WO2024077774A1 - 一种多功能重组抗体及其制备方法和应用 - Google Patents

一种多功能重组抗体及其制备方法和应用 Download PDF

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WO2024077774A1
WO2024077774A1 PCT/CN2022/141440 CN2022141440W WO2024077774A1 WO 2024077774 A1 WO2024077774 A1 WO 2024077774A1 CN 2022141440 W CN2022141440 W CN 2022141440W WO 2024077774 A1 WO2024077774 A1 WO 2024077774A1
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antibody
recombinant antibody
amino acid
human
acid sequence
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French (fr)
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娄竞
陈建鹤
苏冬梅
靳征
吕云英
张若兰
裴若辰
欧艳梅
曲啸
谢写
张静
曾淋
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深圳市百士通科技开发有限公司
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Definitions

  • the present invention belongs to the field of biotechnology and relates to a multifunctional recombinant antibody and a preparation method and application thereof, and specifically relates to a multifunctional recombinant antibody that can recognize CD73 and has IL15 function and an application thereof.
  • Antibody drugs have now become a major component of anticancer drugs, but in terms of solid tumors, the efficacy of antibody drugs is still limited, so it is important to find more new solid tumor antigens, but it is also relatively difficult.
  • an important aspect is to relieve the inhibitory effect of the tumor microenvironment (TME) on immune effector cells.
  • CD73 is a 5-primary nucleotide hydrolase that can hydrolyze extracellular adenosine monophosphate (AMP) into adenosine, which is a powerful immunosuppressive molecule that can inhibit the activation of CD8-positive T cells and help cancer cells escape the "hunt" of T cells.
  • AMP adenosine monophosphate
  • CD73 is one of the key components of the formation of an immunosuppressive microenvironment in cancer. It is reported that CD73 is expressed in many different cancers, including colon cancer, lung cancer, pancreatic cancer, ovarian cancer, bladder cancer, leukemia, glioma, glioblastoma, melanoma, thyroid cancer, esophageal cancer, prostate cancer and breast cancer. Moreover, the expression of CD73 in cancer is associated with cell proliferation, migration, neovascularization, invasiveness, metastasis and shorter survival of patients. CD73 activity has also been proposed as a prognostic marker for papillary thyroid carcinoma.
  • CD73 has been shown to regulate tumor cell-cell and cell-matrix interactions
  • CD73 expression and activity have also been associated with reduced T cell responses and have been suggested to be involved in drug resistance. Therefore, CD73 can directly and indirectly regulate the development of cancer, which reflects its potential as a new therapeutic target, and it is highly desirable to inhibit its activity through regulation and develop related therapeutic agents.
  • IL15 is a cytokine expressed by a variety of cells, including monocytes, macrophages, epidermal cells and fibroblasts, but not by T lymphocytes. Unlike many other cytokines, IL15 is generally not secreted from cells to exert its effects, but is localized on specific cell membranes after combining with IL15R ⁇ , thereby stimulating nearby effector cells, mainly NK and CD8+T cells. IL15 is closely related to IL2. After IL15 forms a complex with IL15R ⁇ , it can bind to the ⁇ / ⁇ receptors shared by IL2 to mediate its biological activity.
  • IL15/R ⁇ does not stimulate the proliferation of Treg.
  • IL15-related molecules in the research and development stage for the treatment of malignant tumors.
  • ALT-803 which is the fastest-progressing one, is a complex formed by IL15 and IL15R ⁇ sushi-hFc1.
  • Multiple clinical studies have shown that ALT-803 is effective against a variety of tumors, including melanoma, but it also has serious side effects, mainly including liver damage, hypotension and fever.
  • the purpose of the present invention is to provide a monoclonal antibody that can specifically recognize CD73 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 CD73 protein and have IL15 function.
  • the technical solution adopted by the present invention is: a monoclonal antibody, which can recognize human CD73-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.
  • the present invention prepares a mouse monoclonal antibody capable of specifically recognizing human CD73 protein by immunizing mice with recombinantly expressed human CD73-ECD protein, named 3E10.
  • the amino acid sequences of the heavy chain variable region and the light chain variable region of the mouse monoclonal antibody are obtained through biological analysis.
  • the present invention also claims protection for a recombinant antibody, wherein the recombinant antibody is obtained by humanizing the monoclonal antibody 3E10, the amino acid sequence of the heavy chain variable region of the recombinant antibody is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region of the recombinant antibody is shown in SEQ ID NO: 18.
  • the heavy chain amino acid sequence of the recombinant antibody is shown in SEQ ID NO: 17, and the light chain amino acid sequence of the recombinant antibody is shown in SEQ ID NO: 19.
  • the inventors of the present invention further analyzed the obtained mouse monoclonal antibody 3E10 sequence, replaced the CDR region of the human template, recombined the heavy chain variable region with the human IgG1 constant region, and recombined the light chain variable region with the human kappa chain constant region.
  • the buried residues, the residues that directly interact with the CDR region, and the residues that have an important influence on the conformation of the VL and VH of each antibody were back-mutated, and finally a humanized recombinant antibody was obtained, named hu3E10.
  • the amino acid sequences of the heavy chain variable region and the light chain variable region, the heavy chain amino acid sequence, and the light chain amino acid sequence of the humanized recombinant antibody hu3E10 are shown above.
  • the present invention also claims protection for a multifunctional recombinant antibody, the heavy chain of which includes an antibody functional region that recognizes human CD73, 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 that recognizes human CD73 includes the amino acid sequence of the heavy chain variable region of the recombinant antibody.
  • the IL15 functional region can recognize the functional domain of human IL2/IL15 ⁇ / ⁇ receptor.
  • 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, and further obtained a recombinant antibody that can recognize human CD73 protein and has IL15 function.
  • 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.
  • 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: 24.
  • the inventors of the present invention use a sequence containing a mutated human IgG1 constant region to obtain a multifunctional recombinant antibody with a shorter metabolic cycle, lower toxic side effects and better safety.
  • amino acid sequence of the human IL15 functional region is shown in SEQ ID NO: 21.
  • amino acid sequence of the human IL15 functional region is as shown in SEQ ID NO: 22.
  • the amino acid sequence of the functional region of preferred human IL15 shown in SEQ ID NO: 22 is human IL15Rsushi and human IL15 are linked via (GGGGS) 6 to form a single-chain IL15, namely IL15sc.
  • the non-functional amino acid fragment used to connect the functional regions in the multifunctional recombinant antibody is a GGGGS repeat.
  • the GGGGS repeat is (GGGGS) 3 .
  • the amino acid sequence of the human IgG1 constant region functional domain of the multifunctional recombinant antibody is as shown in SEQ ID NO: 8; the heavy chain amino acid sequence of the multifunctional recombinant antibody is as shown in SEQ ID NO: 23; and the light chain amino acid sequence of the multifunctional recombinant antibody is as shown in SEQ ID NO: 19.
  • the present invention prepares a multifunctional antibody that can recognize CD73 and has IL15 function, named SPGL003.
  • 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: 24; the heavy chain amino acid sequence of the multifunctional recombinant antibody is shown in SEQ ID NO: 26; and the light chain amino acid sequence of the multifunctional recombinant antibody is shown in SEQ ID NO: 19.
  • the multifunctional recombinant antibody prepared using the preferred multifunctional recombinant antibody sequence contains a sequence of a mutated human IgG1 constant region and is named SPGL004; the multifunctional recombinant antibody SPGL004 can not only effectively recognize CD73 in a variety of tumor cells, but also effectively inhibit the growth of a variety of cancer cells, and has a shorter half-life, lower toxicity and good safety.
  • the present invention also claims protection for the nucleotide sequence encoding the monoclonal antibody, or the recombinant antibody, or the multifunctional recombinant antibody.
  • the corresponding nucleotide sequence of the encoding gene can be obtained.
  • 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.
  • the present invention also claims protection for an expression vector comprising the nucleotide sequence.
  • An expression vector containing the nucleotide sequence can be constructed by molecular biological methods.
  • the present invention also claims to protect a host cell comprising the expression vector.
  • the expression vector can be used to construct a host cell capable of effectively expressing the antibody protein through cell biology methods.
  • 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 sequence, or the expression vector, or the host cell in the preparation of a biological agent for treating tumors.
  • 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 sequence, or the expression vector, or the host cell.
  • the present invention also provides a method for preparing the monoclonal antibody or the recombinant antibody or the multifunctional recombinant antibody, comprising the following steps:
  • 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.
  • the expression vector containing the gene fragment of the monoclonal antibody or recombinant antibody or multifunctional recombinant antibody is prepared by molecular biology by inserting the heavy chain or light chain nucleotide sequence of the antibody into the pcDNA3.4 expression vector.
  • the cells used for protein expression are Expi-293F cells.
  • the protein purification method in step (3) is purification by Protein G.
  • the present invention has the following technical effects:
  • the present invention not only prepares a monoclonal antibody that can specifically recognize human CD73 protein, but also provides a humanized recombinant antibody thereof.
  • the inventors further used CD73-specific recombinant antibodies to prepare multifunctional recombinant antibodies that can recognize human CD73 and have IL15 function, which can effectively enhance the killing effect of the original antibody on tumor cells.
  • the present invention also obtains a modified specific CD73 monoclonal antibody-IL15 bifunctional molecule by introducing a mutated human IgG1 constant region. While maintaining its anti-tumor activity in vivo, the molecule can effectively shorten its metabolic cycle, thereby reducing its toxic side effects in vivo and greatly improving safety.
  • the multifunctional recombinant antibody of the present invention can recognize CD73 in a variety of tumor cells and has great application prospects.
  • Figure 2 The results of antibody 3E10 inhibiting CD73 enzyme activity in NCI-H1975 cells.
  • Figure 3 The results of antibody 3E10 binding to mouse and cynomolgus monkey CD73 antigens.
  • Figure 5 The results of human antibody hu3E10 inhibiting the enzymatic activity of tumor cell membrane CD73.
  • Figure 6 The results of the binding of multifunctional recombinant antibodies SPGL003 and SPGL004 to human CD73.
  • Figure 7 shows the results of each group of antibodies binding to CD73 and CD122/132 simultaneously.
  • FIG. 8 Multifunctional recombinant antibodies SPGL003 and SPGL004 inhibit the enzyme activity of tumor cell membrane CD73.
  • Figure 9 shows the results of multifunctional recombinant antibodies SPGL003 and SPGL004 stimulating the proliferation of CTLL2 cells; A is the detection result of SPGL003 and SPGL004, and B is the detection result of IL2.
  • FIG. 10 shows the changes in the volume of MC38 colorectal cancer cell transplanted tumors in each group of mice over time.
  • FIG. 11 shows the changes in the volume of human lung cancer cell H1975 transplanted tumors in each group over time.
  • Figure 12 The results of the multifunctional recombinant antibody SPGL004 combined with HER2 monoclonal antibody inhibiting human breast cancer cell JIMT-1 transplanted tumors.
  • Figure 13 Metabolism of multifunctional recombinant antibodies SPGL003 and SPGL004 in huFcRn transgenic mice.
  • FIG14 is the result of flow cytometry detection of the binding of SPGL004 to CD73 of various tumor cells.
  • Figure 15 Protein status of the multifunctional recombinant antibody SPGL004 after 0 weeks of storage.
  • Figure 16 Protein status of the multifunctional recombinant antibody SPGL004 after being stored at 4°C for 5 weeks.
  • mice Use recombinantly expressed human CD73-ECD protein (purchased from Beijing Biossys Company, Acro BIOSYSTEMS, the amino acid sequence is shown in SEQ ID NO: 1).
  • the soluble human CD73-ECD protein was emulsified with Freund's complete adjuvant (CFA, Sigma) and mixed thoroughly, and then subcutaneously injected into Balb/c mice (50 ⁇ g/mouse); on days 14 and 36, the soluble human CD73-ECD protein was emulsified with Freund's incomplete adjuvant (IFA, Sigma) and mixed thoroughly, and then subcutaneously boosted immunization was performed on Balb/c mice (50 ⁇ g/mouse); on day 50, 50 ⁇ g/mouse of human CD73-ECD protein was injected intraperitoneally for stimulation, and the spleens of mice were taken for fusion experiments 3 to 4 days after the injection.
  • CFA Freund's complete adjuvant
  • IFA Freund's incomplete adjuvant
  • mouse spleen cells and mouse myeloma cells SP2/0 were fused by PEG (PEG1450, Sigma) method using conventional hybridoma technology.
  • the fused cells were evenly suspended in complete culture medium, which was a culture medium composed of RPMI1640 and DMEM F12 culture medium mixed at a ratio of 1:1 and added with 1% glutamine (Gibco), 1% sodium pyruvate (Gibco), 1% MEM-NEAA (minimum essential medium-non-essential amino acid solution, Gibco), 1% penicillin-streptomycin (Gibco), 50 ⁇ M ⁇ -mercaptoethanol (Gibco) and 20% FBS (Gibco).
  • complete culture medium which was a culture medium composed of RPMI1640 and DMEM F12 culture medium mixed at a ratio of 1:1 and added with 1% glutamine (Gibco), 1% sodium pyruvate (Gibco), 1% MEM-NEAA (minimum essential medium-non
  • the method for screening hybridoma wells with positive human CD73-ECD protein activity by indirect enzyme-linked immunosorbent assay is as follows: dilute the recombinant human CD73-ECD protein to 1 ⁇ g/mL with coating solution (50mM carbonate coating buffer, pH 9.6), add 100 ⁇ L/well to the ELISA plate, and coat overnight at 4°C. Wash the plate 3 times with PBST, add 200 ⁇ L/well blocking solution (2% BSA-PBST), place at 37°C for 1h, and then wash the plate once with PBST for use. Add the collected hybridoma supernatant to the blocked ELISA plate in sequence, 100 ⁇ L/well, and place at 37°C for 1h.
  • 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 placed at 37°C for 30 min. After washing the plate five times with PBST, the residual droplets were patted dry on absorbent paper as much as possible, 100 ⁇ L of TMB (KPL) was added to each well, and the plate was placed at room temperature (20 ⁇ 5°C) in the dark for 5 min. 50 ⁇ L 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 binding ability of the test antibody to the target antigen human CD73-ECD protein.
  • HRP-labeled goat anti-mouse IgG secondary antibody purchased from Abcam, catalog number ab6789
  • the hybridoma cell lines obtained by screening were amplified in serum-containing complete medium, and the medium was changed to serum-free medium (SFM medium) by centrifugation to a cell density of 1-2 ⁇ 10 7 cells/mL.
  • SFM medium serum-free medium
  • the cells were cultured at 37°C under 5% CO 2 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 CD73-ECD protein monoclonal antibody named 3E10.
  • Example 2 ELISA method to determine the binding activity of 3E10 and human CD73
  • the residual droplets were patted dry on absorbent paper as much as possible, 100 ⁇ L of TMB (KPL) was added to each well, and the plate was placed at room temperature (20 ⁇ 5°C) in the dark for 5 min. 50 ⁇ L 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 binding ability of the test antibody to the target antigen human CD73.
  • TMB TMB
  • Example 3 Mouse antibody 3E10 inhibits CD73 enzyme activity in NCI-H1975 cells
  • CD73 expressed by tumor cells has enzymatic activity and can decompose AMP into Ado.
  • AMP can inhibit the chemiluminescent reaction caused by ATP, but Ado does not have this effect. Therefore, the activity of CD73 can be detected by detecting the consumption of AMP, thereby detecting the inhibitory effect of monoclonal antibodies on the enzymatic activity of CD73.
  • the experimental results are shown in Figure 2.
  • the IC 50 of antibody 3E10 in inhibiting CD73 enzyme activity of tumor cells NCI-H1975 is 39.89 ng/mL, i.e. 0.27 nM.
  • the experimental method was similar to that of Example 2, except that the human CD73-ECD was replaced with mouse CD73-ECD (amino acid sequence as shown in SEQ ID NO: 2) and cynomolgus monkey CD73-ECD (amino acid sequence as shown in SEQ ID NO: 3), both purchased from Beijing Biopsies Co., Ltd.
  • the binding ability of antibody 3E10 to mouse and cynomolgus monkey CD73 antigens was determined.
  • antibody 3E10 can bind to cynomolgus monkey CD73 protein well, with an EC 50 of 33.63 ng/mL, i.e., 0.22 nM, which is comparable to the binding of human CD73 (EC 50 is 0.20 nM); however, monoclonal antibody 3E10 does not bind to mouse CD73.
  • the heavy chain variable region and light chain variable region of hybridoma 3E10 were obtained by molecular biology related methods, and chimeric antibodies were further constructed.
  • RNA of 3E10 hybridoma cells was extracted by Trizol and reverse transcribed by mRNA to obtain cDNA. Then, cDNA was used as a template to perform PCR with degenerate primers of the heavy chain and light chain of mouse antibodies (Antibody Engineering Volume 1, Edited by Roland Kontermann and Stefan Dübel, the sequence of the combined primers is from page 323). The obtained PCR products were sequenced and analyzed by kabat database to determine that the obtained sequences were the variable region sequences of mouse antibodies.
  • the relevant sequence information is as follows:
  • the 3E10 heavy chain variable region gene sequence is 366bp in length, encoding 122 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO: 4, and the amino acid sequence is shown in SEQ ID NO: 5.
  • the 3E10 light chain variable region gene sequence is 333bp in length, encoding 111 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO: 6, and the amino acid sequence is shown in SEQ ID NO: 7.
  • the amino acid sequences of the variable regions of the light and heavy chains of the 3E10 antibody were analyzed, and the three antigen complementary determining regions (CDRs) and four framework regions (FRs) of the mouse antibody were determined according to the Kabat rule.
  • the amino acid sequences of the heavy chain complementary determining regions of 3E10 are HCDR1: GYSFTSYW (SEQ ID NO: 10), HCDR2: IYPGNSDT (SEQ ID NO: 11), HCDR3: TREGSKGYDVWYFAV (SEQ ID NO: 12, and the amino acid sequences of the light chain complementary determining regions are LCDR1: KSVSTSGYSY (SEQ ID NO: 13), LCDR2: LASNLES (SEQ ID NO: 14), and LCDR3: QHSRELPFT (SEQ ID NO: 15).
  • the humanized templates that best matched the FR regions of the above-mentioned mouse antibodies were selected from the Germline database.
  • the CDR region of the mouse antibody was then transplanted onto the selected humanized template to replace the CDR region of the human template.
  • the heavy chain variable region was then recombined with the human IgG1 constant region (SEQ ID NO: 8), and the light chain variable region was recombined with the human kappa chain constant region (SEQ ID NO: 9).
  • the embedded residues, the residues that directly interacted with the CDR region, and the residues that had an important influence on the conformation of the VL and VH of each antibody were back-mutated, and finally the humanized antibody hu3E10 was obtained.
  • the humanized antibody hu3E10 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 17, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain is shown in SEQ ID NO: 19).
  • the heavy and light chains of the humanized antibody hu3E10 were constructed separately and inserted into the pcDNA3.4 expression vector, which was transfected into Expi-293F cells.
  • the humanized antibody hu3E10 was obtained by Protein G purification, and the molecular weight of each antibody was confirmed to be correct and the purity was >95% by SDS-PAGE electrophoresis and SEC-HPLC.
  • Example 6 ELISA method to determine the binding activity of humanized antibody hu3E10 and human CD73
  • the experimental method refers to Example 2, and the binding activity of humanized antibody hu3E10 and human CD73 is determined.
  • the experimental results are shown in Figure 4.
  • the experimental results are shown in Figure 4.
  • the EC 50 of humanized antibody hu3E10 binding to human CD73 is 5.44 ng/mL, i.e., 0.04 nM, which is significantly lower than that of antibody 3E10 (whose EC 50 is 29.83 ng/mL, i.e., 0.20 nM, as shown in Example 2). This indicates that humanized antibody hu3E10 has good binding activity to human CD73.
  • Example 7 Determination of the inhibitory activity of humanized antibody hu3E10 on tumor cell membrane CD73 enzyme
  • the experimental method refers to Example 3, and the effect of humanized antibody hu3E10 in inhibiting the enzyme activity of tumor cell membrane CD73 is determined.
  • the experimental results are shown in Figure 5.
  • humanized antibody hu3E10 can effectively inhibit the enzymatic activity of tumor cell CD73, with an IC 50 of 27.81 ng/mL, i.e., 0.19 nM, which is higher than the mouse monoclonal antibody 3E10 (IC 50 0.27 nM). This indicates that humanized antibody hu3E10 inhibits the enzymatic activity of tumor cell membrane CD73.
  • Human IL15Rsushi (amino acid sequence SEQ ID NO: 20) and human IL15 (amino acid sequence SEQ ID NO: 21) were linked via (GGGGS) 6 to form a single-chain IL15, i.e., IL15sc, whose amino acid sequence is shown in SEQ ID NO: 22.
  • Heavy chain of SPGL003 The heavy chain sequence of hu3E10 (amino acid sequence as shown in SEQ ID NO: 17) and the human IL15sc sequence were spliced through (GGGGS) 3 to form the heavy chain of SPGL003 (amino acid sequence as shown in SEQ ID NO: 23);
  • the heavy chain of SPGL004 the hu3E10 heavy chain VH sequence (amino acid sequence as shown in SEQ ID NO: 16) is connected to the mutated human IgG1 constant region (amino acid sequence as shown in SEQ ID NO: 24) to form the hu3E10mu heavy chain (amino acid sequence as shown in SEQ ID NO: 25); the hu3E10mu heavy chain and the human IL15sc sequence are spliced through (GGGGS) 3 to form the heavy chain of SPGL004 (amino acid sequence as shown in SEQ ID NO: 26).
  • the light chain sequence is the same as that of hu3E10 (amino acid sequence is shown in SEQ ID NO: 19).
  • the heavy chain and light chain of SPGL003 (or the heavy chain and light chain of SPGL004) were co-transfected into Expi-293F cells, and the multifunctional recombinant antibody SPGL003 (or multifunctional recombinant antibody SPGL004) was purified by Protein G.
  • the molecular weight of each expressed antibody was confirmed to be correct by SDS-PAGE electrophoresis and SEC-HPLC, and the antibody purity was >95%.
  • the prepared multifunctional antibody was quantified and aliquoted, and stored at -80°C for later use.
  • the experimental method refers to Example 2, and the binding activity of the multifunctional recombinant antibodies SPGL003 and SPGL004 to human CD73-ECD was determined, and the experimental results are shown in Figure 6.
  • the EC50 of SPGL004 and SPGL003 binding to human CD73-ECD are 31.42 ng/mL and 34.26 ng/mL, i.e., 0.16 nM and 0.18 nM, respectively, which are consistent with hu3E10 (0.20 nM, as shown in Example 6), indicating that the formation of a bifunctional molecule between hu3E10 and IL15 does not affect its binding to CD73, and the mutation of IgG1 in the bifunctional molecule does not affect its binding to CD73.
  • diluted HRP-labeled SA (Pierce Company) was added and placed at 37°C for 30 minutes. After washing the plate three times with PBST, the residual droplets were patted dry on absorbent paper as much as possible, 100 ⁇ L of TMB (KPL Company) was added to each well, and the plate was placed at room temperature (20 ⁇ 5°C) away from light for 5 minutes. 50 ⁇ L of 2M H2SO4 stop solution was added to each well to terminate the substrate reaction, and the OD value was read at 450 nm by an enzyme reader to analyze the binding ability of the test antibody to human CD122/132.
  • Example 11 Determination of the inhibitory activity of tumor cell membrane CD73 enzyme by multifunctional recombinant antibodies SPGL003 and SPGL004
  • the experimental method refers to Example 3 to determine the effect of multifunctional recombinant antibodies SPGL004 and SPGL003 in inhibiting the enzymatic activity of tumor cell membrane CD73.
  • the experimental results are shown in FIG8 .
  • the IC50s of antibodies SPGL004 and SPGL003 for inhibiting the CD73 enzyme activity of tumor cells NCI-H1975 are 50.99 ng/mL and 53.08 ng/mL, i.e., 0.27 nM and 0.28 nM, respectively.
  • the enzyme inhibition activity is slightly weaker than that of hu3E10 ( EC50 is 0.19 nM).
  • Example 12 Multifunctional recombinant antibodies SPGL003 and SPGL004 stimulate CTLL2 cell proliferation
  • CTLL2 cells were diluted to 5 ⁇ 10 4 /mL with 1640 culture medium containing 10% FBS, and 100 ⁇ L/well was added to the cell culture plate.
  • IL2 was diluted to 30 ng/mL with 1640 culture medium containing 10% FBS, and then 3-fold dilutions were added to the culture plates containing CTLL2 cells;
  • SPGL004 and SPGL003 were diluted to 5000 ng/mL with 1640 culture medium containing 10% FBS, and then 3-fold dilutions were added to the culture plates containing CTLL2 cells;
  • the relative cell number of each well was determined by CCK8, and the EC 50 was calculated to determine the activity of the sample.
  • the experimental results are shown in Figure 9.
  • both multifunctional recombinant antibodies SPGL004 and SPGL003 can stimulate the proliferation of CTLL2 cells, with EC 50 values of 104.8 ng/mL and 99.3 ng/mL, i.e., 0.55 nM and 0.52 nM, respectively, indicating that SPGL004 has the same biological activity as SPGL003.
  • the EC 50 of IL2 is 0.75 ng/mL, i.e., 0.049 nM.
  • Example 13 The toxicity of the multifunctional recombinant antibody SPGL004 in mice is significantly lower than that of SPGL003
  • Antibody 0.5mg/kg 1.0mg/kg 2.0mg/kg 4.0mg/kg SPGL003 100% 50% 0% 0% SPGL004 100% 100% 100% 10%
  • Example 14 Multifunctional recombinant antibody SPGL004 inhibits the growth of mouse colorectal cancer cell MC38 transplanted tumors
  • mice The mouse colon cancer MC38 cells cultured in vitro were collected and the concentration of the cell suspension was adjusted to 1 ⁇ 10 7 cells/mL. Under sterile conditions, 100 ⁇ L of MC38 cell suspension was inoculated subcutaneously on the right rib of C57BL/6 mice. The diameter of the subcutaneous transplanted tumor of mice was measured with a vernier caliper. After the average tumor volume grew to 100-200 mm 3 , the animals were randomly divided into groups, 6 per group.
  • SPGL004 was administered at 1.0 mg/kg and 0.5 mg/kg
  • SPGL003 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.
  • the diameter of the transplanted tumor was measured 3 times a week, and the mice were weighed at the same time.
  • the calculation formula for tumor volume (TV) is (where a and b represent length and width, respectively):
  • the relative tumor volume (RTV) was calculated based on the measurement results.
  • the calculation formula was (where V0 is the tumor volume measured at d0 of group administration; Vt is the tumor volume at each measurement):
  • the evaluation index of antitumor activity is the relative tumor proliferation rate T/C (%), which is calculated as follows (where TRTV: RTV of the treatment group; CRTV: RTV of the negative control group):
  • T/C (%) (TRTV/CRTV) ⁇ 100;
  • TGI (%) 100-T/C (%).
  • SPGL003 exhibited lethal toxicity at a dose of 1.0 mg/kg, while SPGL004 exhibited lethal toxicity at a dose of 4.0 mg/kg, indicating that SPGL004 containing Fc mutations significantly improved its safety without affecting its anti-tumor activity in vivo.
  • Example 15 Multifunctional recombinant antibody SPGL004 inhibits the growth of human lung cancer cell NCI-H1975 transplanted tumors in nude mice
  • SPGL004 exhibited strong anti-tumor activity in human lung cancer cells.
  • the TGI of SPGL004 was 59.5%, 58.3% and 44.3%, respectively.
  • Example 16 Multifunctional recombinant antibody SPGL004 combined with HER2 monoclonal antibody inhibits the growth of human breast cancer cell JIMT-1 transplanted tumors in nude mice
  • the single-drug group HER2 monoclonal antibody (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 SPGL004, twice a week, by intraperitoneal injection, for a total of 6 times.
  • the diameter of the transplanted tumor was measured twice a week, and the weight of the mice was weighed at the same time. The rest is the same as Example 14. The results are shown in Figure 12.
  • the TGI of trastuzumab alone is 55.3%
  • the TGI of trastuzumab combined with 1.0 mg/kg and 0.3 mg/kg doses of SPGL004 are 90.3% and 71.7%, respectively, which are significantly higher than trastuzumab alone, indicating that SPGL004 and trastuzumab have a good synergistic anti-tumor effect.
  • Example 18 SPGL004 detects the expression of CD73 in various tumor cells
  • the binding ability of SPGL004 to various human tumor cells was determined by flow cytometry fluorescence activated cell sorting (FACS).
  • Various tumor cells were cultured in vitro, and 100 ⁇ L of SPGL004 at a concentration of 2000 ng/mL was used as the primary antibody and incubated with 1 ⁇ 10 5 tumor cells suspended in 100 ⁇ L RPMI-1640 serum-free medium (purchased from Gibco, Catalog No. 22400089) at 4°C for 1 h. The cells were washed twice with PBS to remove unbound SPGL004, and then the cells were incubated with 100 ⁇ L, 2 ⁇ g/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, and finally the cells were resuspended in 100 ⁇ L PBS.
  • the binding affinity of SPGL004 to various cells was determined by flow cytometry, and the obtained MFI values were compared with the MFI values of the control group (without SPGL004 incubation), and the ratio indicated the binding strength of SPGL004.
  • SPGL004 can bind to a variety of 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), indicating that the above-mentioned tumors may become indications for SPGL004.
  • human lung cancer cells H1975, Calu-3, A549, H322, H292
  • JIMT-1 human breast cancer cells
  • A498 human renal cancer cells
  • A431 human skin cancer cells
  • TSKgel G3000SWXL column (TSK) was used on HPLC Ultimate 3000 (Thermo) chromatograph.
  • the mobile phase was PBS (pH 7.4), the constant flow rate was 0.8 mL/min, and the sample volume was 100 ⁇ g/100 ⁇ L.
  • the 280 nM absorption peak integration method was used to calculate the content (%) of the target protein in the total protein to indicate the purity. The results are shown in Figures 15, 16 and 17.

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Abstract

提供一种能够特异性识别人 CD73 蛋白的单克隆抗体及其人源化的重组抗体，以及进行重构得到既能够识别人 CD73，又具有IL15 功能的多功能抗体，能够有效增强原抗体对肿瘤细胞的杀伤作用。还提供一种改构的特异性 CD73 单抗-IL15 双功能分子，通过引入突变的人IgG1恒定区得到，可降低抗体的毒性，提高安全性

Description

一种多功能重组抗体及其制备方法和应用 技术领域
本发明属于生物技术领域，涉及一种多功能重组抗体及其制备方法和应用，具体涉及一种能够既识别CD73又具有IL15功能的多功能重组抗体及其应用。
背景技术
抗体药物目前已经成为抗癌药物中的一个主要组成部分，但是在实体瘤方面，抗体药物的疗效还是有限的，因此寻找更多的新型实体瘤抗原很重要，但是也相对困难。对于实体瘤治疗而言，要克服提高的疗效，很重要的一个方面就是解除肿瘤微环境(TME)对免疫效应细胞的抑制作用。CD73是5-主核苷酸水解酶，可将细胞外腺苷一磷酸(AMP)水解为腺苷，而腺苷是一种功能强大的免疫抑制性分子，能够抑制CD8阳性T细胞的活化，进而帮助癌细胞逃逸T细胞的“追杀”，可以说它是癌症中免疫抑制微环境形成的关键成分之一。据报道，CD73在许多不同的癌症中均有表达，包括结肠癌、肺癌、胰腺癌、卵巢癌、膀胱癌、白血病、神经胶质瘤、胶质母细胞瘤、黑色素瘤、甲状腺癌、食道癌、前列腺癌和乳腺癌等。而且，CD73在癌症中的表达与细胞增殖、迁移、新血管形成、侵袭性、转移和患者更短的生存相关。也有人提出CD73活性可作为甲状腺乳头状癌的预后标志物。虽然已经表明CD73可调节肿瘤细胞与细胞和细胞与基质间的相互作用，但CD73的表达和活性也与T细胞反应降低有关，并且有人提出其涉及抗药性。因此，CD73可以直接和间接地调控癌症的发展，这一点体现了其作为新的治疗靶标的潜力，因此通过调控抑制其活性以及研究出相关治疗剂是非常令人期望的。
IL15是一种由多种细胞表达的细胞因子，这些细胞包括单核细胞、巨噬细胞、表皮细胞和成纤维细胞等，但T淋巴细胞则不表达。与其他多种细胞因子不同，IL15一般不分泌出细胞发挥作用，而是与IL15Rα组合后定位在特定的 细胞膜，从而刺激附近的效应细胞，主要是NK和CD8+T细胞。IL15与IL2关系密切，IL15与IL15Rα形成复合物后可以与IL2共用的β/γ受体结合，介导其生物学活性。在抗肿瘤作用方面，IL15比IL2的一个优势是IL15/Rα不会刺激Treg的增殖。目前，有多种IL15相关的分子在研发阶段用于治疗恶性肿瘤。其中进展最快的是ALT-803，其分子由IL15和IL15Rαsushi-hFc1形成的复合物。多个临床研究显示，ALT-803对包括黑色素瘤在内的多种肿瘤有效，但同时会出现严重的毒副作用，主要包括肝功能损害，低血压和发烧等。
开发一种能够杀伤多种癌症细胞且毒副作用小的药物有重要的意义。
发明内容
针对上述问题，本发明的目的是提供一种够特异性识别CD73蛋白的单克隆抗体、分泌所述单克隆抗体的杂交瘤细胞；以及由所述的单克隆抗体经过人源化改造得到重组抗体；以及既能够识别人CD73蛋白，又具有IL15功能的多功能重组抗体。
为实现上述目的，本发明采取的技术方案为：一种单克隆抗体，所述单克隆抗体能够识别人CD73-ECD蛋白；所述单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO：5所示，所述单克隆抗体的轻链可变区的氨基酸序列如SEQ ID NO：7所示。
本发明通过用重组表达的人CD73-ECD蛋白免疫小鼠制备了能够特异性识别人CD73蛋白的鼠源单克隆抗体，命名为3E10。通过生物学分析，得到所述鼠源单克隆抗体的重链可变区及轻链可变区的氨基酸序列。
进一步地，本发明还要求保护一种重组抗体，所述重组抗体由所述的单克隆抗体3E10经过人源化得到，所述重组抗体的重链可变区的氨基酸序列如SEQ ID NO：16所示，所述重组抗体的轻链可变区的氨基酸序列如SEQ ID NO：18所示。
作为本发明的优选实施方式，所述重组抗体的重链氨基酸序列如SEQ ID NO：17所示，所述重组抗体的轻链氨基酸序列如SEQ ID NO：19所示。
本发明发明人对所得到鼠源单克隆抗体3E10序列进行进一步分析，替换人源模板的CDR区，重链可变区再与人IgG1恒定区重组，轻链可变区与人的kappa链恒定区重组，同时以该抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对各抗体的VL和VH的构象有重要影响的残基进行回复突变，最终获得人源化重组抗体，命名为hu3E10。所述人源化重组抗体hu3E10重链可变区及轻链可变区的氨基酸序列、重链氨基酸序、轻链氨基酸序列如上述所示。
进一步地，本发明还要求保护一种多功能重组抗体，所述多功能重组抗体的重链包括识别人CD73的抗体功能区、人IgG1恒定区功能域、IL15功能区，以及用于连接各功能区的非功能性氨基酸片段；所述识别人CD73的抗体功能区的氨基酸序列包括所述重组抗体的重链可变区氨基酸序列。
所述IL15功能区可识别人IL2/IL15β/γ受体的功能域。
本发明发明人进一步改造了上述得到的人源化抗体，将抗体重链序列与具有IL15功能的序列相连接，进一步得到既能够识别人CD73蛋白，又具有IL15功能的重组抗体。
作为本发明的优选实施方式，所述人IgG1恒定区功能域为人IgG1恒定区，氨基酸序列如SEQ ID NO：8所示。
更优选地，所述人IgG1恒定区功能域为突变的人IgG1恒定区，所述突变的人IgG1恒定区氨基酸序列如SEQ ID NO：24所示。
本发明发明人通过使用含有突变的人IgG1恒定区的序列，得到的多功能重组抗体代谢周期更短，毒副作用更低，安全性更佳。
作为本发明的优选实施方式，所述人IL15功能区的氨基酸序列如SEQ ID NO：21所示。
更优选地，所述人IL15功能区的氨基酸序列如SEQ ID NO：22所示。
SEQ ID NO：22所示的优选人IL15的功能区的氨基酸序列为人IL15Rsushi 与人IL15通过(GGGGS) 6连接形成单链IL15，即IL15sc。
作为本发明的优选实施方式，所述多功能重组抗体中用于连接各功能区的非功能性氨基酸片段为GGGGS重复。
更优选地，所述GGGGS重复为(GGGGS) 3。
作为本发明的优选实施方式，所述多功能重组抗体的人IgG1恒定区功能域氨基酸序列如SEQ ID NO：8；所述多功能重组抗体的重链氨基酸序列如SEQ ID NO：23所示；所述多功能重组抗体轻链氨基酸序列如SEQ ID NO：19所示。
本发明制备了一种能够识别CD73并具有IL15功能的多功能抗体，命名为SPGL003。
更优选地，所述多功能重组抗体的人IgG1恒定区功能域为突变的人IgG1恒定区，所述突变的人IgG1恒定区氨基酸序列如SEQ ID NO：24所示；所述多功能重组抗体的重链氨基酸序列如SEQ ID NO：26所示；所述多功能重组抗体轻链氨基酸序列如SEQ ID NO：19所示。
利用所述优选的多功能重组抗体序列制备得到的多功能重组抗体包含突变的人IgG1恒定区的序列，命名为SPGL004；多功能重组抗体SPGL004不仅能够有效识别多种肿瘤细胞中的CD73，能有效抑制多种癌细胞生长，且半衰期更短，毒性更低，安全性好。
进一步地，本发明还要求保护编码所述单克隆抗体，或所述重组抗体，或所述多功能重组抗体的核苷酸序列。
根据提供所述单克隆抗体，或所述重组抗体，或所述多功能重组抗体的氨基酸序列可得到相应的编码基因的核苷酸序列。
作为本发明的优选实施方式，所述单克隆抗体的重链可变区核苷酸序列如SEQ ID NO：4所示，轻链可变区核苷酸序列如SEQ ID NO：6所示。
进一步地，本发明还要求保护包含所述的核苷酸序列的表达载体。
通过分子生物学的方法，可构建得到含有所述的核苷酸序列的表达载体。
进一步地，本发明还要求保护包含所述的表达载体的宿主细胞。
通过细胞生物学的方法，可利用所述表达载体构建得到能够有效表达所述抗体蛋白的宿主细胞。
进一步地，本发明还要求保护所述单克隆抗体，或所述重组抗体，或多功能重组抗体；或所述核苷酸序列，或所述表达载体，或所述宿主细胞在制备用于治疗肿瘤的生物制剂的应用。
进一步地，本发明还要求保护一种生物制剂，所述生物制剂包括所述单克隆抗体，或所述重组抗体，或所述多功能重组抗体；或所述核苷酸序列，或所述表达载体，或所述宿主细胞中的至少一种。
进一步地，本发明还提供了所述单克隆抗体或所述的重组抗体，或所述多功能重组抗体的制备方法，包括以下步骤：
(1)通过人工合成或分子生物学方法得到含所述单克隆抗体或重组抗体或多功能重组抗体的基因片段的表达载体；
(2)将所述表达载体转染至细胞进行蛋白表达；
(3)通过蛋白纯化得到所述单克隆抗体或重组抗体或多功能重组抗体。
通过分别构建含有将上述抗体的重链或轻链序列的表达载体，转染合适的细胞进行表达和纯化，即可获得相应的抗体。
作为本发明的优选实施方式，所述步骤(1)中，通过分子生物学制备含所述单克隆抗体或重组抗体或多功能重组抗体的基因片段的表达载体为：将所述抗体的重链或轻链核苷酸序列插入至pcDNA3.4表达载体。
作为本发明的优选实施方式，所述步骤(2)中，用于蛋白表达的细胞为Expi-293F细胞。
作为本发明的优选实施方式，所述步骤(3)中的蛋白纯化方法为通过Protein G纯化。
本发明与现有技术相比，具有如下技术效果：
本发明不仅制备了一种能够特异性识别人CD73蛋白的单克隆抗体，还提供了其人源化的重组抗体。并在此基础上，发明人进一步利用CD73特异性重组抗体制备了既能够识别人CD73，又具有IL15功能的多功能重组抗体，能够有效增强原抗体对肿瘤细胞的杀伤作用。进一步地，本发明还通过引入突变的人IgG1恒定区，得到一种改构的特异性CD73单抗-IL15双功能分子，该分子在保持体内抗肿瘤活性的同时，可有效缩短其代谢周期，从而降低了其在体内的毒副作用，大大提高了安全性。本发明的多功能重组抗体能够识别多种肿瘤细胞内的CD73，具有巨大的应用前景。
附图说明
图1抗体3E10与人CD73结合的结果。
图2抗体3E10抑制NCI-H1975细胞CD73酶活性的结果。
图3抗体3E10与小鼠、食蟹猴CD73抗原结合的结果。
图4人源抗体hu3E10与人CD73的结合的结果
图5人源抗体hu3E10抑制肿瘤细胞膜CD73酶活性的结果。
图6多功能重组抗体SPGL003、SPGL004与人CD73结合的结果。
图7各组抗体同时结合CD73和CD122/132的结果。
图8多功能重组抗体SPGL003、SPGL004抑制肿瘤细胞膜CD73酶活性。
图9多功能重组抗体SPGL003、SPGL004刺激CTLL2细胞增殖的结果；A为SPGL003、SPGL004的检测结果，B为IL2的检测结果。
图10各组小鼠结直肠癌细胞MC38移植瘤体积随时间变化的结果。
图11各组人肺癌细胞H1975移植瘤体积随时间变化的结果。
图12多功能重组抗体SPGL004联合HER2单抗抑制人乳腺癌细胞JIMT-1移植瘤的结果。
图13多功能重组抗体SPGL003和SPGL004在huFcRn转基因小鼠体内代谢情况。
图14流式检测SPGL004与多种肿瘤细胞CD73结合的结果。
图15多功能重组抗体SPGL004放置0周蛋白情况。
图16多功能重组抗体SPGL004 4℃放置5周蛋白情况。
图17多功能重组抗体SPGL004 37℃放置5周蛋白情况。
具体实施方式
为更好的说明本发明的目的、技术方案和优点，下面将结合附图和具体实施例对本发明作进一步说明。
实施例1单克隆抗体及杂交瘤的制备
1)抗原免疫小鼠：用重组表达的人CD73-ECD蛋白(购自北京百普赛斯公司，Acro BIOSYSTEMS，氨基酸序列如SEQ ID NO：1所示)。
第一天，将可溶性人CD73-ECD蛋白与弗氏完全佐剂(CFA，Sigma公司)乳化充分混匀后，对Balb/c小鼠进行皮下注射(50μg/鼠)；第14、36天，可溶性人CD73-ECD蛋白与弗氏不完全佐剂乳化(IFA，Sigma公司)充分混匀后，对Balb/c小鼠进行皮下加强免疫(50μg/鼠)；第50天，腹腔内注射人CD73--ECD蛋白50μg/鼠激发，注射3～4天后取小鼠脾脏进行融合实验。
2)杂交瘤的制备和筛选：
在小鼠末次冲击免疫后3～4天，使用常规的杂交瘤技术方案，将小鼠脾细胞与小鼠骨髓瘤细胞SP2/0通过PEG(PEG1450，Sigma公司)法融合。融合后的细胞在完全培养基中悬浮均匀，完全培养基即将RPMI1640和DMEM F12培养基1：1混匀后加入1％的谷氨酰胺(Gibco公司)，1％丙酮酸钠(Gibco公司)， 1％MEM-NEAA(最小基本培养基-非必需氨基酸溶液，Gibco公司)，1％青霉素-链霉素(Gibco公司)，50μM的β-巯基乙醇(Gibco公司)及20％FBS(Gibco公司)组成的培养基。按10 5个细胞/100μL/孔，分入96孔培养板中培养过夜。次日，每孔加入100μL含有2×HAT(Sigma公司)的完全培养基，使96孔板内培养液为200μL/孔(含1×HAT)。在7～12天后，收获上清液，通过间接酶联免疫吸附测定法(ELISA)筛选人CD73-ECD蛋白结合活性阳性的杂交瘤孔。
其中，间接酶联免疫吸附测定法筛选人CD73-ECD蛋白活性阳性的杂交瘤孔的方法如下：将重组人CD73-ECD蛋白以包被液(50mM的碳酸盐包被缓冲液，pH 9.6)稀释至1μg/mL，100μL/孔加入酶标板，4℃包被过夜。PBST洗板3次，加入200μL/孔封闭液(2％BSA-PBST)，37℃放置1h后PBST洗板1次待用。将收取的杂交瘤上清液依次加入封闭后的酶标板，100μL/孔，37℃放置1h。PBST洗板3次，加入HRP标记的羊抗小鼠IgG二抗(购自Abcam，货号ab6789)，37℃放置30min；PBST洗板5次后，在吸水纸上尽量拍干残留液滴，每孔加入100μL的TMB(KPL公司)，室温(20±5℃)避光放置5min；每孔加入50μL 2M H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析待测抗体与靶抗原人CD73-ECD蛋白结合能力。
在含血清完全培养基中扩增筛选获得的杂交瘤细胞株，离心换液至无血清培养液(SFM培养基)，使细胞密度为1～2×10 7个/mL，在5％CO 2，37℃条件下培养2周，离心获取培养上清，通过Protein G亲和层析进行纯化，获得鼠源抗人CD73-ECD蛋白单克隆抗体，命名为3E10。
实施例2 ELISA法测定3E10与人CD73的结合活性
实验步骤：预先包被人CD73，以包被液(50mM的碳酸盐包被缓冲液，pH9.6)稀释至5μg/mL包板，4℃过夜；再用5％的脱脂奶粉封闭，37℃，2小时。PBST洗板3次，将以1％BSA-PBST梯度稀释的待测抗体依次加入封闭后的酶标板，100μL/孔，37℃放置1h。PBST洗板3次，加入HRP标记的羊抗小鼠IgG二抗(购自Millipore,货号AP181P)，37℃放置30min；PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μL的TMB(KPL公司)，室温(20±5℃)避光放置5min；每孔加入50μL 2M H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析待测抗体与靶抗原人CD73的结合能力。
实验结果如图1。由图1可得，抗体3E10结合CD73-ECD的EC 50为29.83ng/mL，即0.20nM；说明抗体3E10与CD73-ECD有很好的结合活性。
实施例3鼠源抗体3E10抑制NCI-H1975细胞CD73酶活性
肿瘤细胞表达的CD73具有酶活性，可以将AMP分解为Ado。AMP可以抑制ATP引起的化学发光反应，但Ado则没有该作用。因此，可以通过检测AMP的消耗来检测CD73的活性，从而检测单抗对CD73酶活性的抑制作用。
实验步骤：人肿瘤细胞NCI-H1975以30000cells/孔接种96孔细胞培养板(Corning,Cat#3599)，于CO 2细胞培养箱培养过夜，次日，吸弃培养上清，加入待检单抗或对照单抗，50μL/孔，系列稀释(10000ng/mL起始，3倍比稀释共10个梯度，最低浓度至0.5ng/mL，)，37℃孵育30分钟，再加入50μL/孔不含血清的培养基配制的800μM AMP(Sigma,cat#A1752)溶液，37℃孵育3小时。吸25μL/孔细胞反应液入荧光检测板(White Opaque 96-well Microplate，PerkinElmer,Cat#6005290)，再加入25μL/孔不含血清培养基配制的80μM ATP溶液，加入50μL/孔CTG溶液(CellTiter Glo，Promega,cat#G7573)，室温孵育5分钟后用化学发光检测仪(BioTech公司)检测荧光值。
实验结果如图2。抗体3E10抑制肿瘤细胞NCI-H1975的CD73酶活性的IC 50为39.89ng/mL，即0.27nM。
实施例4抗体3E10与小鼠、食蟹猴CD73抗原的交叉活性测定
实验方法与实施例2相似，将其中的人CD73-ECD分别替换为小鼠CD73-ECD(氨基酸序列如SEQ ID NO：2所示)和食蟹猴CD73-ECD(氨基酸序列如SEQ ID NO：3所示)，均购自北京百普赛斯公司。测定抗体3E10与小鼠、食蟹猴CD73抗原的结合能力。
实验结果如图3。由图3可得，抗体3E10能很好的结合食蟹猴CD73蛋白，EC 50为33.63ng/mL，即0.22nM，与人CD73的结合相当(EC 50为0.20nM)；但单抗3E10不结合小鼠CD73。
实施例5人源化抗体hu3E10的制备
本实施例通过分子生物学的相关方法获取杂交瘤3E10的重链可变区和轻链可变区，并进一步构建嵌合抗体。
通过Trizol提取3E10杂交瘤细胞的RNA并进行mRNA反转录获取cDNA，随后以cDNA为模板，分别用鼠源抗体的重链和轻链简并引物(《Antibody Engineering》Volume 1，Edited by Roland Kontermann and Stefan Dübel，组合引物的序列来自第323页)进行PCR，对所获得的PCR产物进行测序并通过kabat数据库分析，确定所获得的序列为鼠源抗体的可变区序列。相关序列信息如下：
3E10重链可变区基因序列，全长为366bp，编码122个氨基酸残基，核苷酸序列如SEQ ID NO：4所示，氨基酸序列如SEQ ID NO：5所示；3E10轻链可变区基因序列全长333bp，编码111个氨基酸残基，核苷酸序列如SEQ ID NO：6所示，氨基酸序列如SEQ ID NO：7所示。
对3E10抗体轻链可变区和重链可变区的氨基酸序列进行分析，依据Kabat规则确定鼠源抗体的3个抗原互补决定区(CDR)和4个框架区(FR)。3E10重链互补决定区的氨基酸序列为HCDR1：GYSFTSYW(SEQ ID NO：10)、HCDR2：IYPGNSDT(SEQ ID NO：11)、HCDR3：TREGSKGYDVWYFAV(SEQ ID NO：12，轻链互补决定区的氨基酸序列为LCDR1：KSVSTSGYSY(SEQ ID NO：13)、LCDR2：LASNLES(SEQ ID NO：14)和LCDR3：QHSRELPFT(SEQ ID NO：15)。
在Germline数据库中选取与上述各鼠源抗体FR区匹配最好的人源化模板。然后将鼠源抗体的CDR区移植到所选择的人源化模板上，替换人源模板的CDR区，重链可变区再与人IgG1恒定区(SEQ ID NO：8)重组，轻链可变区与人的kappa链恒定区(SEQ ID NO：9)重组，同时以该抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对各抗体的VL和VH的构象有重要影响的残基进行回复突变，最终获得人源化抗体hu3E10。
所述人源化抗体hu3E10(重链可变区氨基酸序列如SEQ ID NO：16所示，重链氨基酸序列如SEQ ID NO：17所示，轻链可变区氨基酸序列如SEQ ID NO：18所示，轻链氨基酸序列如SEQ ID NO：19所示)。
分别构建人源化抗体hu3E10的重链和轻链至pcDNA3.4表达载体，转染Expi-293F细胞，通过Protein G纯化获得人源化抗体hu3E10，并通过SDS-PAGE电泳及SEC-HPLC确定各抗体分子量大小正确及纯度>95％。
实施例6 ELISA法测定人源化抗体hu3E10与人CD73的结合活性
实验方法参考实施例2，测定人源化抗体hu3E10与人CD73的结合活性，实验结果如图4所示。
实验结果如图4所示，人源化抗体hu3E10与人CD73结合的EC 50为5.44ng/mL，即0.04nM，明显低于抗体3E10(其EC 50为29.83ng/mL，即0.20nM，如实施例2所示)。说明人源化抗体hu3E10与人CD73具有良好的结合活性。
实施例7人源化抗体hu3E10抑制肿瘤细胞膜CD73酶活性测定
实验方法参考实施例3，测定人源化抗体hu3E10抑制肿瘤细胞膜CD73酶活性的效果，实验结果如图5所示。
由图5可得，人源化抗体hu3E10可以高效抑制肿瘤细胞CD73的酶活性，IC 50为27.81ng/mL，即0.19nM，活性高于小鼠源单抗3E10(IC 50 0.27nM)。说明人源化抗体hu3E10抑制肿瘤细胞膜CD73酶活性。
实施例8多功能重组抗体的制备
1.人IL15的功能区：将人IL15Rsushi(其氨基酸序列SEQ ID NO：20)与人IL15(其氨基酸序列SEQ ID NO：21)通过(GGGGS) 6连接形成单链IL15，即IL15sc，其氨基酸序列SEQ ID NO：22所示。
2.识别人CD73的抗体功能区：
①SPGL003的重链：将hu3E10重链序列(氨基酸序列如SEQ ID NO：17所示)与人IL15sc序列通过(GGGGS) 3拼接，形成SPGL003的重链(氨基酸序列如SEQ ID NO：23所示)；
②SPGL004的重链：hu3E10重链VH序列(氨基酸序列如SEQ ID NO： 16所示)与突变的人IgG1恒定区(氨基酸序列如SEQ ID NO：24所示)连接，形成hu3E10mu重链(氨基酸序列如SEQ ID NO：25所示)；hu3E10mu重链与人IL15sc序列通过(GGGGS) 3拼接，形成SPGL004的重链(氨基酸序列如SEQ ID NO：26所示)。
③轻链同hu3E10的轻链序列(氨基酸序列如SEQ ID NO：19所示)。
3.多功能重组抗体的制备
将SPGL003的重链、轻链(或SPGL004的重链、轻链)共转染转染Expi-293F细胞，通过Protein G纯化可获得多功能重组抗体SPGL003(或多功能重组抗体SPGL004)。通过SDS-PAGE电泳及SEC-HPLC确定所表达各抗体分子量正确，且抗体纯度>95％。制备好的多功能抗体定量后分装，并冻存于-80℃备用。
实施例9多功能重组抗体SPGL004结合CD73-ECD
实验方法参考实施例2，测定多功能重组抗体SPGL003、SPGL004分别与人CD73-ECD的结合活性，实验结果如图6所示。
由如图6可得，SPGL004和SPGL003结合人CD73-ECD的EC 50分别为31.42ng/mL和34.26ng/mL，即0.16nM和0.18nM，与hu3E10一致(0.20nM，如实施例6所示),说明hu3E10与IL15形成双功能分子后不影响其与CD73的结合，而且双功能分子中IgG1的突变也不影响其与CD73的结合。
实施例10多功能重组抗体SPGL004同时结合CD73-ECD和CD122/132
实验方法：预先包被人CD73-ECD蛋白，以包被液(50mM的碳酸盐包被缓冲液，pH 9.6)稀释至2μg/mL包板，4℃，过夜；再用5％的脱脂奶粉封闭，37℃，2小时；PBST洗板3次，将以1％BSA-PBST梯度稀释待测抗体至1μg/mL，加入封闭后的酶标板，100μL/孔，37℃放置1h。PBST洗板3次，加入梯度稀释的生物素标记的CD122/132(购自北京百普赛斯公司)，37℃，1小时，PBST洗板3次后加入稀释的HRP标记的SA(Pierce公司)，37℃放置30min；PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μL的TMB(KPL公司)，室温(20±5℃)避光放置5min；每孔加入50μL 2M H 2SO 4终止液终止 底物反应，酶标仪450nm处读取OD值，分析待测抗体与人CD122/132的结合能力。
实验结果如图7。如图7可得，在本实验条件下，抗体SPGL004和SPGL003结合人CD122/132蛋白的EC 50分别为15.85ng/mL和16.84ng/mL，即0.08nM和0.09nM，说明多功能重组抗体SPGL004和SPGL003均有同时结合CD73和CD122/132的良好活性；相对的，对照组的hu3E10抗体虽然能够结合CD73，但不能结合CD122/132。
实施例11多功能重组抗体SPGL003、SPGL004抑制肿瘤细胞膜CD73酶活性测定
实验方法参考实施例3，测定多功能重组抗体SPGL004和SPGL003抑制肿瘤细胞膜CD73酶活性的效果。实验结果如图8所示。
由图8可得，抗体SPGL004和SPGL003抑制肿瘤细胞NCI-H1975的CD73酶活性的IC 50分别为50.99ng/mL和53.08ng/mL，即0.27nM和0.28nM，酶抑制活性稍弱于hu3E10(EC 50为0.19nM)。
实施例12多功能重组抗体SPGL003、SPGL004刺激CTLL2细胞增殖
实验方法：CTLL2细胞以含10％FBS的1640培养液稀释至5×10 4/mL，100μL/孔加入细胞培养板。将IL2以含10％FBS的1640培养液稀释至30ng/mL，后3倍比稀释共8个梯度后分别加入上述含CTLL2细胞的培养板；将SPGL004和SPGL003以含10％FBS的1640培养液稀释至5000ng/mL，后3倍比稀释共8个梯度后分别加入上述含CTLL2细胞的培养板；CO 2细胞培养箱培养72小时后，以CCK8测定各孔的相对细胞数，并计算EC 50，判定样品的活性。实验结果如图9所示。
由图9A可得，多功能重组抗体SPGL004和SPGL003均可以刺激CTLL2细胞的增殖，EC 50分别为104.8ng/mL和99.3ng/mL，即0.55nM和0.52nM，说明SPGL004具有与SPGL003一致的生物学活性。作为对照，由图9B可得，IL2的EC 50为0.75ng/mL，即0.049nM。
实施例13多功能重组抗体SPGL004较SPGL003在小鼠体内的毒性明显下降
实验方法：多功能重组抗体SPGL004和SPGL003于第1天、第3天和第5天，腹腔注射C57BL/6小鼠(维通利华公司)共3次，注射体积为0.2mL/次，SPGL004和SPGL003均按0.5mg/kg、1mg/kg、2mg/kg和4mg/kg剂量给药，每天观察实验小鼠的死亡情况，对照组给药同体积的PBS。结果如表1所示。
表1各剂量组实验动物存活率
抗体 0.5mg/kg 1.0mg/kg 2.0mg/kg 4.0mg/kg
SPGL003 100％ 50％ 0％ 0％
SPGL004 100％ 100％ 100％ 10％
如表1所示，在实验第7天，SPGL003样品4.0mg/kg组和2.0mg/kg组所有实验动物死亡(存活率0％)；1.0mg/kg组仍然有50％实验动物死亡，0.5mg/kg组则无实验动物死亡；SPGL004样品4.0mg/kg组出现90％实验动物死亡，但其余剂量组均无实验动物死亡。这些结果说明，Fc突变后，SPGL004对实验小鼠的毒性明显下降。
实施例14多功能重组抗体SPGL004抑制小鼠结直肠癌细胞MC38移植瘤生长
实验方法：收集体外培养的小鼠结肠癌MC38细胞，将细胞悬液浓度调整为1×10 7cells/mL。在无菌条件下，接种100μL MC38细胞悬液于C57BL/6小鼠右侧肋部皮下。小鼠皮下移植瘤用游标卡尺测量移植瘤直径，待平均肿瘤体积生长至100-200mm 3后将动物随机分组，6只/组。SPGL004按1.0mg/kg、0.5mg/kg给药，SPGL003按0.5mg/kg、0.25mg/kg给药，对照组给等量PBS，每周腹腔注射给药3次，给药体积0.2mL/次，连续给药2周。整个实验过程中，每周3次测量移植瘤直径，同时称小鼠体重。肿瘤体积(tumor volume，TV)的计算公式为(其中，a、b分别表示长、宽)：
TV＝1/2×a×b2。
根据测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为(其中，V 0为分组给药d0时测量所得肿瘤体积；V t为每一次测量时的肿瘤体积)：
RTV＝V t/V 0。
抗肿瘤活性的评价指标为相对肿瘤增殖率T/C(％)，计算公式如下(其中，TRTV：治疗组RTV；CRTV：阴性对照组RTV)：
T/C(％)＝(TRTV/CRTV)×100；
抑瘤率TGI(％)＝100-T/C(％)。
测定结果如图10所示。由图10可得，SPGL004和SPGL003均体现了较强的抗肿瘤活性，SPGL003在0.5mg/kg和0.25mg/kg剂量下，TGI分别为60.6％和22.9％，SPGL004在1.0和0.5mg/kg剂量下，TGI分别为72.2％、46.1％。
参考实施例13，SPGL003在1.0mg/kg剂量下体现致死毒性，SPGL004在4.0mg/kg剂量下才体现致死毒性，说明含有Fc突变的SPGL004在不影响体内抗肿瘤活性的同时，安全性明显提高。
实施例15多功能重组抗体SPGL004抑制人肺癌细胞NCI-H1975移植瘤在裸小鼠体内的生长
实验方法：收集体外培养的人肺癌NCI-H1975细胞，将细胞悬液浓度调整为8×10 7cells/mL。在无菌条件下，接种100μL细胞悬液于裸小鼠右侧肋部皮下。待肿瘤细胞在小鼠皮下形成实体肿瘤，用游标卡尺测量移植瘤直径，待平均肿瘤体积生长至100-200mm 3后将动物随机分组。多功能重组抗体SPGL004按1.0mg/kg、0.3mg/kg和0.1mg/kg剂量，每周腹腔给药3次，共给药6次。整个实验过程中，每周2次测量移植瘤直径，同时称量小鼠体重。其余同实施例14。结果如图11所示。
由图11可得，SPGL004在人肺癌细胞中体现了较强的抗肿瘤活性。SPGL004 在1.0mg/kg、0.3mg/kg和0.1mg/kg剂量下，TGI分别为59.5％、58.3％和44.3％。
实施例16多功能重组抗体SPGL004联合HER2单抗抑制人乳腺癌细胞JIMT-1移植瘤在裸小鼠体内的生长
实验方法：收集体外培养的人乳腺癌JIMT-1细胞，将细胞悬液浓度调整为8×10 7cells/mL。在无菌条件下，接种100μL细胞悬液于裸小鼠右侧肋部皮下。待肿瘤细胞在小鼠皮下形成实体肿瘤，用游标卡尺测量移植瘤直径，待平均肿瘤体积生长至50-100mm 3后将动物随机分组。单药组HER2单抗(曲妥珠)按照20mg/kg剂量，联合组由固定剂量20mg/kg曲妥珠分别联合1.0mg/kg和0.3mg/kg的SPGL004给药，每周2次，腹腔注射给药，共6次。整个实验过程中，每周2次测量移植瘤直径，同时称量小鼠体重。其余同实施例14。结果如图12所示。
由图12可得，曲妥珠单药的TGI为55.3％，曲妥珠联合1.0mg/kg、0.3mg/kg剂量SPGL004的TGI分别为90.3％和71.7％，明显高于曲妥珠单药，说明SPGL004与曲妥珠具有良好的协同抗肿瘤作用。
实施例17多功能重组抗体SPGL003和SPGL004在huFcRn转基因小鼠体内代谢情况
实验方法：采用人FcRn转基因小鼠测定SPGL003和SPGL004的药代动力学。8只小鼠分两组，分别按1mg/kg剂量，腹腔注射SPGL003和SPGL004，2小时、6小时、24小时、48小时和72小时后取血，获取血清。血清作适当稀释后，采用ELISA法测定血药浓度，方法基本同实施例11。结果如图13所示。
由图13可得，Fc突变的SPGL004较Fc野生型的SPGL003在人FcRn转基因小鼠体内的代谢明显加快，半衰期分别为9小时和22小时，相差显著。
实施例18SPGL004检测多种肿瘤细胞CD73的表达
通过流式细胞荧光分选技术(Fluorescence activated Cell Sorting，FACS)的方法测定SPGL004对多种人肿瘤细胞的结合力。
体外培养多种肿瘤细胞，将100μL浓度为2000ng/mL的SPGL004作为一抗，分别与悬浮于100μL RPMI-1640无血清培养基(购自Gibco公司，货号22400089)中的1×10 5个肿瘤细胞于4℃孵育1h，PBS洗涤细胞两次以去除未结合的SPGL004，再将细胞与100μL、2μg/mL、Alexa Fluor 488标记的抗人IgG荧光二抗(购自Thermo，invitrogen公司，货号A11013)于4℃孵育30min，PBS洗涤细胞两次以去除未结合的二抗，最后将细胞重悬在100μL PBS中，通过流式细胞仪测定SPGL004对各种细胞的结合亲和力，所得MFI数值，与对照组(未孵育SPGL004)MFI值比较，比值表示SPGL004的结合强度。
由图14可得，SPGL004可以结合多种人肺癌细胞(H1975、Calu-3、A549、H322、H292)，结合人乳腺癌细胞(JIMT-1)，结合人肾癌细胞(A498)，结合人皮肤癌细胞(A431)，表明上述多种肿瘤可能成为SPGL004的适应症。
实施例19 SPGL004稳定性检测
采用分子筛高效液相色谱(SEC-HPLC)检测SPGL004放置不同时间后纯度的变化，考察SPGL004的稳定性。
实验方法：采用TSKgel G3000SWXL色谱柱(TSK公司)，在HPLC Ultimate3000(Thermo公司)色谱仪上进行。检测流动相为PBS(pH 7.4)，恒定流速0.8mL/min，上样量为100μg/100μL。采用280nM吸收峰积分法计算目标蛋白在总蛋白中的含量(％)来表示纯度。结果如图15、图16和图17所示。
由图15、图16和图17可得，SPGL004在PBS溶液中，0周是纯度为89.7％，4℃放置5周后，纯度为87.8％，变化率<3％；37℃放置5周后，纯度为87.6％，变化率<3％，表明SPGL004在PBS缓冲液中，4℃和37℃条件下均保持稳定，SPGL004具有良好的稳定性。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案，而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细地说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。

Claims (10)

  1. 一种单克隆抗体，其特征在于，所述单克隆抗体能够识别人CD73-ECD蛋白；所述单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO：5所示，所述单克隆抗体的轻链可变区的氨基酸序列如SEQ ID NO：7所示。
  2. 一种重组抗体，其特征在于，所述重组抗体由权利要求1所述的单克隆抗体经过人源化得到，所述重组抗体的重链可变区的氨基酸序列如SEQ ID NO：16所示，所述重组抗体的轻链可变区的氨基酸序列如SEQ ID NO：18所示。
  3. 一种多功能重组抗体，其特征在于，所述多功能重组抗体的重链包括识别人CD73的抗体功能区、人IgG1恒定区功能域、人IL15的功能区，以及用于连接各功能区的非功能性氨基酸片段；所述识别人CD73的抗体功能区的氨基酸序列包括如权利要求2所述重组抗体的重链可变区氨基酸序列。
  4. 如权利要求3所述的多功能重组抗体，其特征在于，所述人IgG1恒定区功能域为突变的人IgG1恒定区，所述突变的人IgG1恒定区氨基酸序列如SEQ ID NO：24所示；所述多功能重组抗体重链氨基酸序列如SEQ ID NO：26所示；所述多功能重组抗体轻链氨基酸序列如SEQ ID NO：19所示。
  5. 编码如权利要求1所述单克隆抗体，或如权利要求2所述重组抗体，或如权利要求3或4所述多功能重组抗体的核苷酸序列。
  6. 一种表达载体，其特征在于，所述表达载体包含如权利要求5所述的核苷酸序列。
  7. 一种宿主细胞，其特征在于，所述宿主细胞包含如权利要求6所述的表达载体。
  8. 如权利要求1所述单克隆抗体，或如权利要求2所述重组抗体，或如权利要求3或4所述多功能重组抗体；或如权利要求5所述的核苷酸序列，或如权利要求6所述的表达载体，或如权利要求7所述的宿主细胞在制备用于治疗肿瘤的生物制剂的应用。
  9. 一种生物制剂，其特征在于，所述生物制剂包括如权利要求1所述单克隆抗体，或如权利要求2所述重组抗体，或如权利要求3或4所述多功能重组抗体；或如权利要求5所述的核苷酸序列，或如权利要求6所述的表达载体，或如权利要求7所述的宿主细胞中的至少一种。
  10. 如权利要求1所述单克隆抗体或如权利要求2所述的重组抗体，或如权利要求3或4所述多功能重组抗体的制备方法，其特征在于，包括以下步骤：
    (1)通过人工合成或分子生物学方法得到含所述单克隆抗体或重组抗体或多功能重组抗体的基因片段的表达载体；
    (2)将所述表达载体转染细胞进行蛋白表达；
    (3)通过蛋白纯化得到所述单克隆抗体或重组抗体或多功能重组抗体。
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