier caliper, and the animals were randomly divided into groups after the average tumor volume grew to 100-200 mm 3 / week for 2 weeks. During the entire experiment, the diameter of the transplanted tumor was measured 3 times a week, and the body weight of the mice was also measured. The formula for calculating the tumor volume (TV) is:

[0163] TV = 1 / 2 x a x b2

[0164] where a and b represent the length and width, respectively. The relative tumor volume (RTV) was calculated according to the measured results, and the formula is: RTV = Vt / V0. Where V0 is the tumor volume measured at the time of grouping (i.e. d0), and Vt is the tumor volume at each measurement. The evaluation index of anti-tumor activity is the relative tumor proliferation rate T / C (%), and the formula is as follows:

[0165] T / C (%) = (TRTV / CRTV) x 100

[0166] TGI (%) = 100 - T / C (%)

[0167] TRTV: treated group RTV; CRTV: negative control group RTV.

[0168] Results as shown in Table 2, SPGL008 and SPGL007 both exhibited strong anti-tumor activity, TGI were 76.2% and 69.4% respectively, with no significant difference (p>0.05), indicating that Fc mutation of SPGL008 did not affect the anti-tumor activity in vivo. Figure 11 Example 15 SPGL008 inhibits the growth of human lung cancer cell NCI H1975 xenografts

[0169] Human lung cancer NCI-H1975 cells cultured in vitro were collected, and the cell suspension concentration was adjusted to 8 x 10 7 / ml. Under sterile conditions, 100 μl of cell suspension was inoculated subcutaneously on the right flank of nude mice. When the tumor cells formed solid tumors subcutaneously in mice, the diameters of the xenografts were measured with a vernier caliper, and the animals were randomly divided into groups when the average tumor volume grew to 50-100 mm 3 The animals were intraperitoneally injected with drugs 3 times a week for a total of 6 times at doses of 1.0 mg / kg, 0.3 mg / kg and 0.1 mg / kg. During the entire experiment, the diameters of the xenografts were measured twice a week, and the body weights of the mice were measured. The rest was the same as in Example 14.

[0170] Results as shown in Table 2, SPGL008 and SPGL007 both exhibited strong anti-tumor activity, TGI were 76.2% and 69.4% respectively, with no significant difference (p>0.05), indicating that Fc mutation of SPGL008 did not affect the anti-tumor activity in vivo.

[0171] Figure 12 Results as shown in Table 2, SPGL008 and SPGL007 both exhibited strong anti-tumor activity, TGI were 76.2% and 69.4% respectively, with no significant difference (p>0.05), indicating that Fc mutation of SPGL008 did not affect the anti-tumor activity in vivo.

[0172] Example 16 SPGL008 combined with HER2 monoclonal antibody inhibits the growth of human breast cancer cell JIMT-1 xenografts in nude mice

[0173] Human breast cancer JIMT-1 cells cultured in vitro were collected, and the cell suspension concentration was adjusted to 8 x 10 7 / ml. Under sterile conditions, 100 μl of cell suspension was inoculated subcutaneously on the right flank of nude mice. When the tumor cells formed solid tumors subcutaneously in mice, the diameters of the xenografts were measured with a vernier caliper, and the animals were randomly divided into groups when the average tumor volume grew to 50-100 mm 3 ​The animals were then randomly grouped. The single drug group HER2 mAb (trastuzumab) was administered at a dose of 20 mg / kg, and the combination group was administered with a fixed dose of 20 mg / kg trastuzumab combined with 1.0 mg / kg and 0.3 mg / kg of SPGL008, respectively, twice a week, intraperitoneally, for a total of 6 times. During the entire experiment, the diameter of the transplanted tumors was measured twice a week, and the body weight of the mice was measured. The rest was the same as in Example 15.

[0174] The results are shown in Figure 13 Table 2. The trastuzumab group showed a weak anti-tumor effect, with a TGI of 55%, indicating that JIMT-1 is a trastuzumab-resistant tumor; the TGI of the combination of SPGL008 at a dose of 1.0 mg / kg and trastuzumab reached 90%, and 50% (3 / 6) of the tumors completely disappeared (CR); even at a dose of 0.3 mg / kg, the combination of SPGL008 and trastuzumab had a TGI of 72%, which was significantly higher than that of trastuzumab alone.

[0175] Example 17 Metabolism of SPGL008 in huFcRn Transgenic Mice

[0176] The pharmacokinetics of SPGL007 and SPGL008 were determined using human FcRn transgenic mice. The method was as follows: 8 mice were divided into two groups and injected intraperitoneally with SPGL007 and SPGL008 at a dose of 1 mg / kg, respectively. Blood was taken at 2 hours, 6 hours, 24 hours, 48 hours, and 72 hours, and serum was obtained. After appropriate dilution, the serum was determined for drug concentration by ELISA, basically the same as in Example 11.

[0177] The results are shown in Figure 14 Table 3. The Fc mutant SPGL008 was metabolized significantly faster than the Fc wild-type SPGL007 in human FcRn transgenic mice, with half-lives of 10 hours and 18 hours, respectively, with a significant difference.

[0178] Example 18 Detection of CD276 Expression on Various Tumor Cells by SPGL008

[0179] Flow cytometry was used for detection, basically the same as in Example 3.

[0180] The results are shown in Figure 15 Table 4. SPGL008 can bind to various human lung cancer cells (H1975, Calu-3, A549, H322, H292), human breast cancer cells (JIMT-1), human renal cancer cells (A498), and human skin cancer cells (A431), suggesting that the above-mentioned various tumors can be indications for SPGL008.

[0181] Example 19 SPGL008 has good stability

[0182] SEC-HPLC was used to detect the purity change of SPGL008 after different time of storage, and the stability of SPGL008 was investigated. The method was as follows:

[0183] TSKgel G3000SWXL column (TSK company) was used on HPLC Ultimate 3000 (Thermo company) chromatograph. The detection mobile phase was PBS (pH 7.4) with constant flow rate of 0.8 ml / min, and the sample volume was 100 ug / 100 ul. The content (%) of target protein in total protein was calculated by 280 nM absorption peak integration method to represent the purity.

[0184] The results are shown in Figure 16 , Figure 17 and Figure 18 , the purity of SPGL008 in PBS solution was 84.9% at 0 week, after 5 weeks of storage at 4℃, the purity was 84.0%, the change rate was <2%; after 5 weeks of storage at 37℃, the purity was 83.9%, the change rate was <2%, indicating that SPGL008 remained stable in PBS buffer at 4℃ and 37℃.

[0185] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not limited to the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

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

7.

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

22.

3. A multifunctional recombinant antibody, characterized in that, The heavy chain of the multifunctional recombinant antibody comprises an antibody functional region recognizing human CD276, a human IgG1 constant region functional domain, a functional region of human IL15, and a non-functional amino acid fragment for connecting the functional regions; The amino acid sequence of the antibody functional region recognizing human CD276 comprises the amino acid sequence of the heavy chain variable region of the recombinant antibody of claim 2; The human IgG1 constant region functional domain is a mutant human IgG1 constant region, and the amino acid sequence of the mutant human IgG1 constant region is shown as SEQ ID NO: 25; The amino acid sequence of the heavy chain of the multifunctional recombinant antibody is shown as SEQ ID NO: 31; The amino acid sequence of the light chain of the multifunctional recombinant antibody is shown as SEQ ID NO:

24.

4. A nucleotide, characterized in that, The nucleotide sequence encodes the monoclonal antibody of claim 1, or the recombinant antibody of claim 2, or the multifunctional recombinant antibody of claim 3.

5. An expression vector, characterized by, The expression vector comprises the nucleotide of claim 4.

6. A host cell, characterized in that, The host cell comprises the expression vector of claim 5.

7. Use of the monoclonal antibody of claim 1, or the recombinant antibody of claim 2, or the multifunctional recombinant antibody of claim 3; or the nucleotide of claim 4, or the expression vector of claim 5, or the host cell of claim 6 in the preparation of a biological agent for treating colorectal cancer, lung cancer and / or breast cancer.

8. A biological agent, characterized in that, The biological agent comprises at least one of the monoclonal antibody of claim 1, or the recombinant antibody of claim 2, or the multifunctional recombinant antibody of claim 3; or the nucleotide of claim 4, or the expression vector of claim 5, or the host cell of claim 6.

9. A method for preparing the monoclonal antibody according to claim 1 or the recombinant antibody according to claim 2, or the multifunctional recombinant antibody according to claim 3, wherein The method comprises the following steps: (1) obtaining an expression vector containing a gene fragment of the monoclonal antibody or the recombinant antibody or the multifunctional recombinant antibody by artificial synthesis or molecular biology methods; (2) transfecting cells with the expression vector for protein expression; (3) obtaining the monoclonal antibody or the recombinant antibody or the multifunctional recombinant antibody by protein purification.

Images