A multifunctional recombinant antibody and its preparation method and application

Abstract

The present invention discloses a multifunctional recombinant antibody and its preparation method and application. The present invention not only provides a monoclonal antibody that can specifically recognize human HER3 protein, but also provides its humanized recombinant antibody, and further reconstructs on this basis to obtain a multifunctional antibody that can recognize human HER3 and has IL15 function, which can effectively enhance the killing effect of the original antibody on tumor cells. At the same time, the present invention also obtains a reconstructed specific HER3 monoclonal antibody-IL15 bifunctional molecule by introducing a mutated human IgG1 constant region, which can effectively reduce the toxicity of the antibody and improve safety. The antibody of the present invention can recognize HER3 protein in a variety of tumor cells, and then has a killing effect on a variety of tumor cells. At the same time, the reconstructed multifunctional recombinant antibody has the advantages of a short drug metabolism cycle, low toxicity and high safety, and has great application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a multifunctional recombinant antibody for treating tumors, a preparation method thereof, and an application thereof; in particular, it relates to a multifunctional recombinant antibody capable of recognizing HER3 and having IL15 function, and an application thereof. Background Art

[0002] Human epidermal receptor (HER) proteins belong to the receptor tyrosine kinase family and play a role in both normal and tumor cells. HER3, as a member of the HER family, differs from other members in having little or no intracellular tyrosine kinase activity, and its ligands include neuregulin 1 (NRG-1) and neuregulin 2 (NRG-2). HER3 is ubiquitously expressed in various cancers, including breast, ovarian, colon, gastric, lung, skin, and pancreatic cancers, and its high expression is also associated with disease progression and / or poor prognosis. In recent years, as the importance of HER3 in tumor progression and drug resistance has gradually become apparent, HER3 has received increasing attention. The clinical efficacy of existing drugs targeting HER3 is not significant enough, and the potential for subsequent development is relatively small.

[0003] IL15 is a cytokine expressed by a variety of cell types, including monocytes, macrophages, epidermal cells, and fibroblasts, but not by T lymphocytes. Unlike many other cytokines, IL15 is not typically secreted outside cells to exert its effects. Instead, it binds to IL15Rα and localizes to specific cell membranes, thereby stimulating nearby effector cells, primarily NK and CD8+ T cells. IL15 is closely related to IL2. The complex formed by IL15 and IL15Rα binds to the β / γ receptors shared by IL2, mediating its biological activity. One advantage of IL15 over IL2 in terms of anti-tumor effects is that IL15 / Rα does not stimulate the proliferation of Tregs. Currently, several IL15-related molecules are in development for the treatment of malignancies. The most advanced of these is ALT-803, which consists of a complex of IL15 and IL15Rαsushi-hFc1. Multiple clinical studies have shown that ALT-803 is effective against a variety of tumors, including melanoma, but it can also cause serious toxic side effects, mainly including liver damage, hypotension, and fever.

[0004] How to improve the effectiveness of anti-tumor drugs targeting HER3 and develop a safe and effective drug is of great significance. Summary of the Invention

[0005] In response to the above problems, the purpose of the present invention is to provide a monoclonal antibody that can specifically recognize HER3 protein, a hybridoma cell that secretes the monoclonal antibody; and a recombinant antibody obtained by humanizing the monoclonal antibody; as well as a multifunctional recombinant antibody that can both recognize human HER3 protein and have human IL15 function.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is: a monoclonal antibody that can specifically recognize the HER3 protein; the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 8.

[0007] The present invention immunizes mice with recombinantly expressed human HER3-ECD protein and produces and screens suitable hybridomas to generate a murine monoclonal antibody, designated SPGA08-158, that effectively binds to the HER3 protein. This antibody effectively blocks NRG1 binding to HER3. Biological analysis revealed that the heavy chain variable region of the monoclonal antibody encodes 118 amino acid residues, while the light chain variable region encodes 107 amino acid residues.

[0008] Furthermore, the present invention also claims protection for a recombinant antibody, which is obtained by humanizing the monoclonal antibody SPGA08-158, the amino acid sequence of the heavy chain variable region of the recombinant antibody is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the recombinant antibody is shown in SEQ ID NO: 19.

[0009] As a preferred embodiment of the present invention, the heavy chain amino acid sequence of the recombinant antibody is shown in SEQ ID NO: 18, and the light chain amino acid sequence of the recombinant antibody is shown in SEQ ID NO: 20.

[0010] The inventors further analyzed the variable region sequence of the murine monoclonal antibody SPGA08-158 and analyzed the amino acid sequences of the light and heavy chain variable regions of the monoclonal antibody. The three complementarity determining regions (CDRs) and four framework regions (FRs) of the murine antibody were determined according to the Kabat rules. Humanized templates that best matched the FR regions of each murine antibody were selected from the Germline database. The CDR regions of the murine antibody were then transplanted onto the selected humanized templates, replacing the CDR regions of the human templates. The heavy chain variable region was then recombined with a human IgG1 constant region (SEQ ID NO: 15), and the light chain variable region was recombined with a human kappa chain constant region (SEQ ID NO: 16). Based on the three-dimensional structure of the antibody, backmutations were performed on buried residues, residues that directly interact with the CDR regions, and residues that significantly influence the VL and VH conformations of each antibody. The resulting humanized recombinant antibody was named huSPGA08-158. The amino acid sequences of the heavy chain variable region and light chain variable region, the heavy chain amino acid sequence, and the light chain amino acid sequence of the humanized recombinant antibody huSPGA08-158 are shown above.

[0011] Furthermore, the present invention also claims protection for a multifunctional recombinant antibody, the heavy chain of which comprises an antibody functional region that recognizes human HER3 and an antibody functional region that recognizes human IL15, and a non-functional amino acid fragment for connecting the functional regions;

[0012] The heavy chain variable region of the antibody functional region that recognizes human HER3 includes complementary determining regions VH-CDR1, VHCDR2, and VH-CDR3, and the VH-CDR1, VHCDR2, and VH-CDR3 include amino acid sequences consistent with the VH-CDR1, VHCDR2, and VH-CDR3 of the light chain variable region shown in SEQ ID NO. 17. The antibody functional region that recognizes human HER3 includes a human IgG1 constant region functional domain, and the sequence of the human IgG1 constant region functional domain is shown in SEQ ID NO: 15 or 25;

[0013] The amino acid sequence of the functional region of human IL15 is shown in SEQ ID NO: 23; the non-functional amino acid fragment used to connect the functional regions is (GGGGS)3;

[0014] The light chain variable region of the multifunctional recombinant antibody includes complementary determining regions VL-CDR1, VL-CDR2 and VL-CDR3, and the VL-CDR1, VL-CDR2 and VL-CDR3 include amino acid sequences consistent with VL-CDR1, VL-CDR2 and VL-CDR3 of the light chain variable region shown in SEQ ID NO.19.

[0015] As one embodiment of the present invention, the amino acid sequence of the heavy chain of the multifunctional recombinant antibody is shown as SEQ ID NO: 24 or SEQ ID NO: 27; the amino acid sequence of the light chain of the multifunctional recombinant antibody is shown as SEQ ID NO: 20.

[0016] The IL15 functional region is a functional domain that can recognize human IL2 / IL15β / γ receptors.

[0017] The inventors of the present invention further modified the humanized antibody obtained above by connecting the antibody heavy chain sequence with a sequence having IL15 function, thereby obtaining a recombinant antibody that can recognize human HER3 protein and has IL15 function.

[0018] As a preferred embodiment of the present invention, the human IgG1 constant region functional domain is a human IgG1 constant region, and the amino acid sequence is shown in SEQ ID NO: 15.

[0019] 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.

[0020] The inventors of the present invention further modified the obtained multifunctional recombinant antibody by using a sequence containing a mutated human IgG1 constant region, thereby obtaining a multifunctional recombinant antibody with a shorter metabolic cycle, lower toxicity and better safety.

[0021] As a preferred embodiment of the present invention, the amino acid sequence of the IL15 functional region is shown in SEQ ID NO: 22.

[0022] More preferably, the amino acid sequence of the human IL15 functional region is shown in SEQ ID NO: 23.

[0023] The amino acid sequence of the preferred IL15 functional region is human IL15Rsushi and human IL15 connected by a (GGGGS)6 linker to form a single-chain IL15, namely IL15sc. This linker plays an important role in constructing a stable and biologically active fusion protein, ensuring correct protein folding, maintaining biological activity, and increasing protein yield.

[0024] As a preferred embodiment of the present invention, the non-functional amino acid fragment used to connect the functional regions in the multifunctional recombinant antibody is a GGGGS repeat.

[0025] More preferably, the GGGGS repeat is (GGGGS)3.

[0026] As a preferred embodiment of the present invention, the amino acid sequence of the human IgG1 constant region functional domain of the multifunctional recombinant antibody is shown in SEQ ID NO: 15; the heavy chain amino acid sequence of the multifunctional recombinant antibody is shown in SEQ ID NO: 24; and the light chain amino acid sequence of the multifunctional recombinant antibody is shown in SEQ ID NO: 20.

[0027] The present invention prepares a multifunctional antibody capable of recognizing HER3 and having IL15 function, which is named SPGL013.

[0028] More preferably, the human IgG1 constant region functional domain of the multifunctional recombinant antibody 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; the amino acid sequence of the multifunctional recombinant antibody heavy chain is shown in SEQ ID NO: 27; and the amino acid sequence of the multifunctional recombinant antibody light chain is shown in SEQ ID NO: 20.

[0029] The multifunctional recombinant antibody prepared using the preferred multifunctional recombinant antibody sequence contains a mutated human IgG1 constant region sequence and is named SPGL014; the multifunctional recombinant antibody SPGL014 can not only effectively recognize HER3 in various tumor cells, but also effectively inhibit the growth of various cancer cells, and has a shorter half-life, lower toxicity and good safety.

[0030] Furthermore, the present invention also claims protection for the nucleotide encoding the monoclonal antibody, or the recombinant antibody, or the multifunctional recombinant antibody.

[0031] According to the amino acid sequence of the monoclonal antibody, the recombinant antibody, or the multifunctional recombinant antibody, the corresponding nucleotide sequence of the encoding gene can be obtained.

[0032] As a preferred embodiment of the present invention, the nucleotide sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO: 5, and the nucleotide sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO: 7.

[0033] Furthermore, the present invention also claims protection for an expression vector comprising the nucleotide sequence.

[0034] An expression vector containing the nucleotide sequence can be constructed by molecular biological methods.

[0035] Furthermore, the present invention also claims protection for a host cell comprising the expression vector.

[0036] The expression vector can be used to construct a host cell capable of effectively expressing the antibody protein through cell biology methods.

[0037] Furthermore, the present invention also claims protection for the use of the monoclonal antibody, or the recombinant antibody, or the multifunctional recombinant antibody; or the nucleotide, or the expression vector, or the host cell in the preparation of a biological agent for treating tumors.

[0038] Furthermore, the present invention also claims protection for a biological preparation, which comprises at least one of the monoclonal antibody, or the recombinant antibody, or the multifunctional recombinant antibody; or the nucleotide, or the expression vector, or the host cell.

[0039] Furthermore, the present invention also provides a method for preparing the monoclonal antibody, the recombinant antibody, or the multifunctional recombinant antibody, comprising the following steps:

[0040] (1) obtaining an expression vector containing the gene fragment of the monoclonal antibody, recombinant antibody or multifunctional recombinant antibody by artificial synthesis or molecular biological methods;

[0041] (2) transfecting the expression vector into cells for protein expression;

[0042] (3) Obtaining the monoclonal antibody, recombinant antibody, or multifunctional recombinant antibody by protein purification.

[0043] The corresponding antibodies can be obtained by constructing expression vectors containing the heavy chain or light chain sequence of the above antibodies, transfecting appropriate cells for expression and purification.

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

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

[0046] As a preferred embodiment of the present invention, the protein purification method in step (3) is purification by Protein G.

[0047] Compared with the prior art, the present invention has the following technical effects:

[0048] The present invention provides a monoclonal antibody that can specifically recognize human HER3 protein, and also provides a humanized recombinant antibody thereof. On this basis, the inventors further used the HER3-specific recombinant antibody to prepare a multifunctional recombinant antibody that can not only recognize human HER3 but also have IL15 function, which can effectively enhance the killing effect of the original antibody on tumor cells. At the same time, the present invention also obtains a modified specific HER3 monoclonal antibody-IL15 bifunctional molecule by introducing a mutated human IgG1 constant region. While maintaining its anti-tumor activity in vivo, the bifunctional molecule can effectively shorten its metabolic cycle, thereby reducing its toxic side effects in the body and greatly improving its safety. The multifunctional recombinant antibody of the present invention can recognize HER3 in a variety of tumor cells and has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Results of antibody SPGA08-158 binding to human HER3.

[0050] Figure 2 The antibody SPGA08-158 inhibits the binding of NRG1 to human HER3.

[0051] Figure 3 Results of antibody SPGA08-158 binding to mouse and cynomolgus monkey HER3 antigens.

[0052] Figure 4 Results of binding of human antibody huSPGA08-158 to human HER3

[0053] Figure 5 The human antibody huSPGA08-158 inhibits the binding of NRG1 to human HER3.

[0054] Figure 6 Results of the binding of multifunctional recombinant antibodies SPGL013 and SPGL014 to human HER3.

[0055] Figure 7 The results of multifunctional recombinant antibodies SPGL013 and SPGL014 simultaneously binding to human HER3 and CD122 / 132.

[0056] Figure 8 The results of multifunctional recombinant antibodies SPGL013 and SPGL014 inhibiting NRG1 binding to HER3.

[0057] Figure 9 The results of multifunctional recombinant antibodies SPGL013 and SPGL014 stimulating the proliferation of CTLL2 cells.

[0058] Figure 10The results of the multifunctional recombinant antibody SPGL014 inhibiting mouse colorectal cancer cell MC38 xenograft tumors.

[0059] Figure 11 The results of the multifunctional recombinant antibody SPGL014 inhibiting human lung cancer cell HCC827 xenografts.

[0060] Figure 12 The results of the multifunctional recombinant antibody SPGL014 inhibiting the growth of human breast cancer cell JIMT-1 xenografts.

[0061] Figure 13 Metabolism of the multifunctional recombinant antibodies SPGL013 and SPGL014 in huFcRn transgenic mice.

[0062] Figure 14 Flow cytometry analysis of the binding of SPGL014 to HER3 in various tumor cells. DETAILED DESCRIPTION

[0063] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0064] Example 1 Preparation of monoclonal antibodies and hybridomas

[0065] 1) Antigen immunization of mice: Balb / c mice were routinely subcutaneously immunized with recombinantly expressed human HER3-ECD protein (purchased from AcroBIOSYSTEMS, Beijing).

[0066] On the first day, the soluble human HER3-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, the soluble human HER3-ECD protein was emulsified with Freund's incomplete adjuvant (IFA, Sigma) and thoroughly mixed, and then subcutaneously boosted with 50 μg / mouse. On day 50, human HER3-ECD protein was injected intraperitoneally at 50 μg / mouse for stimulation, and the spleens of the mice were harvested 3 to 4 days after the injection for fusion experiments.

[0067] 2) Preparation and screening of hybridomas:

[0068] 3-4 days after the last shock immunization of mice, mouse spleen cells were fused with mouse myeloma cells SP2 / 0 by PEG (PEG1450, Sigma) method using conventional hybridoma technology protocol. The fused cells were evenly suspended in complete culture medium, which was a mixture of RPMI1640 and DMEM F12 culture medium in a ratio of 1:1, followed by the addition of 1% glutamine (Gibco), 1% sodium pyruvate (Gibco), 1% MEM-NEAA (minimal essential medium-non-essential amino acid solution, Gibco), 1% penicillin-streptomycin (Gibco), 50 μM β-mercaptoethanol (Gibco) and 20% FBS (Gibco). The cells were then incubated at 10 5 Cells were plated at 100 μL / well in a 96-well plate and cultured overnight. The next day, 100 μL of complete culture medium containing 2× HAT (Sigma) was added to each well, bringing the total volume of culture medium in the 96-well plate to 200 μL / well (containing 1× HAT). After 7-12 days, the supernatant was harvested and screened for hybridoma wells positive for human HER3-ECD protein binding activity using an indirect enzyme-linked immunosorbent assay (ELISA).

[0069] The indirect enzyme-linked immunosorbent assay (ELISA) method for screening hybridoma wells positive for human HER3-ECD protein activity is as follows: recombinant human HER3-ECD protein is diluted to 1 μg / mL in coating buffer (50 mM carbonate coating buffer, pH 9.6), and 100 μL / well is added to the ELISA plate and coated overnight at 4°C. The plate is washed three times with PBST, and then 200 μL / well of blocking buffer (2% BSA-PBST) is added. The plate is incubated at 37°C for 1 hour, followed by one wash with PBST. The collected hybridoma supernatant is then added to the blocked ELISA plate at 100 μL / well and incubated at 37°C for 1 hour. The plate was washed three times with PBST, and HRP-labeled goat anti-mouse IgG secondary antibody (purchased from Abcam, catalog number ab6789) was added and incubated at 37°C for 30 min. After washing the plate five times with PBST, any residual droplets were patted dry on absorbent paper as much as possible, and 100 μL of TMB (KPL) was added to each well. The plate was incubated at room temperature (20 ± 5°C) in the dark for 5 min. The substrate reaction was terminated by adding 50 μL of 2 M H2SO4 stop solution to each well. The OD value was read at 450 nm on a microplate reader to analyze the binding ability of the test antibody to the target antigen, human HER3-ECD protein.

[0070] The hybridoma cell lines obtained by screening were expanded in serum-containing complete medium, and the medium was changed to serum-free medium (SFM medium) by centrifugation to make the cell density 1-2×10 7The cells were cultured at 37°C under 5% CO2 for 2 weeks, and the culture supernatant was obtained by centrifugation. The supernatant was purified by Protein G affinity chromatography to obtain a mouse anti-human HER3-ECD protein monoclonal antibody named SPGA08-158.

[0071] Example 2 ELISA determination of the binding activity of SPGA08-158 to human HER3

[0072] The binding ability of mouse antibodies to human HER3-ECD protein was determined by indirect enzyme-linked immunosorbent assay.

[0073] Experimental steps: Pre-coat the plate with human HER3-ECD protein, dilute it to 2 μg / mL with coating solution (50 mM carbonate coating buffer, pH 9.6), and coat the plate overnight at 4°C; then block with 5% skim milk powder at 37°C for 2 hours; wash the plate three times with PBST, and add the antibody to be tested, which is graded diluted in 1% BSA-PBST, to the blocked ELISA plate at 100 μL / well and incubate at 37°C for 1 hour. The plate was washed three times with PBST, and HRP-labeled goat anti-mouse IgG secondary antibody (Millipore) was added and incubated at 37°C for 30 min. After washing the plate three times with PBST, any residual droplets were patted dry on absorbent paper as much as possible, and 100 μL of TMB (KPL) was added to each well. The plate was incubated at room temperature (20 ± 5°C) in the dark for 5 min. The substrate reaction was terminated by adding 50 μL of 2 M H2SO4 stop solution to each well. The OD value was read at 450 nm on a microplate reader to analyze the binding ability of the test antibody to the target antigen, human HER3-ECD protein.

[0074] The experimental results are as follows Figure 1 .Depend on Figure 1 The antibody SPGA08-158 binds to the EC of HER3-ECD. 50 It is 20.35 ng / mL, i.e. 0.14 nM, indicating that the antibody SPGA08-158 has good binding activity with HER3-ECD.

[0075] Example 3 Antibody SPGA08-158 inhibits NRG1 binding to human HER3-ECD

[0076] Experimental procedures: Pre-coat the plate with human HER3-ECD protein diluted to 2 μg / mL in coating buffer (50 mM carbonate coating buffer, pH 9.6) at 4°C overnight. Block the plate with 5% skim milk powder at 37°C for 2 hours. Wash the plate three times with PBST and then block again with 5% skim milk powder at 37°C for 2 hours. Wash the plate three times with PBST. Add the test antibody SPGA08-158, serially diluted in 1% BSA, to the blocked plate at 100 μL / well and incubate at 37°C for 1 hour. The plate was washed three times with PBST, and biotin-labeled NRG1 (Beijing Biopsies Co., Ltd., amino acid sequence as shown in SEQ ID NO: 2) diluted to 200 ng / mL was added and incubated at 37°C for 1 h. After washing the plate three times with PBST, any residual droplets were patted dry on absorbent paper as much as possible, 100 μL of diluted SA-HRP (THERMO Co., Ltd.) was added to each well, and the plate was incubated at 37°C for 30 min. After washing the plate three times with PBST, 100 μL / well of TMB was added and the plate was incubated at room temperature (20±5°C) in the dark for 5 min. 50 μL of 2M H2SO4 stop solution was added to each well to terminate the substrate reaction, and the OD value was read at 450 nm on a microplate reader to analyze the ability of the test antibody to block NRG1 binding to HER3.

[0077] The experimental results are as follows Figure 2 NRG1 can exert its biological function by binding to HER3. Figure 2 Available, IC 50 It is 361.6 ng / mL, or 2.41 nM, indicating that the antibody SPGA08-158 can effectively block the binding of NRG1 to HER3.

[0078] Example 4 Determination of cross-reactivity between antibody SPGA08-158 and mouse and cynomolgus macaque HER3 antigens The experimental method was similar to that of Example 2, except that human HER3 was replaced with mouse HER3-ECD (Beijing Biopsies Co., Ltd., with an amino acid sequence as shown in SEQ ID NO: 3) and cynomolgus macaque HER3-ECD (Beijing Sino Biological Co., Ltd., with an amino acid sequence as shown in SEQ ID NO: 4), respectively, and the binding ability of antibody SPGA08-158 to mouse and cynomolgus macaque HER3 antigens was determined.

[0079] The experimental results are as follows Figure 3 .Depend on Figure 3 The antibody SPGA08-158 can bind well to cynomolgus monkey HER3-ECD protein, EC 50 The binding affinity of HER3-ECD is 19.49 ng / mL, or 0.13 nM, which is comparable to that of human HER3-ECD (EC 50 was 0.14 nM); but did not bind to mouse HER3-ECD.

[0080] Example 5 Preparation of humanized antibody huSPGA08-158

[0081] RNA from SPGA08-158 hybridoma cells was extracted using Trizol and reverse transcribed to obtain cDNA. Using the cDNA as a template, PCR was performed using degenerate primers for the heavy and light chains of a mouse antibody ("Antibody Engineering," Volume 1, edited by Roland Kontermann and Stefan Dübel; primer sequences are from page 323). The PCR products were sequenced and analyzed using the Kabat database to confirm that the sequences obtained were the variable region sequences of the mouse antibody. The relevant sequence information is as follows:

[0082] The SPGA08-158 antibody heavy chain variable region gene sequence is 354 bp in length, encoding 118 amino acid residues. The amino acid sequence is shown in SEQ ID NO: 6, and the corresponding nucleotide sequence is shown in SEQ ID NO: 5; the SPGA08-158 antibody light chain variable region gene sequence is 321 bp in length, encoding 107 amino acid residues. The amino acid sequence is shown in SEQ ID NO: 8, and the corresponding nucleotide sequence is shown in SEQ ID NO: 7.

[0083] The amino acid sequences of the light and heavy chain variable regions of the SPGA08-158 antibody were analyzed, and the three antigenic complementarity determining regions (CDRs) and four framework regions (FRs) of the murine antibody were determined according to the Kabat rules. The amino acid sequence of the SPGA08-158 heavy chain CDR is HCDR1: GYTFTNYYIY (SEQ ID NO: 9), HCDR2: INPSNGGT (SEQ ID NO: 10), and HCDR3: TRDLGYYAMDY (SEQ ID NO: 11). The amino acid sequence of the light chain CDR is LCDR1: QDIKNYLN (SEQ ID NO: 12), LCDR2: YTSRLQS (SEQ ID NO: 13), and LCDR3: QQGETLPWT (SEQ ID NO: 14).

[0084] Humanized templates that best matched the FR regions of each of the aforementioned murine antibodies were selected from the Germline database. The CDR regions of the murine antibodies were then transplanted onto the selected humanized templates, replacing the CDR regions of the human templates. The heavy chain variable region was then recombined with the human IgG1 constant region (SEQ ID NO: 15), and the light chain variable region was recombined with the human kappa chain constant region (SEQ ID NO: 16). Simultaneously, based on the three-dimensional structure of the antibodies, backmutations were performed on buried residues, residues that directly interact with the CDR regions, and residues that significantly influence the VL and VH conformations of each antibody. Ultimately, the humanized antibody huSPGA08-158 was obtained.

[0085] The heavy chain variable region amino acid sequence of the humanized antibody huSPGA08-158 is shown in SEQ ID NO: 17, the heavy chain amino acid sequence is shown in SEQ ID NO: 18, the light chain variable region amino acid sequence is shown in SEQ ID NO: 19, and the light chain amino acid sequence is shown in SEQ ID NO: 20.

[0086] The heavy and light chains of the humanized antibody huSPGA08-158 were constructed and inserted into the pcDNA3.4 expression vector, transfected into Expi-293F cells, and purified by Protein G. SDS-PAGE electrophoresis and SEC-HPLC confirmed that the molecular weight of each antibody was correct and the purity was >95%.

[0087] Example 6 ELISA determination of the binding activity of humanized antibody huSPGA08-158 to human HER3

[0088] The experimental method refers to Example 2, and the binding activity of humanized antibody huSPGA08-158 to human HER3 is determined. The experimental results are as follows: Figure 4 shown.

[0089] Depend on Figure 4 The humanized antibody huSPGA08-158 binds to HER3. 50 The value was 4.779 ng / mL, i.e. 0.03 nM, which was significantly lower than that of antibody SPGA08-158 (its EC 50 The binding activity of the humanized antibody huSPGA08-158 to HER3 was 20.35 ng / mL, i.e., 0.14 nM (see Example 2), indicating that the humanized antibody huSPGA08-158 has good binding activity to HER3.

[0090] Example 7 Humanized Antibody huSPGA08-158 Inhibits NRG1 Binding to HER3

[0091] The experimental method refers to Example 3, and the effect of humanized antibody huSPGA08-158 in inhibiting NRG1 binding to HER3 is determined. The experimental results are as follows: Figure 5 shown.

[0092] Depend on Figure 5 Available, IC of humanized antibody huSPGA08-158 50 The activity was 430.1 ng / mL, or 2.87 nM, which was comparable to that of the mouse monoclonal antibody SPGA08-158 (IC 50 2.41 nM), indicating that the humanized antibody huSPGA08-158 can effectively inhibit the binding of NRG1 to HER3.

[0093] Example 8 Preparation of multifunctional recombinant antibodies

[0094] 1. Antibody functional region recognizing human IL15: Human IL15Rsushi (amino acid sequence shown in SEQ ID NO: 21) and human IL15 (amino acid sequence shown in SEQ ID NO: 22) were linked via (GGGGS)6 to form a single-chain IL15, i.e., IL15sc, whose amino acid sequence is shown in SEQ ID NO: 23.

[0095] 2. Multifunctional recombinant antibodies:

[0096] ① SPGL013 heavy chain: The huSPGA08-158 heavy chain sequence (amino acid sequence shown in SEQ ID NO: 18) was spliced ​​with the human IL15sc sequence via (GGGGS)3 to form the SPGL013 heavy chain (amino acid sequence shown in SEQ ID NO: 24);

[0097] ②SPGL014 heavy chain: The huSPGA08-158 heavy chain VH sequence (amino acid sequence shown in SEQ ID NO: 17) was linked to a mutated human IgG1 constant region (amino acid sequence shown in SEQ ID NO: 25) to form the huSPGA08-158mu heavy chain (amino acid sequence shown in SEQ ID NO: 26); the huSPGA08-158mu heavy chain was spliced ​​with the human IL15sc sequence via (GGGGS)3 to form the SPGL014 heavy chain (amino acid sequence shown in SEQ ID NO: 27).

[0098] ③ The light chain sequence is the same as that of huSPGA08-158 (amino acid sequence shown in SEQ ID NO: 20).

[0099] 3. Preparation of Multifunctional Recombinant Antibodies

[0100] The heavy and light chains of SPGL014 (or SPGL013) were co-transfected into Expi-293F cells and purified using Protein G to obtain the multifunctional recombinant antibody SPGL014 (or SPGL013). SDS-PAGE electrophoresis and SEC-HPLC confirmed the correct molecular weight of each expressed antibody, with a purity of >95%. The prepared multifunctional antibodies were quantified, aliquoted, and stored frozen at -80°C until further use.

[0101] Example 9 Multifunctional recombinant antibody SPGL014 binds to HER3-ECD

[0102] The experimental method refers to Example 2, and the binding activity of the multifunctional recombinant antibodies SPGL013 and SPGL014 to human HER3 is determined. The experimental results are as follows: Figure 6 shown.

[0103] By Figure 6 Available, multifunctional recombinant antibodies SPGL014 and SPGL013 bind to the EC of human HER3-ECD 50 The results were 4.359 ng / mL and 4.981 ng / mL, i.e., 0.023 nM and 0.026 nM, respectively, which were consistent with huSPGA08-158 (EC 50 The results showed that the binding of huSPGA08-158 to IL15 did not affect its binding to HER3, and the mutation of IgG1 in the multifunctional recombinant antibody SPGL014 did not affect its binding to HER3.

[0104] Example 10 Multifunctional recombinant antibody SPGL014 binds to HER3-ECD and CD122 / 132 simultaneously

[0105] Experimental method: Human HER3-ECD protein was diluted to 2 μg / mL with coating solution (50 mM carbonate coating buffer, pH 9.6), and 100 μL / well was added to the ELISA plate and coated at 4°C overnight. The plate was then blocked with 200 μL / well of 5% skim milk powder at 37°C for 2 hours. The plate was washed three times with PBST, and the test antibody was gradiently diluted to 1 μg / mL in 1% BSA-PBST and added to the blocked ELISA plate at 100 μL / well and incubated at 37°C for 1 hour. The plate was washed three times with PBST, and gradient dilutions of biotin-labeled CD122 / 132 (purchased from Beijing Biopsies Co., Ltd.) were added 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, any residual droplets were patted dry on absorbent paper as much as possible, 100 μL of TMB (KPL) was added to each well, and the plate was incubated at room temperature (20±5°C) in the dark for 5 minutes. 50 μL of 2M H2SO4 stop solution was added to each well to terminate the substrate reaction. The OD value was read at 450 nm on a microplate reader to analyze the binding ability of the test antibody to human CD122 / 132.

[0106] The experimental results are as follows Figure 7 .like Figure 7 It can be seen that under the experimental conditions, the EC of antibodies SPGL014 and SPGL013 binding to human CD122 / 132 protein 50 The concentrations of huSPGA08-158 in the control group were 19.04 ng / mL and 18.49 ng / mL, i.e., 0.10 nM and 0.10 nM, respectively. This indicates that the multifunctional recombinant antibodies SPGL014 and SPGL013 both have good activity in binding to HER3 and CD122 / 132 simultaneously. In contrast, the huSPGA08-158 antibody in the control group can bind to HER3 but cannot bind to CD122 / 132.

[0107] Example 11 Multifunctional recombinant antibodies SPGL013 and SPGL014 inhibit NRG1 binding to HER3

[0108] The experimental method refers to Example 3, and the effect of multifunctional recombinant antibodies SPGL014 and SPGL013 in inhibiting NRG1 binding to HER3 was determined. Figure 8 shown.

[0109] Depend on Figure 8 The antibodies SPGL014 and SPGL013 inhibited NRG1 binding to HER3 with IC 50 The blocking activities of SPGL014 and SPGL013 were comparable to those of SPGA08-158 (IC 50 is 2.41nM).

[0110] Example 12 Multifunctional recombinant antibodies SPGL013 and SPGL014 stimulate CTLL2 cell proliferation

[0111] Experimental method: CTLL2 cells were diluted to 5×10 cells / mL in 1640 culture medium containing 10% FBS. 4 / mL, 100μL / well was added to the cell culture plate. IL2 was diluted to 30ng / mL with 1640 culture medium containing 10% FBS, and then diluted 3-fold in a total of 8 gradients and added to the above culture plates containing CTLL2 cells; SPGL014 and SPGL013 were diluted to 5000ng / mL with 1640 culture medium containing 10% FBS, and then diluted 3-fold in a total of 8 gradients and added to the above culture plates containing CTLL2 cells; After 72 hours of incubation in a CO2 cell incubator, the relative cell number of each well was determined by CCK8, and the EC was calculated. 50 , determine the activity of the sample. The experimental results are as follows Figure 9 shown.

[0112] Depend on Figure 9 It was found that both multifunctional recombinant antibodies SPGL014 and SPGL013 could stimulate the proliferation of CTLL2 cells, EC 50 The EC values ​​of IL-2 were 43.55 ng / mL and 39.83 ng / mL, or 0.23 nM and 0.21 nM, respectively, indicating that SPGL014 has the same biological activity as SPGL013. 50 It is 0.9475ng / mL, or 0.059nM.

[0113] Example 13 The toxicity of the multifunctional recombinant antibody SPGL014 in mice is significantly lower than that of SPGL013

[0114] Experimental Methods: Multifunctional recombinant antibodies SPGL014 and SPGL013 were intraperitoneally injected into C57BL / 6 mice (Vitamin Liva) three times on days 1, 3, and 5, with an injection volume of 0.2 mL per injection. SPGL014 and SPGL013 were administered at doses of 0.5 mg / kg, 1 mg / kg, 2 mg / kg, and 4 mg / kg. Mice were observed daily for mortality. A control group received the same volume of PBS. The results are shown in Table 1.

[0115] Table 1 Survival rate of experimental animals in each dose group

[0116] sample 0.5 mg / kg 1.0 mg / kg 2.0 mg / kg 4.0 mg / kg SPGL013 100％ 70％ 20％ 10％ SPGL014 100％ 100％ 100％ 30％

[0117] As shown in Table 1, on the 10th day of the experiment, the survival rates of the experimental animals in the SPGL013 sample 4.0 mg / kg, 2.0 mg / kg, 1.0 mg / kg, and 0.5 mg / kg groups were 10%, 20%, 70%, and 100%, respectively; the survival rates of the experimental animals in the SPGL014 sample 4.0 mg / kg, 2.0 mg / kg, 1.0 mg / kg, and 0.5 mg / kg groups were 30%, 100%, 100%, and 100%, respectively. These results indicate that after the Fc mutation, the toxicity of SPGL014 to experimental mice was significantly reduced compared with SPGL013.

[0118] Example 14 Multifunctional recombinant antibody SPGL014 inhibits the growth of mouse colorectal cancer cell MC38 transplanted tumors

[0119] Experimental method: Mouse colon cancer MC38 cells cultured in vitro were collected and the cell suspension concentration was adjusted to 1×10 7 / mL. Under sterile conditions, 100 μL of MC38 cell suspension was inoculated subcutaneously on the right flank of C57BL / 6 mice. The diameter of the subcutaneous transplanted tumor was measured with a vernier caliper. When the average tumor volume grew to 100-200 mm 3 The animals were then randomly divided into groups of 6 per group. SPGL014 was administered at 1.0 mg / kg and 0.5 mg / kg, and SPGL013 was administered at 0.5 mg / kg and 0.25 mg / kg. The control group was given an equal amount of PBS, intraperitoneally injected 3 times a week with a volume of 0.2 mL / time for 2 consecutive weeks. During the entire experiment, the diameter of the transplanted tumor was measured 3 times a week, and the mice were weighed at the same time. The formula for calculating tumor volume (TV) is (where a and b represent length and width, respectively):

[0120] TV=1 / 2×a×b2.

[0121] The relative tumor volume (RTV) was calculated based on the measurement results. The calculation formula is (where V0 is the tumor volume measured when the group was given, V t is the tumor volume at each measurement):

[0122] RTV=V t / V0.

[0123] The evaluation index of antitumor activity is the relative tumor proliferation rate T / C (%), which is calculated as follows (where TRTV is the RTV of the treatment group; CRTV is the RTV of the negative control group):

[0124] T / C(%)=(TRTV / CRTV)×100;

[0125] Tumor inhibition rate TGI (%) = 100 - T / C (%).

[0126] The results of the test are as follows Figure 10 As shown. Figure 10 It can be seen that both SPGL014 and SPGL013 exhibited strong anti-tumor activity. At doses of 0.5 mg / kg and 0.25 mg / kg, the TGI of SPGL013 were 69.0% and 52.5%, respectively. At doses of 1.0 and 0.5 mg / kg, the TGI of SPGL014 were 71.2% and 58.6%, respectively.

[0127] Referring to Example 13, SPGL013 exhibited lethal toxicity at a dose of 1.0 mg / kg, while SPGL014 exhibited lethal toxicity at a dose of 4.0 mg / kg, indicating that SPGL014 containing Fc mutations significantly improved safety without affecting the anti-tumor activity in vivo.

[0128] Example 15 Multifunctional recombinant antibody SPGL014 inhibits the growth of human lung cancer cell HCC827 transplanted tumors in nude mice

[0129] Experimental method: Human lung cancer HCC827 cells cultured in vitro were collected and the cell suspension concentration was adjusted to 8×10 7 / mL. Under sterile conditions, 100 μL of cell suspension was inoculated subcutaneously on the right flank of nude mice. After the tumor cells formed solid tumors under the mouse skin, the diameter of the transplanted tumor was measured with a vernier caliper. When the average tumor volume grew to 100-200 mm 3 The animals were then randomly divided into groups. SPGL014 was administered at 1.0 mg / kg and 0.5 mg / kg, and SPGL013 was administered at 0.5 mg / kg and 0.25 mg / kg, intraperitoneally 3 times a week for a total of 6 doses. During the entire experiment, the diameter of the transplanted tumor was measured twice a week and the mice were weighed. The rest was the same as in Example 14. The results are shown in Figure 14. Figure 11 shown.

[0130] Depend on Figure 11 Both SPGL014 and SPGL013 demonstrated strong antitumor activity in human lung cancer cells. SPGL013 achieved TGIs of 82.3% and 43.2% at doses of 0.5 mg / kg and 0.25 mg / kg, respectively, while SPGL014 achieved TGIs of 84.2% and 71.1% at doses of 1.0 and 0.5 mg / kg, respectively. This suggests that the Fc-mutated SPGL014 maintains antitumor activity while exhibiting significantly improved in vivo safety.

[0131] Example 16 Multifunctional recombinant antibody SPGL014 inhibits the growth of human breast cancer cell JIMT-1 transplanted tumors in nude mice

[0132] Experimental method: Human breast cancer JIMT-1 cells cultured in vitro were collected and the cell suspension concentration was adjusted to 8×10 7 / mL. Under sterile conditions, 100 μL of cell suspension was inoculated subcutaneously on the right flank of nude mice. After the tumor cells formed solid tumors under the mouse skin, the diameter of the transplanted tumor was measured with a vernier caliper. When the average tumor volume grew to 50-100 mm 3 The animals were then randomly divided into groups. SPGL014 was administered at 1.0 mg / kg and 0.5 mg / kg, and SPGL013 was administered at 0.5 mg / kg and 0.25 mg / kg, intraperitoneally three times per week for a total of six doses. Throughout the experiment, the diameter of the transplanted tumors was measured twice per week, and the mice were weighed. All other procedures were the same as in Example 14. The results are shown in Figure 12.

[0133] Depend on Figure 12 Both SPGL014 and SPGL013 demonstrated strong anti-tumor activity in human breast cancer cells. SPGL013 achieved TGIs of 51.5% and 32% at doses of 0.5 mg / kg and 0.25 mg / kg, respectively, while SPGL014 achieved TGIs of 59% and 52.1% at doses of 1.0 and 0.5 mg / kg, respectively. This suggests that the Fc-mutated SPGL014 maintains anti-tumor activity while exhibiting significantly improved in vivo safety.

[0134] Example 17 Metabolism of Multifunctional Recombinant Antibodies SPGL013 and SPGL014 in huFcRn Transgenic Mice

[0135] Experimental Methods: Human FcRn transgenic mice were used to determine the pharmacokinetics of SPGL013 and SPGL014. Eight mice were divided into two groups and injected intraperitoneally with 1 mg / kg of SPGL013 and SPGL014, respectively. Blood samples were collected 2, 6, 24, 48, and 72 hours later to obtain serum. The serum was appropriately diluted and the blood drug concentration was determined by ELISA, using the same method as in Example 11. The results are shown in Figure 11. Figure 13 shown.

[0136] Depend on Figure 13 It was found that the metabolism of Fc-mutant SPGL014 was significantly accelerated in human FcRn transgenic mice compared with Fc wild-type SPGL013, with half-lives of 11 hours and 35 hours, respectively, showing a significant difference.

[0137] Example 18 SPGL014 detects HER3 expression in various tumor cells

[0138] The binding affinity of SPGL014 to human breast cancer cells MCF-7 was determined by flow cytometry fluorescence activated cell sorting (FACS).

[0139] In this experiment, human breast cancer MCF-7 cells were used as target cells, and 100 μL of 2000 ng / mL SPGL014 was used as the primary antibody and incubated with 1×10 5 MCF-7 cells were incubated at 4°C for 1 h, washed twice with PBS to remove unbound SPGL014, and then incubated with 100 μL of 2 μg / mL Alexa Fluor 488-labeled anti-human IgG fluorescent secondary antibody (purchased from Thermo, Invitrogen, Catalog No. A11013) at 4°C for 30 min. The cells were washed twice with PBS to remove unbound secondary antibody. Finally, the cells were resuspended in 100 μL PBS, and the binding affinity of SPGL014 to the cells was determined by flow cytometry. The obtained MFI value was compared with the MFI value of the control group (not incubated with SPGL014), and the logarithm of the ratio was used to represent the binding intensity of SPGL014.

[0140] As described above, the binding affinity of SPGL014 to other tumor cells was tested, including human breast cancer cells JIMT-1, human skin cancer cells A375, human ovarian cancer cells SKOV3, human colorectal cancer cells HT-29, and human lung cancer cells H1975 and HCC827. Figure 14 shown.

[0141] Depend on Figure 14 It can be seen that SPGL014 can bind to various tumor cells to varying degrees, among which JIMT-1, A375, HCC827 and MCF-7 bind best, followed by H1975 and HT-29, and SKOV3 cells bind the weakest.

[0142] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention 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 invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A monoclonal antibody, characterized in that The monoclonal antibody can specifically recognize HER3 protein; the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:

8.

2. A recombinant antibody, characterized in that The recombinant antibody is obtained by humanizing the monoclonal antibody according to claim 1, the amino acid sequence of the heavy chain variable region of the recombinant antibody is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the recombinant antibody is shown in SEQ ID NO:

19.

3. A multifunctional recombinant antibody, characterized in that: The heavy chain of the multifunctional recombinant antibody includes an antibody functional region that recognizes human HER3 and an antibody functional region that recognizes human IL15, as well as a non-functional amino acid fragment used to connect the functional regions; The heavy chain variable region of the antibody functional region that recognizes human HER3 includes complementary determining regions VH-CDR1, VHCDR2, and VH-CDR3, and the VH-CDR1, VHCDR2, and VH-CDR3 include amino acid sequences consistent with the VH-CDR1, VHCDR2, and VH-CDR3 of the light chain variable region shown in SEQ ID NO.

17. The antibody functional region that recognizes human HER3 includes a human IgG1 constant region functional domain, and the sequence of the human IgG1 constant region functional domain is shown in SEQ ID NO: 15 or 25; The amino acid sequence of the functional region of human IL15 is shown in SEQ ID NO: 23; the non-functional amino acid fragment used to connect the functional regions is (GGGGS)3; The light chain variable region of the multifunctional recombinant antibody includes complementary determining regions VL-CDR1, VL-CDR2 and VL-CDR3, and the VL-CDR1, VL-CDR2 and VL-CDR3 include amino acid sequences consistent with VL-CDR1, VL-CDR2 and VL-CDR3 of the light chain variable region shown in SEQ ID NO.

19.

4. The multifunctional recombinant antibody according to claim 3, wherein The amino acid sequence of the multifunctional recombinant antibody heavy chain is shown in SEQ ID NO: 24 or SEQ ID NO: 27; the amino acid sequence of the multifunctional recombinant antibody light chain is shown in SEQ ID NO:

20.

5. A nucleotide encoding the monoclonal antibody according to claim 1, the recombinant antibody according to claim 2, or the multifunctional recombinant antibody according to claim 3 or 4.

6. An expression vector, characterized in that The expression vector comprises the nucleotide sequence of claim 5.

7. A host cell, characterized in that The host cell comprises the expression vector according to claim 6.

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

9. A biological agent, characterized in that The biological preparation comprises at least one of the monoclonal antibody according to claim 1, or the recombinant antibody according to claim 2, or the multifunctional recombinant antibody according to claim 3 or 4; or the nucleotide according to claim 5, or the expression vector according to claim 6, or the host cell according to claim 7.

10. A method for preparing the monoclonal antibody according to claim 1, the recombinant antibody according to claim 2, or the multifunctional recombinant antibody according to claim 3 or 4, characterized in that: The following steps are involved: (1) Obtaining an expression vector containing the gene fragment of the monoclonal antibody, recombinant antibody or multifunctional recombinant antibody by artificial synthesis or molecular biological methods; (2) transfecting the expression vector into cells for protein expression; (3) Obtaining the monoclonal antibody, recombinant antibody, or multifunctional recombinant antibody by protein purification.

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