Nothing Special   »   [go: up one dir, main page]

WO2021110181A2 - 可结合cd47的多肽及其应用 - Google Patents

可结合cd47的多肽及其应用 Download PDF

Info

Publication number
WO2021110181A2
WO2021110181A2 PCT/CN2021/075043 CN2021075043W WO2021110181A2 WO 2021110181 A2 WO2021110181 A2 WO 2021110181A2 CN 2021075043 W CN2021075043 W CN 2021075043W WO 2021110181 A2 WO2021110181 A2 WO 2021110181A2
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
seq
polypeptide
vhh
antibody
Prior art date
Application number
PCT/CN2021/075043
Other languages
English (en)
French (fr)
Other versions
WO2021110181A3 (zh
Inventor
吴喜林
吴稚伟
苏艾荣
黄碧莲
Original Assignee
源道隆（苏州）医学科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 源道隆（苏州）医学科技有限公司 filed Critical 源道隆（苏州）医学科技有限公司
Publication of WO2021110181A2 publication Critical patent/WO2021110181A2/zh
Publication of WO2021110181A3 publication Critical patent/WO2021110181A3/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®

Definitions

  • the invention relates to the field of biomedicine. More particularly, it relates to a polypeptide capable of binding CD47 and its application.
  • CD47 is widely expressed on the cell surface of different tissues and is a member of the immunoglobulin superfamily.
  • CD47 protein can send out a "don't eat me” signal by binding to the ligand signal regulatory protein ⁇ (SIRP ⁇ ) on macrophages.
  • SIRP ⁇ ligand signal regulatory protein ⁇
  • Tumor cells can escape the recognition and elimination of the body's immune system through a variety of ways, including overexpression of the cell surface protein CD47.
  • Studies have shown that the use of antibodies to block the CD47-SIRP ⁇ pathway can promote the phagocytosis of tumor cells.
  • Targeting CD47 is a promising anti-tumor direction that can be used to treat patients with solid tumors and lymphomas.
  • the antibody naturally lacks the light chain and consists only of the heavy chain.
  • the heavy chain contains two constant regions (CH2 and CH3), a hinge region and a variable heavy chain domain (VHH, which is the antigen binding site).
  • VHH variable heavy chain domain
  • the relative molecular mass of the heavy chain variable region is about 13KDa, which is only 1/10 of that of conventional antibodies, and the molecular height and diameter are both at the nanometer level. It is the smallest functional antibody fragment currently available. It is called Nanobody (Nb). Due to its high stability (not degraded at 90°C), high affinity, more than 80% homology with human antibodies, and low toxicity and immunogenicity, nano monoclonal antibodies have been widely used recently.
  • Nanobodies are also widely used in antibody screening in the field of cell therapy.
  • the invention obtains alpaca-derived nano monoclonal antibody and its VHH by immunizing alpaca with an antigen, and is used for the treatment of solid tumors and lymphomas.
  • the present invention provides a polypeptide that can bind to CD47, including three complementarity determining regions CDR1-3, CDR1 sequence is or includes one of the sequences shown in SEQ ID NO:1-13, and CDR2 sequence is or includes SEQ One of the sequences shown in ID NO: 14-24, and the CDR3 sequence is or includes one of the sequences shown in SEQ ID NO: 25-34.
  • the polypeptide further includes four framework regions FR1-4, and the FR1-4 and the CDR1-3 are arranged in a staggered manner.
  • the FR1-4 sequence can be designed as shown in SEQ ID NO: 35-38, but the scope of the present invention is not limited to this, and the FR1-4 sequence of the framework region can also be humanized as shown in SEQ ID NO: 39- 42 shown.
  • the specific recognition and binding ability of an antibody is mainly determined by the sequence of the CDR region.
  • the FR sequence has little effect and can be designed according to the species, which is well known in the art.
  • a human, murine, or alpaca-derived FR region sequence can be designed to link the above CDRs to obtain a polypeptide or domain that can bind to human CD47.
  • the polypeptide is a monoclonal antibody.
  • polypeptide is VHH.
  • polypeptide is alpaca-derived VHH or humanized VHH.
  • the CDR sequence of the polypeptide is as follows:
  • CDR2 is SEQ ID NO: 43, where the X at position 4 represents isoleucine, lysine, arginine or threonine, and the X at position 8 represents threonine or isoleucine Acid;
  • the sequence of CDR1 is selected from SEQ ID NO: 1-4, and the sequence of CDR3 is selected from SEQ ID NO: 25-27.
  • the sequence of CDR1 is SEQ ID NO: 1
  • the sequence of CDR2 is SEQ ID NO: 14
  • the sequence of CDR3 is SEQ ID NO: 25; or
  • the sequence of CDR1 is SEQ ID NO: 2
  • the sequence of CDR2 is SEQ ID NO: 15
  • the sequence of CDR3 is SEQ ID NO: 25; or
  • the sequence of CDR1 is SEQ ID NO: 3
  • the sequence of CDR2 is SEQ ID NO: 16 and the sequence of CDR3 is SEQ ID NO: 25; or
  • the sequence of CDR1 is SEQ ID NO: 3
  • the sequence of CDR2 is SEQ ID NO: 16 and the sequence of CDR3 is SEQ ID NO: 26; or
  • the sequence of CDR1 is SEQ ID NO: 4
  • the sequence of CDR2 is SEQ ID NO: 17
  • the sequence of CDR3 is SEQ ID NO: 27.
  • the CDR sequence of the polypeptide is as follows:
  • CDR2 is SEQ ID NO: 44, wherein X at position 6 is arginine or threonine;
  • CDR1 is selected from SEQ ID NO: 5-8; and the sequence of CD3 is selected from SEQ ID NO: 28-30.
  • the CDR sequence of the polypeptide is as follows:
  • the sequence of CDR1 is SEQ ID NO: 5
  • the sequence of CDR2 is SEQ ID NO: 18
  • the sequence of CDR3 is SEQ ID NO: 28; or
  • the sequence of CDR1 is SEQ ID NO: 5
  • the sequence of CDR2 is SEQ ID NO: 18 and the sequence of CDR3 is SEQ ID NO: 29; or
  • the sequence of CDR1 is SEQ ID NO: 6, the sequence of CDR2 is SEQ ID NO: 18, and the sequence of CDR3 is SEQ ID NO: 28; or
  • the sequence of CDR1 is SEQ ID NO: 7
  • the sequence of CDR2 is SEQ ID NO: 18
  • the sequence of CDR3 is SEQ ID NO: 28; or
  • the sequence of CDR1 is SEQ ID NO: 8
  • the sequence of CDR2 is SEQ ID NO: 19
  • the sequence of CDR3 is SEQ ID NO: 30.
  • the CDR sequence of the polypeptide is as follows:
  • the sequence of CDR1 is selected from SEQ ID NO: 9 and 10; and the sequence of CD2 is selected from SEQ ID NO: 20 and 21.
  • the CDR sequence of the polypeptide is as follows:
  • the sequence of CDR1 is SEQ ID NO: 9
  • the sequence of CDR2 is SEQ ID NO: 20
  • the sequence of CDR3 is SEQ ID NO: 31; or
  • the sequence of CDR1 is SEQ ID NO: 10
  • the sequence of CDR2 is SEQ ID NO: 21
  • the sequence of CDR3 is SEQ ID NO: 31.
  • the CDR sequence of the polypeptide is as follows:
  • the sequence of CDR1 is SEQ ID NO: 11
  • the sequence of CDR2 is SEQ ID NO: 22
  • the sequence of CDR3 is SEQ ID NO: 32; or
  • the sequence of CDR1 is SEQ ID NO: 12
  • the sequence of CDR2 is SEQ ID NO: 23
  • the sequence of CDR3 is SEQ ID NO: 33;
  • the sequence of CDR1 is SEQ ID NO: 13
  • the sequence of CDR2 is SEQ ID NO: 24
  • the sequence of CDR3 is SEQ ID NO: 34.
  • the present invention also provides the application of the above-mentioned polypeptides in tumor treatment drugs.
  • the present invention also provides the nucleic acid coding sequence of the above-mentioned polypeptide.
  • the nucleic acid coding sequence is a DNA coding sequence or an RNA coding sequence.
  • the nucleic acid coding sequence is present in a gene expression frame.
  • the present invention develops nanobody drugs for solid tumors and lymphomas.
  • the nanobody VHH that specifically binds to human CD47 is screened and identified. Its CDR sequence, and the construction of humanized VHH-huFc1 (C47NB); at the same time, flow cytometry was used to evaluate the binding of humanized antibody to CD47 protein on the cell surface.
  • the invention provides potential new nanobody drugs for the clinical treatment of tumors.
  • Figure 1 is the detection curve of antiserum titer one week after the 3rd and 4th immunization of alpaca with CD47;
  • Figure 2 is a flow cytometric diagram showing the binding of serum from CD47 to the CD47 protein on the surface of multiple myeloma cells 8226 one week after the fourth immunization.
  • the abscissa indicates that the antibody in the serum binds to the cell surface CD47 protein
  • the ordinate indicates that the commercially available direct-labeled antibody binds to the cell surface CD47 protein.
  • Figure 3 is an electrophoresis diagram of PCR products amplified by the CD47-VHH phage antibody library as a template
  • Figure 4 is the panning identification of the CD47-VHH phage antibody library, where A is the ELISA detection statistics of the phage library for CD47 protein panning; B is the second round (2 nd ) and third round (3 rd ) panning The latter phage antibody library selects 40 and 46 clones respectively for phage ELISA detection statistics;
  • Figure 5 is a statistical chart of ELISA detection of VHH antibodies expressed in prokaryotic cells. Each point represents a clone. The ordinate is OD450 for human CD47/OD450 for blank control. A ratio greater than 5.0 is defined as positive;
  • FIGS 6 and 7 are flow charts showing the binding of 19 VHH antibodies prepared by the present invention to CD47 on the surface of PRMI8226 cells.
  • CD47-his His-tag
  • CD47-rFc Rabbit Fc
  • the emulsified mixture of 250 ⁇ g CD47-rFc protein and 250 ⁇ l Freund's complete adjuvant was used to immunize the Bactrian alpaca, and the immunization was boosted with CD47-rFc protein and 250 ⁇ l Freund's incomplete adjuvant on the 14, 28 and 42 days.
  • the titer of the antiserum was detected by ELISA, the test plate was coated with CD47-his protein at a concentration of 0.5 ⁇ g/ml, and 100 ⁇ l of serially diluted antiserum or purified antibody was added to each well (control is alpaca serum before immunization), 37°C Incubate for 1.5 hours, wash twice, add 1:10000 diluted horseradish peroxidase labeled Goat anti-Llamma IgG (H+L) secondary antibody to each well, incubate at 37°C for 1 hour, wash 4-6 times, add 100 ⁇ l TMB substrate was incubated at 37°C for 10min, 50 ⁇ l 0.2M H 2 SO 4 was used to stop the reaction, and the OD 450nm was measured.
  • the ELISA test serum titer is defined as the highest dilution factor when the OD450 is 2.1 times or more of the blank control and greater than 0.2.
  • the results are shown in Figure 1.
  • the antiserum titers of the 3 and 4 vaccines were 1.09 ⁇ 10 6 and 3.28 ⁇ 10 6, respectively . It can be seen that the antigen can induce alpaca to produce high-titer antiserum specific to human CD47 protein.
  • Collect 200ml of the immunized alpaca's peripheral blood use the lymphocyte separator (GE Ficoll-Paque Plus) to separate the alpaca's PBMC, according to the TRIzol operation manual, extract RNA, and use oligo (dT) to reverse the cDNA, through primers Amplification, and molecular cloning techniques, clone the alpaca's VHH gene into the phagemid plasmid, transform TG1 bacteria, and obtain the VHH phage library.
  • PCR was used to amplify the VHH target gene of the immunized CD47 alpaca.
  • the target band is 500bp and the size is in line with expectations ( Figure 3), indicating that the CD47-VHH phage antibody library It contains the VHH gene.
  • Thirty-three clones were selected for sequencing. The sequencing results showed that the diversity of the tested sequences was 93.9%; the comparison results showed that most of the different sequences were in the CDR binding region.
  • the constructed CD47-VHH phage antibody library has a volume of 1.35 ⁇ 10 9 , a positive rate of 100%, a sequence diversity of 93.9%, and an effective insertion rate (In frame rate) of greater than 95%.
  • the bacteria transformed with VHH-phagemid were recovered from the phage antibody library and precipitated with PEG/NaCl.
  • the phage antibody library was enriched three times with CD47-His protein coated with 50 ⁇ g/ml.
  • the enriched phage was eluted, transformed, plated, and a single clone was picked for the ELISA binding identification of the phage and CD47 protein.
  • the clone with a binding reading value> 1.0 was sequenced and cloned into the expression vector phv13, and then expressed in SS320 cells. Production of nano monoclonal antibodies.
  • the library after panning was tested for binding to CD47 protein.
  • the results of phage ELISA showed that the binding reading of CD47-VHH phage library and CD47 protein before enrichment was 0.33, and the readings of phage library after one, two and three rounds of enrichment were 0.49, 1.73, 3.34 ( Figure 4A).
  • 40 and 46 clones were selected from the library after the second and third rounds of enrichment for single phage ELISA detection.
  • MOCK is the PBS control
  • the neg group is the negative control, that is, the prokaryotic expression supernatant control without antibody
  • the positive is the positive control, that is, the positive antibody control that binds to the CD47 membrane protein.
  • VHH antibodies can bind to PRMI8226 cells, of which IAP-114, 118, 121, 129, 132, 140 and 148 have higher binding capacity. Similar results were obtained using humanized VHH antibodies. It can be seen that the above-mentioned VHH antibodies have the ability to target tumor cells. At the same time, it is possible to block the CD47 molecules on the surface of tumor cells to promote the phagocytosis of macrophages, thereby achieving the effect of treating or inhibiting tumor growth. Therefore, these 19 VHH Antibodies have the potential to become new antibody drugs for the treatment of tumors.
  • 19 VHH antibody sequences can also be applied to CAR (Chimeric Antigen Receptor, antigen chimeric receptors).
  • the VHH sequence is fused to the third or fourth generation CD28-4- 1BB-CD3zeta molecular sequence constitutes) cell therapy for the treatment of tumors.
  • VHH can be used for ADC (Antibody-drug conjugate) therapy by coupling drugs or coupled with isotopes for antibody-dependent molecular imaging diagnosis.
  • Adeno-associated virus vector (AAV) is derived from non-pathogenic wild-type adeno-associated virus. Due to its good safety, wide range of host cells (dividing and non-dividing cells), low immunogenicity, it takes a long time to express foreign genes in vivo It is regarded as one of the most promising gene transfer vectors and has been widely used in gene therapy and vaccine research worldwide.
  • the AAV Helper-Free virus packaging system was purchased from Cell Biolabs, San Diego USA. Insert the above-mentioned DNA coding sequence of VHH into the pAAV-MCS plasmid by molecular cloning technology; after the successful construction is proved by sequencing, the constructed plasmid pAAV-Ab and pHelper and pAAV-DJ plasmids are in a 1:1:1 mass ratio AAV-293T cells were co-transfected with PEI transfection reagent. After transfection, the supernatant was collected at 48, 72, 96 and 120 hours, and concentrated with 5xPEG8000 (sigma), and finally purified with 1.37g/ml cesium chloride. The purified AAV was dissolved in PBS, and stored at -80°C after identification and aliquots.
  • mice received intramuscular injection of AAV-VVH (1x10 11 gc/100 ⁇ l), and AAV-GFP was used as the control group.
  • AAV-VVH has a therapeutic effect on multiple myeloma.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • General Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

本发明涉及一种可结合人CD47蛋白的多肽，包括3个互补决定区CDR1-3，CDR1序列为或包括SEQ ID NO:1-13所示序列之一，CDR2序列为或包括SEQ ID NO:14-24所示序列之一，CDR3序列为或包括SEQ ID NO:25-34所示序列之一。本发明针对CD47这一肿瘤免疫治疗靶点，通过制备CD47蛋白，免疫羊驼、利用噬菌体库展示纳米单抗的平台技术等，筛选到特异性结合CD47的纳米抗体VHH，并构建了人源化抗体；同时利用流式细胞术检测方法鉴定人源化抗体与细胞表面CD47蛋白的结合。本发明为多种癌症的临床治疗提供潜在的纳米抗体新药。

Description

可结合CD47的多肽及其应用 技术领域
本发明涉及生物医药领域。更特别地，涉及一种可结合CD47的多肽及其应用。
背景技术
CD47广泛表达于不同组织细胞表面，是免疫球蛋白超家族成员。CD47蛋白通过与巨噬细胞上的配体信号调节蛋白α(SIRPα)发结合，可发出“别吃我”信号。肿瘤细胞可通过多种途径逃避机体免疫系统的识别和清除,其中即包括过表达细胞表面蛋白CD47。研究表明，使用抗体阻断CD47-SIRPα通路可促使肿瘤细胞被吞噬，靶向CD47是一个极具潜力的抗肿瘤方向，可用于治疗实体瘤和淋巴瘤患者。
1993年，一种来源于羊驼科的新型天然抗体被发现。该抗体天然缺失轻链而只由重链组成，其重链包含两个恒定区(CH2和CH3)、一个铰链区和一个重链可变区(Variable heavy chain domain，VHH，即抗原结合位点)，该重链可变区的相对分子质量约为13KDa，仅为常规抗体的1/10，且分子高度和直径均在纳米级别，是目前可获得的最小的功能性抗体片段，因此又被称为纳米单抗(Nanobody，Nb)。因纳米单抗稳定性高(90℃条件下仍不会降解)、亲和力高、与人源抗体同源性超过80％、毒性和免疫原性均较低等特点，最近纳米单抗被广泛用于免疫诊断试剂盒研发、影像学研发以及针对肿瘤、炎症、传染病和神经系统疾病等领域的抗体药物研发。此外随着细胞治疗领域的快速发展，纳米抗体也广泛应用于细胞治疗领域的抗体筛选。
治疗性抗体药物发展的趋势从鼠源、人鼠嵌合、人源化再到全人源，再到近年得到广泛关注的纳米抗体，纳米抗体的研发得到高速发展。2018年9月首个纳米抗体药物获批。目前国内外有多个纳米抗体药物在临床研究阶段。我们期望通过免疫羊驼，获得高亲和力的靶向CD47的治疗性纳米抗体。
发明内容
本发明通过用抗原免疫羊驼，获取羊驼源纳米单抗及其VHH，用于实体瘤及淋巴瘤的治疗。基于这些研究，本发明提供了一种可结合CD47的多肽，包括3个互补决定区CDR1-3，CDR1序列为或包括SEQ ID NO:1-13所示序列之一，CDR2序列为或包括SEQ ID NO:14-24所示序列之一，CDR3序列为或包括SEQ ID NO:25-34所示序列之一。
在一个具体实施方案中，所述多肽还包括4个框架区FR1-4，所述FR1-4与所述CDR1-3交错排列。例如，可将FR1-4序列设计为如SEQ ID NO:35-38所示，但本发明的范围不限于此，也可将框架区FR1-4序列人源化为如SEQ ID NO:39-42所示。抗体的特异性识别和结合能力主要由CDR区序列决定，FR序列影响不大，可根据物种来设计，这是本领域公知的。例如，可设计人源、鼠源或羊驼源的FR区序列来连接上述CDR，从而得到一个可结合人CD47的多肽或结构域。
在一个优选实施方案中，所述多肽为单克隆抗体。
在一个优选实施方案中，所述多肽为VHH。
在一个优选实施方案中，所述多肽为羊驼源的VHH或人源化的VHH。
在一个具体实施方案中，所述多肽的CDR序列如下：
I)CDR2的序列为SEQ ID NO:43,其中，第4位的X表示异亮氨酸、赖氨酸、精氨酸或苏氨酸，并且第8位的X表示苏氨酸或异亮氨酸；并且
II)CDR1的序列选自SEQ ID NO:1-4，并且CDR3的序列选自SEQ ID NO:25-27。
优选地，CDR1的序列为SEQ ID NO:1，CDR2的序列为SEQ ID NO:14并且CDR3的序列为SEQ ID NO:25；或
CDR1的序列为SEQ ID NO:2，CDR2的序列为SEQ ID NO:15并且CDR3的序列为SEQ ID NO:25；或
CDR1的序列为SEQ ID NO:3，CDR2的序列为SEQ ID NO:16并且CDR3的序列为SEQ ID NO:25；或
CDR1的序列为SEQ ID NO:3，CDR2的序列为SEQ ID NO:16并且CDR3的序列为SEQ ID NO:26；或
CDR1的序列为SEQ ID NO:4，CDR2的序列为SEQ ID NO:17并且CDR3的序列为SEQ ID NO:27。
在另一个具体实施方案中，所述多肽的CDR序列如下：
I)CDR2的序列为SEQ ID NO:44，其中第6位的X为精氨酸或苏氨酸；并且
II)CDR1的序列选自SEQ ID NO:5-8；并且CD3的序列选自SEQ ID NO:28-30。
优选地，所述多肽的CDR序列如下：
CDR1的序列为SEQ ID NO:5，CDR2的序列为SEQ ID NO:18并且CDR3的序列为SEQ ID NO:28；或
CDR1的序列为SEQ ID NO:5，CDR2的序列为SEQ ID NO:18并且CDR3的序列为SEQ ID NO:29；或
CDR1的序列为SEQ ID NO:6，CDR2的序列为SEQ ID NO:18并且CDR3的序列为SEQ ID NO:28；或
CDR1的序列为SEQ ID NO:7，CDR2的序列为SEQ ID NO:18并且CDR3的序列为SEQ ID NO:28；或
CDR1的序列为SEQ ID NO:8，CDR2的序列为SEQ ID NO:19并且CDR3的序列为SEQ ID NO:30。
在另一个具体实施方案中，所述多肽的CDR序列如下：
I)CDR3的序列为SEQ ID NO:31；并且
II)CDR1的序列选自SEQ ID NO:9和10；并且CD2的序列选自SEQ ID NO:20和21。
优选地，所述多肽的CDR序列如下：
CDR1的序列为SEQ ID NO:9，CDR2的序列为SEQ ID NO:20并且CDR3的序列为SEQ ID NO:31；或
CDR1的序列为SEQ ID NO:10，CDR2的序列为SEQ ID NO:21并且CDR3的序列为SEQ  ID NO:31。
在另一个具体实施方案中，所述多肽的CDR序列如下：
CDR1的序列为SEQ ID NO:11，CDR2的序列为SEQ ID NO:22并且CDR3的序列为SEQ ID NO:32；或
CDR1的序列为SEQ ID NO:12，CDR2的序列为SEQ ID NO:23并且CDR3的序列为SEQ ID NO:33；
CDR1的序列为SEQ ID NO:13，CDR2的序列为SEQ ID NO:24并且CDR3的序列为SEQ ID NO:34。
本发明还提供了上述多肽在肿瘤治疗药物中的应用。
本发明还提供了上述多肽的核酸编码序列。
在一个实施方案中，所述核酸编码序列为DNA编码序列或RNA编码序列。
在一个具体实施方案中，所述核酸编码序列存在于基因表达框中。
本发明针对实体瘤和淋巴瘤进行纳米抗体药物开发，通过制备人CD47蛋白、免疫羊驼、利用噬菌体库展示纳米单抗的平台技术等，筛选到特异性结合人CD47的纳米抗体VHH，鉴定了其CDR序列，并构建了人源化的VHH-huFc1(C47NB)；同时利用流式细胞术评估人源化抗体与细胞表面CD47蛋白的结合。本发明为肿瘤的临床治疗提供潜在的纳米抗体新药。
附图说明
图1为CD47第3和4次免疫羊驼一周后的抗血清效价检测曲线；
图2为CD47第4次免疫一周后的血清与多发性骨髓瘤细胞8226细胞表面CD47蛋白结合的流式结果图。其中，横坐标表示血清中抗体结合细胞表面CD47蛋白，纵坐标为市售直标抗体结合细胞表面CD47蛋白。
图3为CD47-VHH噬菌体抗体文库为模板扩增的PCR产物的电泳图；
图4为CD47-VHH噬菌体抗体文库的淘选鉴定，其中，A为噬菌体文库针对CD47蛋 白淘选后ELISA检测统计图；B为第二轮(2 nd)和第三轮(3 rd)淘选后的噬菌体抗体文库分别挑选40个和46个克隆进行噬菌体ELISA检测统计图；
图5为原核表达的VHH抗体的ELISA检测统计图，每个点代表一个克隆，纵坐标为针对人CD47的OD450/空白对照的OD450，比值大于5.0定义为阳性；
图6和7为本发明制备的19个VHH抗体与PRMI8226细胞表面CD47结合的流式图。
具体实施方式
1.免疫原的制备
我们依据NCBI网站上人CD47蛋白序列和基因序列信息，分析并设计了可有效诱导羊驼产生针对细胞表面CD47蛋白的特异性抗体的多肽CD47，在C端连接His-tag(CD47-his)或兔Fc(CD47-rFc)用于后续纯化及检测。
2.羊驼免疫与抗血清的获得
用250μg CD47-rFc蛋白与250μl弗氏完全佐剂的乳化混合物对双峰羊驼进行初免，在第14天、28天、42天用CD47-rFc蛋白与250μl弗氏不完全佐剂加强免疫3次，第2次和第3次免疫1周后，采血检测抗血清滴度；第4次免疫1周后，采血200ml用于噬菌体抗体库的构建。
抗血清效价通过ELISA检测，用浓度为0.5μg/ml的CD47-his蛋白包被检测板，每孔加入梯度稀释的抗血清或者纯化的抗体100μl(对照为免疫前羊驼血清)，37℃孵育1.5h，洗涤2次，每孔加入1：10000稀释的辣根过氧化物酶标记的Goat anti-Llamma IgG(H+L)二抗，37℃孵育1h，洗涤4-6次后，加100μl TMB底物，37℃孵育10min，50μl 0.2M的H 2SO 4中止反应，测定OD 450nm。ELISA检测血清效价规定为在OD450是空白对照的2.1倍以上并且大于0.2的最高稀释倍数。
结果如图1所示，3免和4免的抗血清效价分别为1.09×10 6和3.28×10 6。由此可见，该抗原可诱导羊驼产生特异性针对人CD47蛋白的高滴度抗血清。
为了进一步验证该高滴度的羊驼抗血清是否能有效结合细胞表面CD47蛋白，进行流 式细胞检测。将不同稀释浓度的抗血清和免疫前血清分别与人多发性骨髓瘤细胞RPMI8226细胞共同孵育60min，洗涤细胞后加入荧光二抗Alexa Fluor 488 Goat anti Alpaca IgG(H+L),4℃孵育30min后洗涤细胞，上机检测。流式细胞术结果显示，CD47免疫的羊驼抗血清可结合细胞表面CD47蛋白(图2)。综上所述，CD47蛋白诱导了高滴度抗血清，同时该抗血清具有结合细胞表面CD47蛋白的能力，可用于流式检测。
3.VHH噬菌体库构建及淘选
收集200ml免疫后羊驼的外周血，利用淋巴细胞分离液(GE Ficoll-Paque Plus)分离获得羊驼的PBMC，根据TRIzol操作手册，提取RNA，并利用oligo(dT)反转为cDNA,通过引物扩增，以及分子克隆等技术，将羊驼的VHH基因克隆至phagemid质粒，转化TG1细菌，得到VHH噬菌体库。为了进一步鉴定CD47-VHH噬菌体库是否构建成功，通过PCR扩增免疫CD47羊驼的VHH目的基因，可以看出目的条带为500bp，大小符合预期(图3)，说明该CD47-VHH噬菌体抗体文库里含有VHH基因。挑选33个克隆进行测序，测序结果显示，所测序列多样性为93.9％；比对结果显示，差异序列大多在CDR结合区。经检测，该构建了一个CD47-VHH噬菌体抗体文库的库容为1.35×10 9，阳性率为100％，序列多样性(Diversity)为93.9％，有效插入率(In frame rate)大于95％。
在M13KO7辅助噬菌体的帮助下，用VHH-phagemid转化的细菌，进行噬菌体抗体库的复苏，并用PEG/NaCl进行沉淀。将包被有50μg/ml的CD47-His蛋白进行三次富集噬菌体抗体库。将富集的噬菌体，洗脱、转化、涂板、挑取单克隆进行噬菌体与CD47蛋白ELISA的结合鉴定，将结合读值>1.0的克隆进行测序，并克隆至表达载体phv13，转化SS320细胞表达生产纳米单抗。
淘选后的文库与CD47蛋白进行结合检测。噬菌体ELISA结果显示，没有富集前的CD47-VHH噬菌体文库与CD47蛋白的结合读值为0.33，经过一轮、二轮、三轮富集后的噬菌体文库读值分别为0.49、1.73、3.34(图4A)。为了进一步验证富集后的文库中结合CD47-VHH蛋白的阳性噬菌体率，从第2、3轮富集后的文库里分别挑选了40、46个克隆进行单个噬菌体ELISA检测。结果显示，第2轮文库里，42.5％的单个噬菌体克隆为阳 性，第3轮文库里89％的噬菌体克隆为阳性，而且结合的平均读值在3.0左右(图4B)，通过CD47蛋白淘选成功地富集了高结合力的CD47-VHH噬菌体文库。
4.VHH原核表达文库的构建及VHH表达
对上述二轮和三轮淘选富集后的2nd-CD47-VHH和3rd-cCD47-VHH噬菌体抗体文库进行PCR扩增；获取并纯化抗体库中所有VHH的基因片段，将VHH的基因片段克隆至原核表达载体，转化SS320菌株，构建VHH的原核表达抗体库；将原核表达抗体库涂布平板，过夜培养，次日随机挑选单克隆菌落600个，使用IPTG诱导表达抗体上清，对抗体上清与CD47蛋白进行ELISA结合检测。
结果显示，有细菌上清与CD47蛋白结合，同时不与空白对照结合，CD47结合的读值/空白对照的读值大于5.0(图5和表1)。将这些序列进行测序比对，剔除重复序列，最终获得52个的VHH抗体序列。进一步的实验证实，这52个VHH抗体中有19个抗体可与细胞表面CD47蛋白结合(SEQ ID NO:1-19)。
表1 19个VHH抗体与CD47蛋白的结合值及其序列
Figure PCTCN2021075043-appb-000001
5.流失细胞法检测VHH抗体与肿瘤细胞的结合
将VHH抗体与PRMI 8226细胞混合孵育，100μl/样品，4℃1h；以0.5％PBSF洗涤两遍后，加入二抗Alexa Fluor 488 goat anti human IgG，4℃30min；以0.5％PBSF洗涤两遍后，上机检测。MOCK为PBS对照；neg组为阴性对照，即不含抗体的原核表达上清对照；positive为阳性对照，即可结合CD47膜蛋白的阳性抗体对照。结果如图6和7所示，流式检测显示，19个VHH抗体均可与PRMI8226细胞结合，其中，IAP-114、118、121、129、132、140和148的结合力较高。使用人源化的VHH抗体亦得到类似的结果。可见，上述VHH抗体具有靶向结合肿瘤细胞的能力，同时有可能通过阻断肿瘤细胞表面的CD47分子，促进巨噬细胞的吞噬作用，从而达到治疗或者抑制肿瘤生长的效果，因此这19个VHH抗体均有潜力成为治疗肿瘤的新型抗体药物。
因为19个VHH能够识别肿瘤细胞表面的CD47分子，因此19个VHH抗体序列也可以应用于CAR(Chimeric Antigen Receptor,抗原嵌合受体,由VHH序列融合第三代或者第四代CD28-4-1BB-CD3zeta分子序列构成)细胞治疗肿瘤的治疗。另外因为19个VHH能识别肿瘤细胞表面的CD47分子，因此VHH可以通过偶联药物用于ADC(Antibody-drug conjugate,抗体偶联药物)治疗或者偶联同位素用于依赖抗体的分子影像诊断等。
为肿瘤的临床治疗提供潜在的纳米新药。
6.使用AAV病毒载体装载的人源化VHH进行体内实验
腺相关病毒载体(AAV)源于非致病的野生型腺相关病毒，由于其安全性好、宿主细胞范围广(分裂和非分裂细胞)、免疫源性低，在体内表达外源基因时间长等特点，被视为最有前途的基因转移载体之一，在世界范围内的基因治疗和疫苗研究中得到广泛应用。
AAV Helper-Free病毒包装系统购于Cell Biolabs,San Diego USA。将上述VHH的DNA编码序列通过分子克隆技术插入到pAAV-MCS质粒；通过测序证明构建成功后，将构建好的质粒pAAV-Ab与pHelper和pAAV-DJ质粒按照质量比1:1:1的方式使用PEI转染试剂共转染AAV-293T细胞。转染后分别于48、72、96和120小时收集上清，并用5xPEG8000(sigma)进行浓缩，最后用1.37g/ml氯化铯进行纯化。纯化的AAV溶解于PBS 里，进行鉴定和分装后保存于-80℃。
使多发性骨髓瘤模型小鼠接受AAV-VVH(1x10 11gc/100μl)肌肉注射，以AAV-GFP为对照组。结果显示，AAV-VVH对多发性骨髓瘤有治疗作用。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种可结合CD47的多肽，其特征在于，包括3个互补决定区CDR1-3，CDR1序列为或包括SEQ ID NO:1-13所示序列之一，CDR2序列为或包括SEQ ID NO:14-24所示序列之一，CDR3序列为或包括SEQ ID NO:25-34所示序列之一。
  2. 根据权利要求1所述的多肽，其特征在于，所述多肽还包括4个框架区FR1-4，所述FR1-4与所述CDR1-3按顺序交错排列。
  3. 根据权利要求2所述的多肽，其特征在于，所述多肽为单克隆抗体。
  4. 根据权利要求2所述的多肽，其特征在于，所述多肽为VHH。
  5. 根据权利要求4所述的多肽，其特征在于，所述多肽为羊驼源的VHH或人源化的VHH。
  6. 权利要求1-5中任一项所述的多肽在检测细胞表面CD47中的应用。
  7. 权利要求1-5中任一项所述的多肽在制备肿瘤治疗药物中的应用。
  8. 权利要求1-5中任一项所述的多肽在制备CAT T细胞治疗剂中的应用。
  9. 权利要求1-5中任一项所述的多肽的核酸编码序列在基因治疗中的应用。
  10. 一种检测细胞表面的CD47的试剂，其特征在于，包含权利要求1-5中任一项所述的多肽。
PCT/CN2021/075043 2019-12-03 2021-02-03 可结合cd47的多肽及其应用 WO2021110181A2 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911216842.X 2019-12-03
CN201911216842.XA CN110862455B (zh) 2019-12-03 2019-12-03 可结合cd47的多肽及其应用

Publications (2)

Publication Number Publication Date
WO2021110181A2 true WO2021110181A2 (zh) 2021-06-10
WO2021110181A3 WO2021110181A3 (zh) 2021-07-29

Family

ID=69658103

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/075043 WO2021110181A2 (zh) 2019-12-03 2021-02-03 可结合cd47的多肽及其应用

Country Status (2)

Country Link
CN (1) CN110862455B (zh)
WO (1) WO2021110181A2 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110862455B (zh) * 2019-12-03 2021-12-21 源道隆（苏州）医学科技有限公司 可结合cd47的多肽及其应用
CN111808193B (zh) * 2020-06-30 2022-04-05 源道隆（苏州）医学科技有限公司 可结合人cd38的纳米抗体及其应用
CN113583979B (zh) * 2021-08-03 2022-11-22 杭州荣谷生物科技有限公司 一种重组溶瘤痘苗病毒、制备方法及其用途

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3470424A4 (en) * 2016-06-08 2020-03-04 Shanghai Jiaotong University School of Medicine SEQUENCE OF HEAVY CHAIN CONSTANT ANTIBODY REGION TO INCREASE AGONIST-ANTIBODY ACTIVITY
CN109096395B (zh) * 2017-06-20 2022-06-24 华兰生物工程股份有限公司 阻断型cd47纳米抗体及其用途
CN110144009B (zh) * 2018-02-14 2020-01-21 上海洛启生物医药技术有限公司 Cd47单域抗体及其用途
CN110003335B (zh) * 2019-04-12 2023-07-04 深圳普瑞金生物药业股份有限公司 Cd47单域抗体、核酸及试剂盒
CN110256565B (zh) * 2019-08-02 2021-03-23 天津大学 抗cd47纳米抗体突变体及其应用
CN110862455B (zh) * 2019-12-03 2021-12-21 源道隆（苏州）医学科技有限公司 可结合cd47的多肽及其应用

Also Published As

Publication number Publication date
WO2021110181A3 (zh) 2021-07-29
CN110862455B (zh) 2021-12-21
CN110862455A (zh) 2020-03-06

Similar Documents

Publication Publication Date Title
CN112601762B (zh) 抗cd47抗体及其应用
CN111051347B (zh) Tigit抗体、其抗原结合片段及医药用途
CN112094343B (zh) 与SARS-CoV-2 RBD结合的羊驼源纳米抗体
WO2021110181A2 (zh) 可结合cd47的多肽及其应用
CN111848798B (zh) 可结合bcma的纳米抗体及其应用
CN112724248A (zh) 可结合SARS-CoV-2的纳米抗体及其应用
CN110713536B (zh) 可结合sftsv的多肽、其核酸编码序列及应用
CN106946989B (zh) 抗cea抗原vhh结构域及含有其的双特异性抗体
WO2021129891A1 (zh) 可结合cd4的多肽及其应用
EP3683234A1 (en) Il-6r antibody and antigen binding fragment thereof and medical use
CN114276452A (zh) 可结合bcma的纳米抗体及其应用
CN110922482B (zh) 可结合cd19的多肽及其应用
CN111808193B (zh) 可结合人cd38的纳米抗体及其应用
CN110950957B (zh) 可结合ctla4的多肽及其应用
CN110950956B (zh) 可结合pd1的多肽及其应用
US20230086530A1 (en) Human cd47-targeting single-domain antibody and use thereof
CN114292329B (zh) 抗cd19抗体及其应用
CN111393527B (zh) 可结合pd-l1的多肽及其应用
CN117843776B (zh) 抗体分子、核酸、制药用途及炎性疾病治疗方法
WO2023143484A1 (zh) 一种抗原结合蛋白及其用途
WO2022037002A1 (zh) 特异性结合糖基化ceacam5的抗体
TW202222836A (zh) 特異性結合糖基化ceacam5的抗體
CN118459591A (zh) 抗ildr2抗体及其用途
CN114249821A (zh) 与SARS-CoV-2 RBD结合的羊驼源纳米抗体
CN114249822A (zh) 与SARS-CoV-2 RBD结合的羊驼源纳米抗体

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21729785

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21729785

Country of ref document: EP

Kind code of ref document: A2