WO2024125352A1 - 一种靶向cd47的单克隆抗体及其应用 - Google Patents

一种靶向cd47的单克隆抗体及其应用 Download PDF

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WO2024125352A1
WO2024125352A1 PCT/CN2023/136641 CN2023136641W WO2024125352A1 WO 2024125352 A1 WO2024125352 A1 WO 2024125352A1 CN 2023136641 W CN2023136641 W CN 2023136641W WO 2024125352 A1 WO2024125352 A1 WO 2024125352A1
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monoclonal antibody
amino acid
antibody
acid sequence
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陈艺丽
谭杰
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上海迈石生物技术有限公司
达石药业(广东)有限公司
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Publication of WO2024125352A1 publication Critical patent/WO2024125352A1/zh

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  • the present invention relates to the technical field of antibody drugs, and in particular to a monoclonal antibody targeting a new antigenic epitope of a CD47 molecule, a method for preparing the antibody, and uses of the antibody.
  • Tumor immunotherapy mainly relies on autoimmunity to kill and eliminate tumor cells by regulating the human immune system and tumor microenvironment.
  • Current tumor immunotherapy is mainly based on restoring the function of T cells, that is, the function of the adaptive immune system, to achieve anti-tumor effects, such as immune checkpoint inhibitors represented by PD-1 and PD-L1 antibodies.
  • immune checkpoint inhibitors represented by PD-1 and PD-L1 antibodies.
  • the immune system and innate immunity can also play a very important role in tumor immunotherapy.
  • CD47 also known as integrin-associated protein (IAP)
  • IAP integrin-associated protein
  • SIRP ⁇ is a ligand of CD47, which is mainly expressed on the surface of myeloid cells such as macrophages.
  • tumor cells can transmit the "don't eat me” signal to macrophages to achieve immune escape.
  • this signaling pathway is blocked with CD47 antibodies, the phagocytic effect of macrophages on tumor cells can be restored, realizing the anti-tumor effect of the innate immune system.
  • Mobilizing macrophages to exert anti-tumor effects by blocking CD47 signals is considered to be an effective supplement to PD-1 antagonists and is expected to become a competitive hotspot in the "post-PD-1 era”.
  • CD47 antibodies have achieved remarkable results in many preclinical studies of tumor treatment.
  • AML acute myeloid leukemia
  • the leukemia cells in the bone marrow and peripheral blood of the CD47 antibody-treated group were significantly reduced, and the mice survived for a long time.
  • the application of CD47 antibody treatment can significantly reduce the tumor volume of mice and improve the tumor The survival rate of transplanted mice; after the combined use of CD47 antibody and rituximab injection, it can even eliminate tumor cells in tumor-bearing mice and achieve a complete cure.
  • CD47 antibodies can significantly inhibit tumor growth and metastasis and prolong the survival of tumor-bearing mice.
  • CD47-SIRP ⁇ signaling pathway has shown good drug development prospects for the CD47 target, but the clinical results of antibodies targeting CD47 have also shown some problems such as insufficient single-drug efficacy and related blood toxicity. Therefore, it is necessary to select CD47 monoclonal antibodies with strong specificity, wide adaptability, excellent efficacy and high safety to overcome some of the problems currently faced by such antibodies in clinical practice.
  • the technical purpose of the present invention is to provide a monoclonal antibody or an immunologically active fragment thereof targeting CD47, which has the characteristics of strong efficacy and low toxicity and is expected to play a role in the treatment of CD47-positive tumors and liver fibrosis and other indications.
  • Another technical purpose of the present invention is to provide a new antigenic epitope of CD47 molecule for use in screening monoclonal antibodies targeting CD47.
  • the present invention provides a monoclonal antibody or an immunologically active fragment thereof targeting CD47, comprising: a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementary determining regions: HCDR1 having an amino acid sequence of SEQ ID NO:5, HCDR2 having an amino acid sequence of SEQ ID NO:6, and HCDR3 having an amino acid sequence of SEQ ID NO:7, or the three complementary determining regions of the heavy chain variable region are mutant sequences having an overall identity of more than 85% compared with the aforementioned amino acid sequences SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7;
  • the light chain variable region includes three complementarity determining regions: LCDR1 with an amino acid sequence of SEQ ID NO:8, LCDR2 with an amino acid sequence of SEQ ID NO:9 and LCDR3 with an amino acid sequence of SEQ ID NO:10, or the three complementarity determining regions of the light chain variable region are mutant sequences that have an overall consistency of more than 85% compared with the aforementioned amino acid sequences SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.
  • the molecular subtype of the monoclonal antibody is human IgG4.
  • the monoclonal antibody is a humanized monoclonal antibody.
  • the heavy chain amino acid sequence of the monoclonal antibody is shown as SEQ ID NO:1
  • the light chain amino acid sequence is shown as SEQ ID NO:2.
  • the present invention provides a polynucleotide encoding the above monoclonal antibody.
  • the polynucleotide sequence encoding the heavy chain of the monoclonal antibody is: SEQ ID NO: 3; and the polynucleotide sequence encoding the light chain of the monoclonal antibody is: It is: SEQ ID NO:4.
  • the polynucleotide sequence used to encode the heavy chain of the monoclonal antibody is a synonymous nucleotide sequence of SEQ ID NO:3
  • the polynucleotide sequence used to encode the light chain of the monoclonal antibody is a synonymous nucleotide sequence of SEQ ID NO:4.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the above monoclonal antibody or an immunologically active fragment thereof, and a pharmaceutically acceptable carrier.
  • the present invention provides use of the above monoclonal antibody or its immunologically active fragment or the above pharmaceutical composition in preparing a drug.
  • the drug is a tumor treatment drug or a liver fibrosis treatment drug.
  • the tumor is a CD47 positive tumor, including but not limited to ovarian cancer, colon cancer, breast cancer, lung cancer, head and neck tumors, bladder cancer, colorectal cancer, pancreatic cancer, non-Hodgkin's lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granulocytic lymphocytic leukemia, adult T-cell leukemia, multiple myeloma, (malignant) melanoma, leiomyoma, leiomyosarcoma, glioma, glioblastoma, myeloma, monocytic leukemia, B cell-derived leukemia, T cell-derived leukemia, B cell-derived lymphoma, T cell-derived lymphoma, endo
  • the liver fibrosis is liver fibrosis caused by non-alcoholic steatohepatitis (NASH).
  • NASH non-alcoholic steatohepatitis
  • the present invention provides a biological molecule that can specifically bind to a conformational epitope of CD47, wherein the epitope is composed of all or part of the amino acid residues G44, R45, D46, I47, T49, D51, A53, L54, K56, S57, T58, V59, P60, T61 and D62 on the CD47 protein.
  • the monoclonal antibody of the present invention shows good binding and blocking activity with CD47, and can inhibit tumor growth in animal models. In cynomolgus monkeys, the monoclonal antibody of the present invention does not induce obvious decrease in hemoglobin or platelets, showing good safety.
  • different forms of genetically engineered antibodies can be transformed and produced in eukaryotic cells and any expression system, and can be used clinically for the treatment of various CD47-positive tumors, liver fibrosis and other related diseases.
  • FIG1 Schematic diagram of the structure of the monoclonal antibody (DS003) targeting CD47 of the present application.
  • Figure 2 Reducing SDS-PAGE electrophoresis of CD47 antibody molecules.
  • FIG. 3 Antigen-antibody binding capacity determination.
  • Figure 4 Antibody blocking ability assay.
  • Figure 5 In vitro phagocytosis assay.
  • Figure 6 Screening and detection of phage library.
  • A Detection of positive phages by nitrocellulose membrane;
  • B Binding reaction at phage level.
  • Figure 7 Purity analysis of humanized CD47 antibody.
  • A Non-reduced and reduced SDS-PAGE;
  • B HPLC-SEC analysis of antibody purity.
  • FIG. 8 Octet-based antibody affinity assay.
  • A Antigen-antibody binding response curve
  • B Affinity constant of humanized CD47 antibody.
  • Figure 9 Determination of antibody blocking ability.
  • A Antibody blocking curve;
  • B EC 50 of antibody blocking activity.
  • Figure 10 In vitro phagocytosis assay.
  • A Flow cytometry detection of antibody-induced phagocytosis;
  • B phagocytic index of macrophages.
  • Figure 11 Results of red blood cell agglutination test.
  • FIG. 12 In vivo efficacy evaluation of CD47 antibody DS003.
  • Figure 13 Repeated dosing of DS003 does not induce significant anemia or anemic platelet responses in cynomolgus monkeys.
  • Figure 14 Use Fortebio Octet to determine whether the antigenic epitopes of DS003 and Hu5F9 overlap.
  • Figure 15 The antigen binding epitope of DS003 binding to CD47 protein was analyzed using cryo-electron microscopy.
  • the main content of the present invention is the screening of humanized monoclonal antibodies (example DS003) targeting CD47 and the evaluation of their drugability, pharmacological efficacy and safety.
  • mice were immunized with human CD47 antigen, and the spleen of mice with the highest antibody titer was taken to extract total spleen RNA, which was reverse transcribed to form cDNA; using cDNA as a template, primers were used to amplify the full set of mouse antibody heavy and light chain variable region genes; the antibody gene was inserted into the phage vector, and the antibody could be displayed on the surface of the phage in the form of Fab along with the phage structural protein. In this way, a high-quality mouse phage immune library with large storage capacity and high diversity was successfully constructed. After completing three rounds of solid and liquid phase panning of the phage library, the enriched phages were screened again.
  • phage clones were selected from each 5550pfu solid phase and liquid phase panning phage. These clones were verified again by phage single-point ELISA, and the antibody gene was amplified from the positive phage and sent for sequencing to obtain multiple unique and new CD47 antibody sequences.
  • the eukaryotic expression vector of the obtained antibody was constructed, and the protein of the mouse CD47 antibody was successfully expressed. After preliminary verification of the purity and molecular size of the antibodies, they were tested for species cross-reactivity, and a mouse CD47 monoclonal antibody that met the requirements and could bind to both human and cynomolgus macaque CD47 was obtained. HPLC-SEC and DSF methods confirmed that these antibody molecules had high purity and thermal stability.
  • ELISA and FCM were used to evaluate the binding activity and blocking activity of these antibodies at the protein and cellular levels.
  • the red blood cell agglutination reaction was used to detect whether these antibody molecules had the adverse effect of causing red blood cell aggregation, showing that two antibodies were positive for red blood cell agglutination, while the remaining five antibodies had no red blood cell agglutination phenomenon.
  • Based on biomembrane interferometry technology a comparison of the antigenic epitopes of the remaining five CD47 antibodies showed that three of them had The first and second groups of antibodies had similar antigen binding epitopes, and the remaining two had similar antigen binding epitopes, while the epitopes of the two groups of antibodies were quite different.
  • the ability of CD47 antibodies to mediate macrophage phagocytosis was evaluated. At a single concentration, all five antibodies were able to significantly promote the occurrence of phagocytic reactions.
  • mouse CD47 monoclonal antibody #003 was selected for humanization.
  • the mouse CD47 antibody was humanized using the "framework region rotation" method. This method overlaps the six CDR regions of the mouse antibody with multiple human antibody framework regions. The mutual combination of each framework region during the splicing process makes the humanized phage antibody library have a high diversity.
  • the literature was consulted to select human antibody germline genes that can be used for antibody humanization. The antibodies obtained by humanization with germline genes have low immunogenicity to the human body; then the NCBI IgBlast database was used to annotate the human antibody germline gene framework region and the CDR region of the mouse CD47 antibody based on the amino acid numbering and CDR region definition criteria of the Kabat method.
  • the FR region of the human antibody gene and the CDR region of the mouse CD47 antibody were successfully spliced through three rounds of PCR to form a complete antibody variable region gene, which was then connected to the phage vector using the Kunkel mutation method to construct a phage library.
  • the library was panned using both solid phase and liquid phase methods, and the phages enriched in the first round of panning were screened. From the 10,000 pfu phages screened, 96 CD47 antibody-positive phage clones were picked. These clones were tested again by single-point ELISA, and the genes of the antibodies displayed by the phages that were still positive were amplified and sequenced. Finally, 7 humanized antibodies were obtained, which retained the CDR region of the original mouse antibody and had 100% of the human framework region.
  • DS003 a humanized CD47 antibody, was selected, and IgG4 was selected as the subtype of this antibody molecule.
  • Necessary mutations were made to DS003 to increase the stability of the molecule.
  • the efficacy results in nude mice showed that DS003 could significantly inhibit the growth of tumor cells and prolong the survival time of mice.
  • the safety evaluation results in cynomolgus monkeys showed that DS003 did not cause obvious toxic side effects such as anemia or anemia.
  • DS002 is a humanized monoclonal antibody molecule targeting CD47 with strong efficacy, low toxicity and good development potential, and is expected to play a role in the treatment of CD47-positive tumors and liver fibrosis and other indications.
  • hCD47-ECD extracellular region of human CD47
  • hCD47-ECD https://www.ncbi.nlm.nih.gov/genbank
  • pCAT1.0 proprietary eukaryotic cell expression vector
  • HEK293F cells Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
  • the hCD47-ECD-Fc protein in the cell culture supernatant was separated and purified using Protein A affinity chromatography (GE Lifesciences), and a high-concentration protein solution was obtained by ultrafiltration concentration.
  • the obtained hCD47-ECD-Fc fusion protein molecules were analyzed using ultramicrospectrophotometer, SDS-PAGE, HPLC-SEC and other means.
  • mice (Shanghai Southern Model Animal Center) were immunized with hCD47-ECD-Fc fusion protein antigen (made in the laboratory), and the spleen of the mouse with the highest antibody titer was taken to extract the total spleen RNA, which was reverse transcribed to form cDNA; using cDNA as a template, primers were used to amplify the full set of mouse antibody heavy and light chain variable region genes; the antibody genes were inserted into a phage vector (laboratory M13MP18 (New England Biolabs)), the antibody can be displayed on the surface of the phage in the form of Fab along with the phage structural protein. A high-quality mouse phage immune library with large storage capacity and high diversity was successfully constructed.
  • the enriched phages were screened again. Multiple phage clones were selected from each 5550pfu solid-phase and liquid-phase panning phage. These phage clones were verified again by phage single-point ELISA, and the antibody genes were amplified from the positive phages for sequencing and obtained multiple unique and new CD47 antibody sequences. The correctly sequenced antibody plasmid was filtered and sterilized and transfected into HEK293F cells. After 7 days of shaking culture, the supernatant was collected and purified using a Protein A column. The purified protein was verified by reduced SDS-PAGE ( Figure 2), indicating that the heavy and light chain bands were of the correct size.
  • Example 2 ELISA reaction to determine the binding of antigen and mouse CD47 antibody
  • hCD47-ECD-Fc was coated overnight, and the mouse CD47 antibody was diluted 2-fold to 7 concentrations starting from 100nM, using goat anti-mouse IgG-HRP as the color substrate and TMB as the color developing solution.
  • the absorbance was read at OD450nm, and the data was processed to obtain the antigen-antibody binding curve ( Figure 3).
  • the antigen-antibody binding was concentration-dependent, and the binding reached saturation when the antibody concentration was around 100nM, and the binding force of each antibody to the antigen was at the nM level.
  • Example 3 ELISA reaction to determine the blocking activity of mouse CD47 antibodies
  • hSIRP ⁇ -ECD-Fc purchased from Beijing Sino Biological Technology Co., Ltd.
  • mouse CD47 antibody was diluted in series and incubated with biotinylated hCD47-ECD-Fc for 30 minutes.
  • Streptavidin-HRP was used as a color substrate
  • TMB Thermo Scientific
  • the absorbance was read at OD450nm.
  • the data was processed to obtain a blocking curve ( Figure 4). All mouse antibodies had good blocking activity, and the blocking activity of two antibodies was stronger than that of the control antibody Hu5F9 (prepared according to the sequence searched at http://www.umabs.com ).
  • Daudi cells (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were labeled with CFSE (Thermo Scientific) and seeded in 96-well plates at 1*10 4 per well.
  • 4*10 4 mouse macrophages RAW264.7 (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were added and co-cultured for 2 hours in the presence of 10 ⁇ g/ml CD47 antibody.
  • Human macrophages (prepared in the laboratory) were infected with PE-labeled anti-mouse and anti-human CD11b antibodies (BD Biosciences).
  • CFSE and PE double-positive cells were detected by flow cytometry to represent the phagocytic status of the cells. The results showed that each CD47 antibody had a significant promoting phagocytic effect (Figure 5).
  • the mouse antibody was humanized, and its CDR regions were fixed and assembled with the human antibody backbone region in the correct reading frame to form a phage library, from which 7 clones with sequence differences were selected for the next step of experimental verification ( Figure 6).
  • the correctly sequenced antibody plasmid was filtered and sterilized and then transfected into HEK293F cells. After 7 days of shaking culture, the supernatant was collected and purified using a Protein A column. The purified protein was verified by reduced and non-reduced SDS-PAGE and analyzed by HPLC-SEC ( Figure 7). The heavy and light chain bands were of correct size and the antibody purity was good, so it could be used for the next experiment.
  • hSIRP ⁇ -ECD-Fc was coated overnight, and the humanized CD47 antibody was diluted in series and incubated with biotinylated hCD47-ECD-Fc for 30 minutes. Streptavidin-HRP was used as the color substrate and TMB was used as the color developing solution. The absorbance was read at OD450nm, and the data was processed to obtain the blocking curve (Figure 9). The results showed that the humanized antibody maintained the blocking activity of the original mouse antibody.
  • Daudi cells were labeled with CFSE and seeded in 96-well plates at 1*10 4 per well.
  • 4*10 4 mouse macrophages RAW264.7 (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) labeled with cell proliferation dye eFluor TM 670 were added. They were co-cultured for 2 hours in the presence of 10 ⁇ g/ml CD47 antibody. The phagocytosis was detected by flow cytometry. The results showed that the humanized CD47 antibody maintained the activity of the original mouse antibody and had a significant phagocytic effect (Figure 10).
  • Red blood cells were collected, washed three times with PBS, and added to a U-bottom 96-well cell culture plate after being diluted to a density of 2%.
  • Humanized CD47 antibody was added after being diluted 2-fold in a concentration gradient starting from 3.75 ⁇ M and incubated at 37°C and 5% CO2 for 2 hours. The results showed that the Hu5F9 positive control antibody could induce red blood cell agglutination, while the humanized antibody did not ( Figure 11).
  • Example 11 Tumor inhibition effect of DS003 in nude mice
  • the humanized variant #2-10 of the mouse antibody #2 was selected and constructed on the invariable region of human IgG4 kappa. Two mutation sites, F234A and L235A, were introduced into the Fc segment of the IgG4 subtype to further inactivate the functional effect of IgG4.
  • the mutant IgG4 #2-10 antibody was named DS003 (heavy chain amino acid sequence: SEQ ID NO: 1; light chain amino acid sequence: SEQ ID NO: 2, structure see Figure 1).
  • the inhibition of Daudi cell growth by DS003 was evaluated in nude mice. The mice with tumors were divided into PBS, isotype-G4, DS003, and Hu5F9-G4 (wt) groups, with 8 mice in each group.
  • mice were administered 3 times a week for 3 consecutive weeks, and the dosage of each antibody was 10 mg/kg.
  • the growth curve of mouse tumors is shown in Figure 12.
  • the tumor volume of mice in the PBS group reached 2000 mm 3 on the 20th day after the start of administration, and the mice were euthanized.
  • the changes in the tumor volume of mice show that CD47 antibodies can still effectively inhibit the growth of tumor cells in nude mice, and this inhibitory effect mainly comes from the phagocytic effect of macrophages induced by the blocking of CD47 protein on the surface of tumor cells by CD47 antibodies.
  • Example 12 Long-term toxicity study of repeated administration in cynomolgus monkeys for 5 weeks
  • 40 cynomolgus monkeys were randomly divided into 4 groups according to sex and weight, namely, blank control group, DS003 low-dose group, DS003 medium-dose group, and DS003 high-dose group, with 10 monkeys in each group, half of which were female and half were male.
  • the DS003 low-dose, medium-dose and high-dose groups of cynomolgus monkeys were given 10, 30 and 100 mg/kg of DS003, respectively.
  • the administration volume of each group was 10 mL/kg, and the corresponding administration concentrations were 1, 3 and 10 mg/mL.
  • the blank control group was given the same administration volume of DS003 preparation buffer.
  • the drug was administered by intravenous drip for 30 minutes, once a week, for 5 consecutive weeks, and the recovery period was 6 weeks.
  • repeated administration of DS003 did not cause obvious anemia or anemic platelet reactions in cynomolgus monkeys.
  • Example 13 Study on the antigenic epitope of DS003 binding to CD47 protein
  • FIG. 14 shows the real-time signal of the change in the wavelength of the interference spectrum caused by the change in the thickness of the biofilm layer as the antibody and antigen bind.
  • the first antibody is DS003
  • the second antibody Hu5F9 but not DS003 itself, can produce a significant binding signal. Therefore, the epitopes between the two groups of antibodies DS003 and Hu5F9 are inconsistent.
  • the DS003 antibody was digested with papain, and the antibody Fab fragment was obtained after two-step purification by protein A affinity chromatography and molecular sieve (Superdex 200 increase 10/300G) chromatography.
  • the antibody Fab fragment was co-incubated with the antigen at a ratio of 1:1.5 at 4°C for 1 hour and then purified by molecular sieve chromatography to obtain the antigen/antibody complex.
  • An appropriate amount of the complex was spread on a gold grid treated with glow discharge, then cast into liquid ethane for rapid freezing, prepared into a glassy sample, and transferred to a cryo-electron microscope for observation and data collection.

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Abstract

本发明涉及一种靶向CD47的单克隆抗体及其应用。所述单克隆抗体包括：重链可变区和轻链可变区，其中，重链可变区包括三个互补决定区：氨基酸序列为SEQ ID NO:5的HCDR1、氨基酸序列为SEQ ID NO:6的HCDR2和氨基酸序列为SEQ ID NO:7的HCDR3，轻链可变区包括三个互补决定区：氨基酸序列为SEQ ID NO:8的LCDR1、氨基酸序列为SEQ ID NO:9的LCDR2和氨基酸序列为SEQ ID NO:10的LCDR3。所述单克隆抗体结合在CD47蛋白全新的抗原表位上，其在显示出与CD47的良好的结合及阻断活性的同时，还显示出良好的安全性，特别是血液安全性。

Description

一种靶向CD47的单克隆抗体及其应用 技术领域
本发明涉及抗体药物技术领域，具体来讲涉及针对CD47分子新抗原表位的单克隆抗体，该抗体的制备方法以及该抗体的用途。
背景技术
肿瘤免疫疗法主要是通过调节人体免疫系统和肿瘤微环境，依靠自身免疫来杀伤和清除肿瘤细胞。目前的肿瘤免疫疗法主要是基于恢复T细胞的功能即适应性免疫系统的功能来达到抗肿瘤效应，如以PD-1、PD-L1抗体为代表的免疫检查点抑制剂。而免疫系统作为统一的整体，固有免疫在肿瘤免疫疗法中也能起到十分重要的作用。
CD47又被称为整合素相关蛋白(IAP)，是免疫球蛋白超家族的一员，在淋巴瘤、血液病与绝大部分实体瘤细胞表面高表达，且它的过表达与肿瘤不良预后有关。SIRPɑ是CD47的一个配体，主要表达在髓系细胞如巨噬细胞的表面。当CD47与SIRPɑ相互作用后，肿瘤细胞便可向巨噬细胞传递“不要吃我”信号，实现免疫逃逸。当用CD47抗体阻断这一信号通路时，便可恢复巨噬细胞对肿瘤细胞的吞噬作用，实现固有免疫系统的抗肿瘤效应。通过阻断CD47信号来调动巨噬细胞发挥抗肿瘤作用被认为是对PD-1拮抗剂的有效补充，有望成为“后PD-1时代”的竞争热点。
阻断型CD47抗体在许多肿瘤治疗的临床前研究中取得显著成效，如在急性髓细胞白血病AML的小鼠模型中，CD47抗体治疗组骨髓及外周血中的白血病细胞明显减少，并使小鼠得以长期生存；在人非霍奇金淋巴瘤Raji小鼠模型中，应用CD47抗体治疗后可以明显缩小小鼠荷瘤体积并提高肿瘤 移植小鼠的生存率；CD47抗体与利妥昔单抗注射液联合应用后，甚至可以清除荷瘤小鼠体内的肿瘤细胞，达到完全治愈的效果。在乳腺癌、恶性胶质瘤等多种实体瘤的体外实验中，CD47抗体均可显著抑制肿瘤的生长和转移并延长荷瘤小鼠的生存期。
同时，在非酒精性脂肪性肝炎(NASH)导致的肝脏纤维化的发病过程中，巨噬细胞SIRPα和坏死细胞CD47同时上调，形成坏死细胞对巨噬细胞的免疫逃逸(Shi,et al.SCIENCE TRANSLATIONAL MEDICINE.2022；14(672):23 Nov 2022)。而通过CD47抗体或SIRPα抗体治疗，可以阻断这种免疫逃逸，促进坏死细胞的清除，同时也可以抑制肝脏星型细胞，从而延缓肝脏纤维化的进展。该研究表明，CD47-SIRPα通路除了在癌症免疫逃逸发挥作用，在NASH的发病过程和治疗方面也发挥了重要作用。
目前已有靶向CD47的抗体或融合蛋白进入临床研究。早期的临床结果显示，这类抗体在晚期实体瘤的单药治疗试验中有效，有先前经历过多次治疗无效、极其难治的患者应答，然而大多数实体瘤患者对现有的CD47抗体单一疗法没有很好响应，需要开发联合用药。另外这类抗体在临床出现导致的红细胞和血小板枯竭问题也限制了它们的临床应用。
因此，对于CD47-SIRPɑ信号通路的基础研究展示了CD47靶点良好的成药前景，但是靶向CD47的抗体临床结果也出现了一些单药疗效不足及相关的血液毒性等问题。因此，需要选择特异性强、适应性广泛、药效出色、安全性高的CD47单克隆抗体，以克服目前临床上此类抗体所面临的一些问题。
发明内容
本发明的技术目的是提供一种靶向CD47的单克隆抗体或其免疫活性片段，其具有药效强、毒性低的特点，有望在治疗CD47阳性肿瘤和肝纤维化等适应症中发挥作用。
本发明的另一技术目的是提供CD47分子的新的抗原表位用于筛选靶向CD47的单克隆抗体的用途。
一方面，本发明提供一种靶向的CD47的单克隆抗体或其免疫活性片段，其包括：重链可变区和轻链可变区，其中，重链可变区包括三个互补决定区：氨基酸序列为SEQ ID NO:5的HCDR1、氨基酸序列为SEQ ID NO:6的HCDR2和氨基酸序列为SEQ ID NO:7的HCDR3，或者所述重链可变区的三个互补决定区为与前述氨基酸序列SEQ ID NO:5、SEQ ID NO:6和SEQ ID NO:7相比总体上具有85％以上一致性的突变序列；
轻链可变区包括三个互补决定区：氨基酸序列为SEQ ID NO:8的LCDR1、氨基酸序列为SEQ ID NO:9的LCDR2和氨基酸序列为SEQ ID NO:10的LCDR3，或者所述轻链可变区的三个互补决定区为与前述氨基酸序列SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10相比总体上具有85％以上一致性的突变序列。
在具体实施方式中，所述单克隆抗体的分子亚型为人IgG4。
在具体实施方式中，所述单克隆抗体为人源化的单克隆抗体。
在具体实施方式中，所述单克隆抗体的重链氨基酸序列如SEQ ID NO:1所示，轻链氨基酸序列如SEQ ID NO:2所示。
另一方面，本发明提供编码上述单克隆抗体的多核苷酸。
在具体实施方式中，用于编码上述单克隆抗体的重链的多核苷酸序列为：SEQ ID NO:3；以及用于编码上述单克隆抗体的轻链的多核苷酸序列 为：SEQ ID NO:4。
在具体实施方式中，用于编码所述单克隆抗体的重链的多核苷酸序列为SEQ ID NO:3的同义核苷酸序列，以及用于编码所述单克隆抗体的轻链的多核苷酸序列为SEQ ID NO:4的同义核苷酸序列。
另一方面，本发明提供一种药物组合物，其包含治疗有效量的上述单克隆抗体或其免疫活性片段，以及药学上可接受的载体。
另一方面，本发明提供上述单克隆抗体或其免疫活性片段或上述药物组合物在制备药物中的用途。
在具体实施方式中，所述药物为肿瘤治疗药物或肝脏纤维化治疗药物。
在具体实施方式中，所述肿瘤为CD47阳性肿瘤，包括但不限于卵巢癌、结肠癌、乳腺癌、肺癌、头颈部肿瘤、膀胱癌、结肠直肠癌、胰腺癌、非霍奇金氏淋巴瘤、急性淋巴细胞性白血病、慢性淋巴细胞白血病、急性髓细胞样白血病、慢性髓细胞性白血病、多毛细胞白血病(HCL)、T细胞幼淋巴细胞白血病(T-PLL)、大粒淋巴细胞白血病、成人T细胞白血病、多发性骨髓瘤、(恶性)黑色素瘤、平滑肌瘤、平滑肌肉瘤、神经胶质瘤、胶质母细胞瘤、骨髓瘤、单核细胞白血病、B细胞衍生白血病、T细胞衍生白血病、B细胞衍生淋巴瘤、T细胞衍生淋巴瘤、子宫内膜癌、肾癌、(良性)胎记瘤、前列腺癌、甲状腺癌、子宫颈癌、胃癌、肝癌、和实体肿瘤。
在具体实施方式中，所述肝脏纤维化为非酒精性脂肪性肝炎(NASH)导致的肝脏纤维化。
又一方面，本发明提供一种生物分子，能特异性结合CD47的构象表位，所述表位由CD47蛋白上的G44、R45、D46、I47、T49、D51、A53、L54、K56、S57、T58、V59、P60、T61和D62全部或部分氨基酸残基组成。
有益效果
本发明的单克隆抗体显示出与CD47的良好的结合及阻断活性，可以在动物模型上抑制肿瘤的生长。在食蟹猴中，本发明的单克隆抗体没有诱发明显的血红蛋白或血小板的降低，显示了良好的安全性。利用此抗体部分或全基因，可在真核细胞及任何表达系统中改造和生产不同形式的基因工程抗体，可在临床上用于多种CD47阳性肿瘤、肝脏纤维化等相关疾病的治疗。
附图说明
图1：本申请的靶向CD47的单克隆抗体(DS003)的结构示意图。
图2：CD47抗体分子的还原SDS-PAGE电泳图。
图3：抗原抗体结合能力测定。
图4：抗体阻断能力测定。
图5：体外吞噬实验。
图6：噬菌体库的筛选和检测。(A)通过硝酸纤维素膜检测阳性噬菌体；(B)噬菌体水平的结合反应。
图7：人源化CD47抗体纯度鉴定。(A)非还原型及还原型SDS-PAGE；(B)HPLC-SEC分析抗体的纯度。
图8：Octet测定抗体的亲和力。(A)抗原抗体结合响应曲线；(B)人源化CD47抗体的亲和力常数。
图9：抗体阻断能力测定。(A)抗体阻断曲线；(B)抗体阻断活性的EC50。
图10：体外吞噬实验。(A)流式检测抗体促吞噬情况；(B)巨噬细胞的吞噬指数。
图11：红细胞凝集实验结果。
图12：CD47抗体DS003的体内药效评价。
图13：DS003的重复给药在食蟹猴中不引起明显的贫血或贫血小板反应。
图14：用Fortebio Octet确定DS003与Hu5F9的抗原表位是否重合。
图15：用低温电镜解析DS003结合CD47蛋白的抗原结合表位。
具体实施方式
以下通过具体实施例来详细描述本发明的技术方案，然而这些实施方式不用于限制本发明的范围。
本发明的主要内容是靶向CD47的人源化单克隆抗体(实例DS003)的筛选和成药性、药理药效及安全性评估。
首先，用人CD47抗原对BALB/c小鼠进行免疫，取抗体效价最高的小鼠脾脏，提取总的脾脏RNA，反转录形成cDNA；以cDNA为模板，用引物扩增出全套鼠源抗体重、轻链可变区基因；将抗体基因插入到噬菌体载体上，抗体便可以Fab的形式随噬菌体结构蛋白展示在噬菌体的表面。由此成功构建了一个库容较大、多样性较高的优质鼠源噬菌体免疫文库。在完成三轮噬菌体库的固相和液相淘选后，对富集到的噬菌体进行再次筛选。从各5550pfu固相、液相淘选所得噬菌体中，分别挑选多个噬菌体克隆。将这些克隆再次通过噬菌体单点ELISA验证，从结果为阳性的噬菌体中扩增出抗体基因送测序并得到多个具有独特性的、全新的CD47抗体序列。构建所得抗体的真核表达载体，成功表达出鼠源CD47抗体的蛋白。在初步验证抗体的纯度和分子大小后，对其进行种属交叉反应性检测，得到符合要求即能同时结合人和食蟹猴CD47的鼠源CD47单克隆抗体。利用HPLC-SEC及DSF法确认了这些抗体分子均具有较高的纯度和热稳定性。利用ELISA和FCM完成了这些抗体在蛋白水平和细胞水平的结合活性、阻断活性的评价。通过红细胞凝集反应检测这些抗体分子是否具有引起红细胞聚集的不良作用，显示两个抗体为红细胞凝集阳性，而其余5个抗体则无红细胞凝集现象。基于生物膜干涉技术，对剩余5个CD47抗体的抗原表位进行比较表明有3个抗 体具有相似的抗原结合表位，其余2个具有相似的抗原结合表位，而前后两组抗体的表位差异较大。最后对CD47抗体介导巨噬细胞吞噬作用的能力进行评估，在单一浓度下，这5个抗体均能够明显促进吞噬反应的发生。
经过对抗体的表达量、纯度、热稳定性、结合活性、阻断活性、安全性、表位、体外药效的实验结果进行综合比较后，选择鼠源CD47单克隆抗体#003进行人源化。
采用“骨架区轮换”的方法对鼠源CD47抗体进行人源化，该方法是将鼠源抗体的6个CDR区与多条人抗体骨架区重叠，拼接过程中各骨架区的相互组合，使得人源化噬菌体抗体文库具有较高的多样性。首先进行文献的查阅，挑选出可以用于抗体人源化的人抗体胚系基因，用胚系基因人源化得到的抗体对人体的免疫原性较低；随后用NCBI IgBlast数据库，基于Kabat法的氨基酸编号和CDR区定义准则，对人抗体胚系基因骨架区和鼠源CD47抗体的CDR区进行标注。通过三轮PCR将人抗体基因的FR区和鼠源CD47抗体的CDR区成功拼接，形成完整的抗体可变区基因，随后利用Kunkel突变法将其与噬菌体载体连接，构建成噬菌体文库。利用固相和液相两种方式淘选文库，将第一轮淘选富集到的噬菌体进行筛选，从筛选的10000pfu噬菌体中，各挑取96个CD47抗体阳性的噬菌体克隆。通过单点ELISA对这些克隆再次检验，将测试结果仍为阳性的噬菌体所展示的抗体的基因扩增后进行序列测定。最终，得到7个人源化抗体，这些抗体既保留了原始鼠源抗体的CDR区又具备100％的人骨架区。
将人源化CD47抗体表达为蛋白后，利用HPLC-SEC测定纯度，结果显示这些抗体几乎都符合抗体药物纯度大于95％的要求。对抗体的热稳定性检测和Tm值测定的实验结果表明这些人源化抗体均具有较好的热稳定性。对抗体在蛋白水平和细胞水平的结合及阻断活性的评价结果指出各组抗体分子间存在活性上的差别，但差别均未跨越数量级。利用巨噬细胞的吞噬 反应对人源化抗体的体外药效评价结果显示，这些抗体均能显著促进巨噬细胞对肿瘤细胞的吞噬。在评价人源化CD47抗体安全性的红细胞凝集实验中，均未产生红细胞凝集的现象。总体而言，用“骨架区轮换”方法得到的人源化抗体，具有较好的成药性，并且能够保持亲本鼠源抗体的活性。
选择DS003这个人源化CD47抗体，选择IgG4作为这个抗体分子的亚型，对DS003进行了必要的突变以增加分子的稳定性。裸鼠体内的药效结果显示DS003能够显著抑制肿瘤细胞的生长，延长小鼠的存活时间。食蟹猴体内的安评结果表明，DS003没有造成明显的贫血或贫血小板的毒副作用。
综上所述，DS002是一个药效强、毒性低、开发性好的靶向CD47的人源化单克隆抗体分子，有望在治疗CD47阳性肿瘤和肝纤维化等适应症中发挥作用。
实施例1:鼠源抗体的产生和表达
将人CD47的胞外区(hCD47-ECD)的核苷酸序列(https://www.ncbi.nlm.nih.gov/genbank)与人IgG1的Fc段的核苷酸序列通过GGGS的可变性链接子连在一起，然后克隆到专有的真核细胞表达载体pCAT1.0上，转染HEK293F细胞(中国科学院上海生科院细胞资源中心)，在无血清培养基中震荡培养7天，使用Protein A亲和层析(GE Lifesciences)对细胞培养上清中的hCD47-ECD-Fc蛋白进行分离纯化，通过超滤浓缩得到高浓度蛋白溶液。使用超微量分光光度计、SDS-PAGE、HPLC-SEC等手段对得到的hCD47-ECD-Fc融合蛋白分子进行分析。
用hCD47-ECD-Fc融合蛋白抗原(实验室内自制)对BALB/c小鼠(上海南方模式动物中心)进行免疫，取抗体效价最高的小鼠脾脏，提取总的脾脏RNA，反转录形成cDNA；以cDNA为模板，用引物扩增出全套鼠源抗体重、轻链可变区基因；将抗体基因插入到噬菌体载体上(实验室用 M13MP18(New England Biolabs)制备)，抗体便可以Fab的形式随噬菌体结构蛋白展示在噬菌体的表面。成功构建了一个库容较大、多样性较高的优质鼠源噬菌体免疫文库。
在完成三轮噬菌体库的固相和液相淘选后，对富集到的噬菌体进行再次筛选。从各5550pfu固相、液相淘选所得噬菌体中，分别挑选到多个噬菌体克隆。将这些噬菌体克隆再次通过噬菌体单点ELISA验证，从结果为阳性的噬菌体中扩增出抗体基因送测序并得到多个具有独特性的、全新的CD47抗体序列。测序正确的抗体质粒过滤除菌后转染进HEK293F细胞中，振荡培养7天后，收集上清用Protein A柱进行纯化，纯化后的蛋白进行还原型SDS-PAGE验证(图2)，表明重轻链条带大小正确。
实施例2:ELISA反应测定抗原与鼠源CD47抗体的结合
过夜包被hCD47-ECD-Fc，将鼠源CD47抗体从100nM开始以2倍梯度稀释7个浓度，以羊抗鼠IgG-HRP为显色底物，TMB为显色液，在OD450nm处读取吸光值，数据经处理后得到抗原抗体的结合曲线(图3)。抗原抗体的结合呈现浓度依赖性，抗体浓度在100nM左右时结合达到饱和，各抗体与抗原的结合力均在nM级。
实施例3:ELISA反应测定鼠源CD47抗体的阻断活性
过夜包被hSIRPɑ-ECD-Fc(购买于北京义翘神州科技股份有限公司)，将鼠源CD47抗体梯度稀释后与生物素化的hCD47-ECD-Fc共孵育30min，以streptavidin-HRP(Thermo Scientific)作为显色底物，TMB(Thermo Scientific)为显色液，在OD450nm处读取吸光值，数据经处理后得到阻断曲线(图4)。各鼠源抗体均有良好的阻断活性，其中2个抗体的阻断活性强于对照抗体Hu5F9(按照http://www.umabs.com搜索到的序列制备)。
实施例4:鼠源抗体的体外吞噬反应
将Daudi细胞(中国科学院上海生科院细胞资源中心)用CFSE(Thermo Scientific)标记，每孔1*104个种于96孔板，加入4*104个鼠巨噬细胞RAW264.7(中国科学院上海生科院细胞资源中心)，在10μg/ml CD47抗体存在的情况下共培养2小时，用PE标记的抗鼠和人CD11b抗体(BD Biosciences)感染人巨噬细胞(实验室制备)，通过流式细胞术检测CFSE和PE双阳性的细胞即可代表细胞的吞噬情况，结果显示各CD47抗体具有明显的促吞噬的作用(图5)。
实施例5:抗体人源化
对鼠源抗体进行人源化，固定其各CDR区，以正确的读码框与人源抗体的骨架区区进行组装，形成噬菌体文库，从中淘选了7个序列差异的克隆进行下一步的实验验证(图6)。
实施例6:人源化CD47抗体表达、纯化及质控
测序正确的抗体质粒过滤除菌后转染进HEK293F细胞中，振荡培养7天后，收集上清用Protein A柱纯化，纯化后的蛋白进行还原型及非还原型SDS-PAGE验证、HPLC-SEC分析(图7)，重轻链条带大小正确、抗体纯度较好，可用于下一步的实验。
实施例7:人源化CD47抗体亲和力测定
使用ForteBio的OctetRed96分子非标记相互作用仪测定了hCD47-ECD-his(实验室制备)与人源化CD47抗体的亲和力，结果显示抗体与抗原具有良好的结合能力，亲和力常数均在nM级以上(图8)。
实施例8:人源化CD47抗体阻断活性
过夜包被hSIRPɑ-ECD-Fc，将人源化CD47抗体梯度稀释后与生物素化的hCD47-ECD-Fc共孵育30min，以streptavidin-HRP作为显色底物，TMB为显色液，在OD450nm处读取吸光值，数据经处理后得到阻断曲线(图9)。结果显示人源化后的抗体保持了原始鼠源抗体的阻断活性。
实施例9:人源化抗体的吞噬反应
将Daudi细胞用CFSE标记，每孔1*104个种于96孔板，加入4*104个细胞增殖染料eFluorTM 670标记的鼠巨噬细胞RAW264.7(中国科学院上海生科院细胞资源中心)，在10μg/ml CD47抗体存在的情况下共培养2小时，通过流式细胞术检测吞噬情况，结果显示人源化CD47抗体保持了原始鼠源抗体的活性，具有明显的促吞噬的作用(图10)。
实施例10:人源化抗体的红细胞凝集反应
收集红细胞，用PBS洗3次，稀释成2％的密度后加入U底96孔细胞培养板，将人源化CD47抗体从3.75μM开始2倍浓度梯度稀释后加入，于37℃、5％CO2静置2小时，结果显示Hu5F9阳性对照抗体可以引起红细胞凝集、而人源化抗体均未引起(图11)。
实施例11:DS003在裸鼠体内的抑瘤效果
选择鼠源抗体#2人源化后的变体#2-10构建到人IgG4kappa的不可变区上，在IgG4亚型的Fc段引入F234A、L235A两个突变位点，以进一步失活IgG4的功能效应，得到的突变型IgG4的#2-10抗体，将其命名为DS003(重链氨基酸序列：SEQ ID NO:1；轻链氨基酸序列：SEQ ID NO:2，结构参见图1)。在裸鼠体内，评价DS003对Daudi细胞生长的抑制。将成瘤后的小鼠分为PBS、isotype-G4、DS003、Hu5F9-G4(wt)组，每组8只小鼠。小鼠每周给药3次，连续给药3周，各抗体的给药剂量均为10mg/kg。小鼠肿瘤的生长曲线如图12所示。PBS组小鼠的肿瘤体积在给药开始后的第20天达到2000mm3，小鼠安乐死。由小鼠肿瘤体积的变化可知，CD47抗体依旧能够有效地抑制肿瘤细胞在裸鼠体内的生长，并且这一抑制作用主要来自CD47抗体对肿瘤细胞表面CD47蛋白的阻断而诱发的巨噬细胞的吞噬效应。
实施例12:食蟹猴5周重复给药长期毒性研究
40只食蟹猴，分性别按体重随机分为空白对照组、DS003低剂量组、DS003中剂量组、DS003高剂量组共4组，每组10只，雌、雄各半。DS003低、中和高剂量组食蟹猴分别给予10、30和100mg/kg的DS003，各组给药体积均为10mL/kg，相应给药浓度为1、3和10mg/mL。空白对照组给予相同给药体积的DS003制剂缓冲液。静脉30分钟滴注给药，每周给药1次，连续给药5周，恢复期6周。从图13可以看到，DS003的重复给药在食蟹猴中不引起明显的贫血或贫血小板反应。
实施例13:DS003结合CD47蛋白的抗原表位研究
首先用Fortebio Octet(美国丹纳赫集团产品)结合的方式研究DS003结合CD47蛋白的抗原表位。将人CD47-his蛋白固化在Ni探针上，以串联的方式依次去结合两个待比较的抗体。图14显示的是随着抗体与抗原的结合，生物膜层厚度的变化，引起的干涉光谱波长变化的实时信号。如图可知，当第一抗体为DS003时，第二抗体Hu5F9，但不是DS003本身，可以产生明显的结合信号。因此DS003与Hu5F9两组抗体间的表位不一致。
另外，用木瓜蛋白酶对DS003抗体进行酶切，经protein A亲和层析和分子筛(Superdex 200 increase 10/300G)层析两步纯化后获得抗体Fab段。将抗体Fab段与抗原以1:1.5的比例在4℃共孵育1小时后通过分子筛层析进行纯化，得到抗原/抗体复合物。取适量复合物铺展到经辉光放电处理的金质载网上，然后投到液态乙烷中快速冷冻，制备成玻璃态样品，转移到低温电镜下面观察并收集数据。经3D重建及优化分析，确定DS003与CD47蛋白上的G44,R45,D46,I47,T49,D51,A53,L54,K56,S57,T58,V59,P60,T61和D62等氨基酸残基相互作用(图15)，这些氨基酸残基分别隶属于C,C`和C``strands。Hu5F9报道的结合部位是FG和BC loop(https://doi.org/10.1016/j.csbj.2021.09.036)，因此两者的抗原表位不同。A  loop,BC loop,C strand,C`strand,C``strand和FG loop据报道都是CD47与SIRPα的结合部位。
氨基酸和核苷酸序列


Claims (10)

  1. 一种靶向的CD47的单克隆抗体或其免疫活性片段，其包括：重链可变区和轻链可变区，其中，
    所述重链可变区包括三个互补决定区：氨基酸序列为SEQ ID NO:5的HCDR1、氨基酸序列为SEQ ID NO:6的HCDR2和氨基酸序列为SEQ ID NO:7的HCDR3，或者所述重链可变区的三个互补决定区为与前述氨基酸序列SEQ ID NO:5、SEQ ID NO:6和SEQ ID NO:7相比总体上具有85％以上一致性的突变序列；
    所述轻链可变区包括三个互补决定区：氨基酸序列为SEQ ID NO:8的LCDR1、氨基酸序列为SEQ ID NO:9的LCDR2和氨基酸序列为SEQ ID NO:10的LCDR3，或者所述轻链可变区的三个互补决定区为与前述氨基酸序列SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10相比总体上具有85％以上一致性的突变序列。
  2. 根据权利要求1所述的单克隆抗体或其免疫活性片段，其中，所述单克隆抗体的分子亚型为人IgG4。
  3. 根据权利要求1所述的单克隆抗体或其免疫活性片段，其中，所述单克隆抗体为人源化的单克隆抗体。
  4. 根据权利要求1所述的单克隆抗体或其免疫活性片段，其中，所述单克隆抗体的重链氨基酸序列如SEQ ID NO:1所示，轻链氨基酸序列如SEQ ID NO:2所示。
  5. 一种编码如权利要求1至4中任一项所述的单克隆抗体的多核苷酸。
  6. 根据权利要求5所述的多核苷酸，其中，用于编码所述单克隆抗体的重链的多核苷酸序列为：SEQ ID NO:3；以及用于编码所述单克隆抗体的轻链的多核苷酸序列为：SEQ ID NO:4，或者
    用于编码所述单克隆抗体的重链的多核苷酸序列为SEQ ID NO:3的同义核苷酸序列，以及用于编码所述单克隆抗体的轻链的多核苷酸序列为SEQ ID NO:4的同义核苷酸序列。
  7. 一种药物组合物，其包含治疗有效量的如权利要求1至4中任一项所述的单克隆抗体或其免疫活性片段，以及药学上可接受的载体。
  8. 如权利要求1至4中任一项所述的单克隆抗体或其免疫活性片段，或如权利要求7所述的药物组合物在制备药物中的用途。
  9. 根据权利要求8所述的用途，其中，所述药物为肿瘤治疗药物或肝脏纤维化治疗药物，特别地，所述肿瘤为CD47阳性肿瘤。
  10. 一种生物分子，其能特异性结合CD47的构象表位，所述表位由CD47蛋白上的G44、R45、D46、I47、T49、D51、A53、L54、K56、S57、T58、V59、P60、T61和D62氨基酸残基组成或由上述氨基酸残基的任意组合组成。
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CN104271757A (zh) * 2012-02-06 2015-01-07 印希彼有限责任公司 Cd47抗体及其使用方法
CN105101997A (zh) * 2013-02-06 2015-11-25 印希彼有限责任公司 不减少血小板和不减少血红细胞的cd47抗体及其使用方法
CN107406503A (zh) * 2014-11-18 2017-11-28 詹森药业有限公司 Cd47抗体、方法和用途
US20200255515A1 (en) * 2016-05-09 2020-08-13 Celgene Corporation Cd47 antibodies and methods of use thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104271757A (zh) * 2012-02-06 2015-01-07 印希彼有限责任公司 Cd47抗体及其使用方法
CN105101997A (zh) * 2013-02-06 2015-11-25 印希彼有限责任公司 不减少血小板和不减少血红细胞的cd47抗体及其使用方法
CN107406503A (zh) * 2014-11-18 2017-11-28 詹森药业有限公司 Cd47抗体、方法和用途
US20200255515A1 (en) * 2016-05-09 2020-08-13 Celgene Corporation Cd47 antibodies and methods of use thereof

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