WO2020073215A1 - 抗BCMA单链抗体scFv及其制备方法和应用 - Google Patents

抗BCMA单链抗体scFv及其制备方法和应用 Download PDF

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WO2020073215A1
WO2020073215A1 PCT/CN2018/109564 CN2018109564W WO2020073215A1 WO 2020073215 A1 WO2020073215 A1 WO 2020073215A1 CN 2018109564 W CN2018109564 W CN 2018109564W WO 2020073215 A1 WO2020073215 A1 WO 2020073215A1
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seq
sequence
chain variable
variable region
region
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PCT/CN2018/109564
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French (fr)
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王媚娘
欧阳冰洁
杨乃波
姜琳
任丙昭
柳阳
赵正琦
李波
侯勇
王飞
葛玉萍
董旋
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深圳华大生命科学研究院
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Priority to PCT/CN2018/109564 priority Critical patent/WO2020073215A1/zh
Priority to JP2021546025A priority patent/JP7126624B2/ja
Priority to CN201880098546.1A priority patent/CN112805302B/zh
Priority to EP18936571.1A priority patent/EP3854815A4/en
Priority to US17/283,976 priority patent/US20210246220A1/en
Publication of WO2020073215A1 publication Critical patent/WO2020073215A1/zh

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    • 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
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30 CD40 or CD95

Definitions

  • the invention relates to the technical field of antibodies, in particular to an anti-BCMA single chain antibody scFv, and a preparation method and application thereof.
  • CAR-T therapy has attracted great attention from researchers since its birth, especially in the treatment of advanced leukemia patients in 2011. It has achieved great success. In 2012, it achieved rapid remission after achieving remission of about 80% of lymphomas. development of.
  • the current CAR-T therapy is mainly based on the second-generation CAR.
  • the treatment method is to isolate T cells from the patient, and use engineered means to equip T cells with genetically modified chimeric antigen receptors (CAR), CAR It is a composite membrane receptor molecule, which includes two functional parts, extracellular and intracellular, and has the function of specifically targeting the target molecule and activating T cells.
  • CAR genetically modified chimeric antigen receptors
  • CAR The extracellular part of CAR is a recombinant receptor (scFv) composed of single-chain variable fragments of antibodies. Through scFv mediation, it specifically targets the target molecule in a way of specific binding of antigen and antibody, activates T cells, and thus exerts a tumor killing effect. .
  • scFv recombinant receptor
  • B cell maturation antigen (B Cell Antigen, BCMA, CD269) is a member of the tumor necrosis factor receptor (TNFR) superfamily and is a non-glycosylated type III integrated membrane protein composed of 185 amino acids.
  • BCMA B cell activation factor
  • APRIL proliferation inducing ligand
  • BCMA was first discovered on the surface of mature B lymphocytes, and it was hardly expressed in other tissue cells, but was highly expressed in malignantly proliferating B lymphocytes (such as myeloma cells and leukemia cells), and mediated downstream signaling pathways. Cell survival, proliferation, metastasis and drug resistance play a key role. These characteristics make it a new drug target for the diagnosis and immunotherapy of multiple myeloma (Multiple Myeloma, MM).
  • LCAR-B38 was submitted by Nanjing Legend Biological Company in December 2017. It is the first BCMA-CART to submit a clinical application in China.
  • a clinical trial data of 35 patients with relapsed or drug-resistant multiple myeloma showed The objective response rate of the therapy reached 100%, and 19 patients achieved a complete response diagnostic standard during the early 4-month median follow-up period.
  • the research of CAR-T therapy for MM targeting BCMA is just in the emerging stage, but the results of preclinical and clinical phase I trials show that the targeted drugs for BCMA can effectively target tumor cells and pass The cytotoxic effect of immune cells or small molecules kills tumor cells. It has significant drug effects and controllable adverse reactions. It is a new target for tumor targeted therapy with great development potential.
  • the invention provides an anti-BCMA single-chain antibody scFv and a preparation method and application thereof.
  • an embodiment provides an anti-BCMA single chain antibody scFv, including a light chain variable region (VL) and a heavy chain variable region (VH),
  • the light chain variable region (VL) includes a framework region (FR) and a complementary structure region (CDR), wherein the complementary structure region (CDR) includes CDR1, the sequence of which is QDISNY (SEQ ID NO: 14), CDR2, the sequence of which is YTS (SEQ ID NO: 16), CDR3, whose sequence is QQYRKLAWT (SEQ ID NO: 18);
  • the above heavy chain variable region (VH) includes a framework region (FR) and a complementary structure region (CDR), in which the complementary structure region (CDR) includes CDR1 with the sequence GGTFSNYW (SEQ ID NO: 21), CDR2 with the sequence TYRGHSDT (SEQ ID NO: 23), CDR3 with the sequence ARGAIYNGYDVLDN (SEQ ID NO: 25); or
  • the light chain variable region (VL) includes a framework region (FR) and a complementary structure region (CDR), wherein the complementary structure region (CDR) includes CDR1, the sequence of which is QDISNY (SEQ ID NO: 28), CDR2, the sequence of which is YTS (SEQ ID NO: 30), CDR3, whose sequence is QQALVVTPFT (SEQ ID NO: 32);
  • the above heavy chain variable region (VH) includes a framework region (FR) and a complementary structure region (CDR), in which the complementary structure region (CDR) includes CDR1, whose sequence is GGTFSNYW (SEQ ID NO: 35), CDR2, whose sequence is TYRGHSDT (SEQ ID NO: 37), CDR3, whose sequence is ARFGMLDN (SEQ ID NO: 39); or
  • the light chain variable region (VL) includes a framework region (FR) and a complementary structure region (CDR), wherein the complementary structure region (CDR) includes CDR1, the sequence of which is QDISNY (SEQ ID NO: 42), CDR2, the sequence of which is YTS (SEQ ID NO: 44), CDR3, whose sequence is QQRLTPSPFT (SEQ ID NO: 46);
  • the above heavy chain variable region (VH) includes a framework region (FR) and a complementary structure region (CDR), in which the complementary structure region (CDR) includes CDR1, its sequence is GGTFSNYW (SEQ ID NO: 49), CDR2, its sequence is TYRGHSDT (SEQ ID NO: 51), CDR3, its sequence is ARNTALLDN (SEQ ID NO: 53).
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • sequence of the above single chain antibody scFv is:
  • an embodiment provides a polynucleotide sequence encoding the anti-BCMA single-chain antibody scFv of the first aspect, including the complementary structure region sequence SEQ ID NO that encodes the light chain variable region (VL) described above: 14.
  • the above polynucleotide sequence includes the nucleotide sequences encoding the complementary structural region sequences SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 32 of the light chain variable region (VL); and the heavy chain encoding The nucleotide sequence of the complementary structure region sequence of the variable region (VH) SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 39; or
  • the above polynucleotide sequence includes the nucleotide sequences encoding the complementary structure region sequences SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 46 of the light chain variable region (VL); and the heavy chain encoding The nucleotide sequences of the variable structure (VH) complementary sequence regions SEQ ID NO: 49, SEQ ID NO: 51 and SEQ ID NO: 53.
  • the polynucleotide sequence includes a nucleotide sequence encoding the light chain variable region (VL) sequence SEQ ID NO: 4; and a core encoding the heavy chain variable region (VH) sequence SEQ ID NO: 3 Nucleotide sequence; or
  • the polynucleotide sequence includes a nucleotide sequence encoding the light chain variable region (VL) sequence SEQ ID NO: 8; and a nucleotide sequence encoding the heavy chain variable region (VH) sequence SEQ ID NO: 7 ;or
  • the polynucleotide sequence includes the nucleotide sequence encoding the light chain variable region (VL) sequence SEQ ID NO: 12; and the nucleotide sequence encoding the heavy chain variable region (VH) sequence SEQ ID NO: 11 .
  • the above polynucleotide sequence is:
  • an embodiment provides an expression vector containing the polynucleotide sequence of the second aspect.
  • an embodiment provides a host cell containing the expression vector of the third aspect, capable of expressing an anti-BCMA single chain antibody scFv.
  • an embodiment provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-BCMA single chain antibody scFv of the first aspect, and a pharmaceutically acceptable carrier, diluent or excipient.
  • an embodiment provides a method for preparing the anti-BCMA single chain antibody scFv of the first aspect, comprising: transforming an expression vector comprising the polynucleotide sequence of the second aspect into an expression host cell, and culturing , Large-scale expression and purification of the above anti-BCMA single chain antibody scFv.
  • the above expression vector is a pComb3XSS vector
  • the above host cells are E. coli XL1-Blue and TG1 strains.
  • an embodiment provides the use of the anti-BCMA single chain antibody scFv of the first aspect in the preparation of BCMA targeted drugs, or the use of immunological detection of BCMA for non-disease diagnosis and treatment purposes.
  • the invention combines the phage display library targeting BCMA with the combination of site-directed mutation and random mutation technology, and combines with the phage display technology to obtain 3 new scFv strains targeting BCMA antigen, which can be used as BCMA-targeting antibodies for in-depth research and development .
  • Example 1 is a graph of electrophoretic detection results of a site-directed mutation secondary library and a random library in Example 1 of the present invention
  • FIG. 2 is a graph showing the results of recombination efficiency of scFv fragments in a phage display library constructed by clone PCR in Example 1 of the present invention, wherein clones 1-36 are clones of the secondary library, and clones 37-72 are clones of the random library;
  • FIG. 3 is a graph showing the affinity results of phage library targeting BCMA after 4 rounds of affinity elutriation by ELISA in Example 2 of the present invention
  • FIG. 4 is a scFv monoclonal PCR screening result targeting BCMA in Example 2 of the present invention, wherein clones 1-36 are clones screened by a random library, clones 37-56 are clones screened by a secondary library, and arrows indicate sequences Mutated clones;
  • FIG. 5 is a graph showing the affinity results of scFv candidate strains targeting BCMA by ELISA in Example 2 of the present invention.
  • the present invention Based on the humanized scFv sequence resistant to BCMA, the present invention performs site-directed mutation and random mutation on the CDR3 regions of its heavy and light chains to artificially synthesize a phage library of scFv resistant to BCMA; The antigen was subjected to multiple rounds of affinity elutriation and screening. Finally, the monoclonal antibody strains obtained by the screening were subjected to sequence analysis and affinity detection. The new anti-BCMA scFv strains can be used as antibodies to BCMA targeted drugs for downstream immunotherapy. Development of therapeutic drugs.
  • the present invention uses the humanized scFv sequence as the backbone, and only artificially transforms its CDR3 region, which can greatly reduce the difficulty of humanizing the antibody in the later stage, and at the same time reduce the impact of the modification on the affinity of the antibody targeting antigen; at the same time,
  • the present invention uses phage display technology in combination to obtain antibody affinity information quickly and intuitively, and obtain multiple new high-affinity scFvs in a relatively short time, providing more ways to develop immunotherapy targeting BCMA , With continued development value.
  • the present invention mutates the CDR3 of its heavy and light chains, and combines with phage display technology to develop three new scFv antibody strains targeting BCMA.
  • the scFv antibody obtained by the invention can efficiently target and bind to BCMA antigen, and is used for the diagnosis or treatment of targeted BCMA.
  • an anti-BCMA single chain antibody scFv including a light chain variable region (VL) and a heavy chain variable region (VH), and the light chain variable region (VL) includes a framework region (FR ) And complementary structure region (CDR), wherein the complementary structure region (CDR) includes CDR1, its sequence is QDISNY (SEQ ID NO: 14), CDR2, its sequence is YTS (SEQ ID NO: 16), CDR3, its sequence is QQYRKLAWT (SEQ ID NO: 18); the heavy chain variable region (VH) includes a framework region (FR) and a complementary structure region (CDR), wherein the complementary structure region (CDR) includes CDR1, and its sequence is GGTFSNYW (SEQ ID NO: 21), CDR2, its sequence is TYRGHSDT (SEQ ID NO: 23), CDR3, its sequence is ARGAIYNG
  • an anti-BCMA single chain antibody scFv includes a light chain variable region (VL) and a heavy chain variable region (VH), wherein the light chain variable region (VL) sequence is SEQ ID NO : 4; heavy chain variable region (VH) sequence is SEQ ID NO: 3; or light chain variable region (VL) sequence is SEQ ID NO: 8; heavy chain variable region (VH) sequence is SEQ ID NO: 7; or the light chain variable region (VL) sequence is SEQ ID NO: 12; the heavy chain variable region (VH) sequence is SEQ ID NO: 11.
  • sequence of an anti-BCMA single chain antibody scFv is SEQ ID NO: 1; or SEQ ID NO: 5; or SEQ ID NO: 9.
  • a technical solution of the present invention claims a polynucleotide sequence encoding the anti-BCMA single-chain antibody scFv of the present invention, including the complementary structure region sequence SEQ ID NO: 14, SEQ ID encoding the light chain variable region (VL)
  • the polynucleotide sequence includes a nucleotide sequence encoding a light chain variable region (VL) sequence SEQ ID NO: 4; and a heavy chain variable region (VH) sequence SEQ ID NO: 3 Nucleotide sequence; or polynucleotide sequence including the nucleotide sequence encoding the light chain variable region (VL) sequence SEQ ID NO: 8; and the core encoding the heavy chain variable region (VH) sequence SEQ ID NO: 7
  • the nucleotide sequence; or the polynucleotide sequence includes the nucleotide sequence encoding the light chain variable region (VL) sequence SEQ ID NO: 12; and the nucleotide sequence encoding the heavy chain variable region (VH) sequence SEQ ID NO: 11 Acid sequence.
  • polynucleotide sequence is: SEQ ID NO: 2; or SEQ ID NO: 6; or SEQ ID NO: 10.
  • a phagemid expression vector which contains the polynucleotide sequence of the present invention.
  • many vectors such as pComb3XSS, pComb3XTT, pComb3HSS, etc., can be used as the expression vector of the polynucleotide sequence of the present invention.
  • the expression vector is a phage display vector pComb3XSS vector (purchased from Wuhan Miaoling Biotechnology Co., Ltd.).
  • a host cell which contains the expression vector of the present invention and can express an anti-BCMA single chain antibody scFv on the surface of a phage.
  • a host cell which contains the expression vector of the present invention and can express an anti-BCMA single chain antibody scFv on the surface of a phage.
  • many cells such as ER2738, SS320, TG1, XL1-Blue, etc. can be used as host cells for the expression vector of the present invention.
  • the host cells are E. coli XL1-Blue strain (purchased from TAKARA) and TG1 strain (purchased from Lucigen, USA).
  • a pharmaceutical composition comprising the anti-BCMA single chain antibody scFv of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition of the present invention can be prepared by a method well known in the art (for example, Remington: The Science and Practice of Pharmacy, 19th. (1995), A. Gennaro et al., Mack Publishing).
  • a method for preparing the anti-BCMA single chain antibody scFv of the present invention includes: transforming an expression vector containing the polynucleotide sequence of the present invention into an expression host cell, culturing, and performing anti-BCMA Large-scale expression and purification of single chain antibody scFv.
  • the expression vector is a phage display vector pComb3XSS vector
  • the host cells are E. coli XL1-Blue strain and TG1 strain.
  • the anti-BCMA single chain antibody scFv of the present invention can be used to prepare anti-BCMA protein monoclonal antibody drugs, and can also be used for immunological detection of BCMA. Therefore, in one embodiment of the present invention, the use of the anti-BCMA single chain antibody scFv of the present invention in the preparation of BCMA targeted drugs or the use of immunological detection of BCMA for non-disease diagnosis and treatment purposes is provided.
  • the heavy chain and light chain variable region genes of synthetic BCMA antibodies were inserted into pComb3XSS vector (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) from the site Sfi I.
  • the primer information for the amplification of antibody fragments is shown in Table 4.
  • Centrifuge the bacterial solution collect the supernatant, add 1/5 volume of PEG / NaCl, mix well, incubate for 30 min on ice, precipitate the phage particles, centrifuge at 12000 rpm for 30 min, discard the supernatant, resuspend the phage particles in PBS, and centrifuge again at 12000 rpm for 5 min.
  • dU-ssDNA uracil-containing single-stranded template DNA
  • M13KO7 M13KO7 was cultured for 1 h
  • the bacterial solution was transferred to 300 ml of medium containing Amp + , kanamycin (Kan + ) and uracil (Uridine), and cultured at 37 ° C. overnight.
  • Centrifuge the bacterial solution collect the supernatant, add 1/4 volume of PEG / NaCl, mix well, incubate for 30min on ice, precipitate the phage particles, centrifuge at 12000rpm for 25min, discard the supernatant, collect the phage particles, use DNA extraction kit, extract dU -ssDNA, and quantify.
  • Each primer dry powder was prepared into 100 ⁇ M working solution, random library primers were mixed into heavy chain primer H3-211 (H3-2, H3-4, H3-9, H3-11 5 ⁇ L each) and light chain primer L3-459 (L3-4 , L3-5, L3-9 5 ⁇ L).
  • the corresponding phosphorylation mutant primers of the secondary library and the random library were mixed and annealed with the dU-ssDNA template.
  • Each library had 1 reaction and each reaction system was 250 ⁇ L, as follows: primers (each library includes light chain and heavy chain) Two primers for the chain) 40 ⁇ L, 10X T4 Buffer 25 ⁇ L, dU-ssDNA template 20 ⁇ L, deionized water to 250 ⁇ L.
  • T4 ligase 5 ⁇ L T4 ligase was added to each system, and the ligation reaction was performed at room temperature for 2h.
  • the ligation product was verified by DNA electrophoresis. As shown in Figure 1, the band was significantly shifted compared to the template, indicating that the scFv was successfully constructed on the vector.
  • Two library storage capacity and random libraries obtained by calculation is greater than 10 8 / ml. All clones were eluted with LB, 5,000g, centrifuged for 5min, the pellet was suspended with 2ml LB, an equal volume of 30% glycerol was added, and frozen at -80.
  • the recombination rates of the library (clone 1-36) and random library (clone 37-72) were 94% and 100%.
  • 56 clones were randomly selected for Sanger sequencing to analyze the diversity of the phage display library; analysis of the scFv sequence showed that 11 of 26 sequences in the secondary library had mutations, and 14 of 25 sequences in the random library had mutations, diversity Reaching 42% and 56%, respectively, can be used for the next step of screening for new antibodies.
  • the precipitate is the phage library with scFv carrying BCMA. After suspending the pellet with PBS, determine its titer, secondary The library is 2.6X 10 12 pfu / ml, and the random library is 3.3X 10 12 pfu / ml.
  • the amplification and rescue method is the same as step (6) in Example 1, the obtained PBS suspension is the amplified phage after the first round of elutriation, stored at 4 °C, and used for the next round of screening; according to the example In the elutriation step of step (1) in 2, the antigen amount is gradually reduced in each round, and elutriation is performed for 3-4 rounds.
  • ELISA plates were coated with 100ng BCMA-Fc antigen and incubated overnight at 4 ° C; pick these 5 monoclonals in 1ml medium and culture at 37 ° C to log phase, add 1mM IPTG to induce overnight; the next day, centrifuge to collect bacterial pellets After centrifugation, centrifuge at 5,000g for 15min to collect supernatant; at the same time, take ELISA plate and add 2% BSA to block at room temperature for 1h; add single-cell crushing supernatant to each well of the experimental group, and add blank TG to crush the supernatant at the control group, incubate at room temperature 2h; PBST 10 times, add mouse anti-HA tag antibody, room temperature for 1h; PBST 3-5 times, add AP labeled anti-mouse IgG antibody, room temperature for 1h; add substrate, react for 10-20min, in a microplate reader Read the absorbance value on the top; when the ratio of the absorbance value to the control well is greater than
  • the three scFv monoclonals are named scFv_20, scFv_43, and scFv_46, respectively.
  • the nucleotide and amino acid sequences are as follows:

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Abstract

提供一种抗BCMA单链抗体scFv及其制备方法，包括结合定点突变和随机突变技术人工合成了靶向BCMA的噬菌体展示库，结合噬菌体展示技术，获得3株靶向BCMA抗原的新型scFv株，可作为靶向BCMA的抗体。

Description

抗BCMA单链抗体scFv及其制备方法和应用 技术领域
本发明涉及抗体技术领域，具体涉及一种抗BCMA单链抗体scFv及其制备方法和应用。
背景技术
在过去的几年里，过继性T细胞免疫治疗已经成为癌症免疫治疗中最有希望的策略之一。CAR-T疗法自诞生起就引起了研究人员的极大关注，尤其在2011年用于白血病晚期病人治疗中取得了巨大的成功，2012年对约80％的淋巴瘤实现缓解后，得到了飞速的发展。目前的CAR-T疗法主要以第二代CAR为主，治疗手段为从患者体内分离出T细胞，使用工程化的手段让T细胞装备上具有基因修饰的嵌合抗原受体(CAR)，CAR是复合膜受体分子，包括细胞外和胞内两个功能部分，同时具有特异性定位到靶标分子和活化T细胞的功能。CAR的胞外部分是抗体单链可变片段组成的重组受体(scFv)，通过scFv介导，以抗原抗体特异性结合的方式特异性定位到靶分子，激活T细胞，从而发挥肿瘤杀伤作用。
在治疗血液病的成功后，人们开始将目光转向实体瘤领域，包括多发性骨髓瘤，乳腺癌，前列腺癌，卵巢癌，胶质瘤，结直肠癌，肝转移，间皮瘤，肾癌，肉瘤，神经细胞癌和胸膜间皮瘤等。2017年被称为细胞治疗元年，法国生物技术公司Cellectis在3月宣布其研究开发的通用型CAR-T疗法UCART123获得了美国FDA研究型新药(IND)一期临床试验批准，这是FDA批准的全球首个以CD123为靶点的同种异体方法的CAR-T临床试验；同年8月，美国FDA批准了Novartis的靶向CD19的CAR-T产品Kymriah，10月，Kite的用于特定类型的大B细胞瘤成人患者治疗的产品Yescarta的上市，这些成果极大地推动了研究者对于细胞免疫治疗研发的热情。作为免疫治疗的最新技术，CAR-T可应用的范围广，前景广阔，预计CAR-T在中国的市场价值超过1000亿。
B细胞成熟抗原(B Cell Maturation Antigen，BCMA，CD269)是肿瘤坏死因子受体(TNFR)超家族的成员，是由185个氨基酸组成的非糖基化的III型整合膜蛋白。BCMA结合B细胞激活因子(BAFF，也称BLys，THANK，TALL-1)和增殖诱导配体(APRIL)，在管理B细胞的成熟和B细胞分化成浆细胞的过程中扮演着至关重要的角色。研究表明，BCMA的过表达会引起蛋白激酶B、MAPK和NF-κB信号的产生，从而增强骨髓瘤细胞的增殖和存活。BCMA最早发现于成熟的B淋巴细胞表面，在其他组织细胞中几乎不表达，而在恶性增殖的 B淋巴细胞(例如骨髓瘤细胞、白血病细胞)中高度表达，并介导的下游信号通路，对细胞的存活、增殖、转移和耐药中起着关键性的作用，这些特性使得它成为用于诊断和免疫治疗多发性骨髓瘤(Multiple Myeloma，MM)的一个新的药物靶点。
随着MM和其他B细胞相关疾病发病率的上升，临床期待在短时间内开发出针对B细胞的特异性疗法，近几年来，针对BCMA的新型肿瘤免疫治疗方法日趋成熟，主要包括CAR-T(Chimeric Antigen Receptor T-Cell Immunotherapy)疗法、双特异性抗体(Bispecific antibody，BsAb)和抗体药物偶联物(Antibody-drug coupling，ADC)。在2017年4月的美国癌症研究协会年会(AACR)上，BCMA一跃成为明星靶点，国内外公司争相开发靶向BCMA的CAR-T疗法，目前已有4类药物进入临床I期，主要有KITE-585，bb2121，LCAR-B38等。LCAR-B38由南京传奇生物公司于2017年12月提交国内临床申请，是国内首个提交临床申请的BCMA-CART，一项35例复发或耐药性多发性骨髓瘤患者参与的临床试验数据显示，该疗法的客观缓解率达到了100％，19名病人在早期为期4个月的中位随访期中有14名达到了完全反应诊断标准。目前，CAR-T疗法用于靶向BCMA的MM的研究处于刚刚兴起的阶段，但临床前及临床I期试验的结果来看，针对BCMA的靶向药物能够有效的靶向肿瘤细胞，并通过免疫细胞或小分子的细胞毒作用杀伤肿瘤细胞，药效显著，不良反应可控，是一个极具发展潜力的肿瘤靶向治疗新靶点。
随着多发性骨髓瘤和其他B细胞相关疾病的蔓延，医药界急需研发针对B细胞的特效疗法，但现有技术对于BCMA抗体药物的开发还集中在采用鼠源传统抗体技术，传统抗体无论是大量表达，还是进行抗体人源化改造都比较困难，耗时长，花费高，并且有效抗体获得率低，严重限制了有效靶向药物的开发。
发明内容
本发明提供一种抗BCMA单链抗体scFv及其制备方法和应用。
根据第一方面，一种实施例中提供一种抗BCMA单链抗体scFv，包括轻链可变区(VL)和重链可变区(VH)，
上述轻链可变区(VL)包括框架区(FR)和互补结构区(CDR)，其中互补结构区(CDR)包括CDR1，其序列为QDISNY(SEQ ID NO：14)，CDR2，其序列为YTS(SEQ ID NO：16)，CDR3，其序列为QQYRKLAWT(SEQ ID NO：18)；上述重链可变区(VH)包括框架区(FR)和互补结构区(CDR)，其中互补结构区(CDR)包括CDR1，其序列为GGTFSNYW (SEQ ID NO：21)，CDR2，其序列为TYRGHSDT(SEQ ID NO：23)，CDR3，其序列为ARGAIYNGYDVLDN(SEQ ID NO：25)；或者
上述轻链可变区(VL)包括框架区(FR)和互补结构区(CDR)，其中互补结构区(CDR)包括CDR1，其序列为QDISNY(SEQ ID NO：28)，CDR2，其序列为YTS(SEQ ID NO：30)，CDR3，其序列为QQALVVTPFT(SEQ ID NO：32)；上述重链可变区(VH)包括框架区(FR)和互补结构区(CDR)，其中互补结构区(CDR)包括CDR1，其序列为GGTFSNYW(SEQ ID NO：35)，CDR2，其序列为TYRGHSDT(SEQ ID NO：37)，CDR3，其序列为ARFGMLDN(SEQ ID NO：39)；或者
上述轻链可变区(VL)包括框架区(FR)和互补结构区(CDR)，其中互补结构区(CDR)包括CDR1，其序列为QDISNY(SEQ ID NO：42)，CDR2，其序列为YTS(SEQ ID NO：44)，CDR3，其序列为QQRLTPSPFT(SEQ ID NO：46)；上述重链可变区(VH)包括框架区(FR)和互补结构区(CDR)，其中互补结构区(CDR)包括CDR1，其序列为GGTFSNYW(SEQ ID NO：49)，CDR2，其序列为TYRGHSDT(SEQ ID NO：51)，CDR3，其序列为ARNTALLDN(SEQ ID NO：53)。
优选地，上述轻链可变区(VL)为：
Figure PCTCN2018109564-appb-000001
上述重链可变区(VH)为：
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGSIFNGYDVLDNWGQGTLVTVSS(SEQ ID NO：3)；或者
上述轻链可变区(VL)为：
Figure PCTCN2018109564-appb-000002
上述重链可变区(VH)为：
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARFGMLDNWGQGTLVTVSS(SEQ ID NO：7)；或者
上述轻链可变区(VL)为：
Figure PCTCN2018109564-appb-000003
上述重链可变区(VH)为：
Figure PCTCN2018109564-appb-000004
优选地，上述单链抗体scFv的序列为：
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLAWTFGQGTKLEIKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGSIFNGYDVLDNWGQGTLVTVSS(SEQ ID NO：1)；或者
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALVVTPFTFGQGTKLEIKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARFGMLDNWGQGTLVTVSS(SEQ ID NO：5)；或者
Figure PCTCN2018109564-appb-000005
根据第二方面，一种实施例中提供一种编码第一方面的抗BCMA单链抗体scFv的多核苷酸序列，包括编码上述轻链可变区(VL)的互补结构区序列SEQ ID NO：14、SEQ ID NO：16和SEQ ID NO：18的核苷酸序列；以及编码上述重链可变区(VH)的互补结构区序列SEQ ID NO：21、SEQ ID NO：23和SEQ ID NO：25的核苷酸序列；或者
上述多核苷酸序列包括编码上述轻链可变区(VL)的互补结构区序列SEQ ID NO：28、SEQ ID NO：30和SEQ ID NO：32的核苷酸序列；以及编码上述重链可变区(VH)的互补结构区序列SEQ ID NO：35、SEQ ID NO：37和SEQ ID NO：39的核苷酸序列；或者
上述多核苷酸序列包括编码上述轻链可变区(VL)的互补结构区序列SEQ ID NO：42、 SEQ ID NO：44和SEQ ID NO：46的核苷酸序列；以及编码上述重链可变区(VH)的互补结构区序列SEQ ID NO：49、SEQ ID NO：51和SEQ ID NO：53的核苷酸序列。
优选地，上述多核苷酸序列包括编码上述轻链可变区(VL)序列SEQ ID NO：4的核苷酸序列；以及编码上述重链可变区(VH)序列SEQ ID NO：3的核苷酸序列；或者
上述多核苷酸序列包括编码上述轻链可变区(VL)序列SEQ ID NO：8的核苷酸序列；以及编码上述重链可变区(VH)序列SEQ ID NO：7的核苷酸序列；或者
上述多核苷酸序列包括编码上述轻链可变区(VL)序列SEQ ID NO：12的核苷酸序列；以及编码上述重链可变区(VH)序列SEQ ID NO：11的核苷酸序列。
优选地，上述多核苷酸序列为：
GACATCCAGATGACCCAGAGCCCTAGCTCACTGAGCGCCAGCGTGGGCGACAGGGTGACCATTACCTGCTCCGCCAGCCAGGACATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTACACCTCCAACCTGCACTCCGGCGTGCCCAGCAGGTTCAGCGGAAGCGGCAGCGGCACCGATTTCACCCTGACCATCTCCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGGAAGCTCGCATGGACTTTCGGCCAGGGCACCAAACTGGAGATCAAGCGTGGTGGAGGAGGTAGCGGAGGAGGCGGGAGCGGTGGAGGTGGCTCTGGAGGTGGCGGAAGCCAGGTGCAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAGCTGCAAGGCCAGCGGCGGCACCTTCAGCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCGGACAGGGCCTGGAGTGGATGGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAAGTTCAAGGGCCGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAACTGAGCAGCCTCAGGAGCGAGGACACCGCTGTGTATTACTGCGCCAGGGGTTCTATTTTCAACGGTTACGACGTTCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC(SEQ ID NO：2)；或者
GACATCCAGATGACCCAGAGCCCTAGCTCACTGAGCGCCAGCGTGGGCGACAGGGTGACCATTACCTGCTCCGCCAGCCAGGACATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTACACCTCCAACCTGCACTCCGGCGTGCCCAGCAGGTTCAGCGGAAGCGGCAGCGGCACCGATTTCACCCTGACCATCTCCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCTCTTGTGGTGACGCCGTTCACTTTCGGCCAGGGCACCAAACTGGAGATCAAGCGTGGTGGAGGAGGTAGCGGAGGAGGCGGGAGCGGTGGAGGTGGCTCTGGAGGTGGCGGAAGCCAGGTGCAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAGCTGCAAGGCCAGCGGCGGCACCTTCAGCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCGGACAGG GCCTGGAGTGGATGGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAAGTTCAAGGGCCGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAACTGAGCAGCCTCAGGAGCGAGGACACCGCTGTGTATTACTGCGCCAGGTTTTAGGGTATGCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC(SEQ ID NO：6)；或者
Figure PCTCN2018109564-appb-000006
根据第三方面，一种实施例中提供一种表达载体，含有第二方面的多核苷酸序列。
根据第四方面，一种实施例中提供一种宿主细胞，含有第三方面的表达载体，能够表达出抗BCMA单链抗体scFv。
根据第五方面，一种实施例中提供一种药物组合物，包含第一方面的抗BCMA单链抗体scFv，以及药学上可接受的载体、稀释剂或赋形剂。
根据第六方面，一种实施例中提供一种制备第一方面的抗BCMA单链抗体scFv的方法，包括：将包含第二方面的多核苷酸序列的表达载体转化至表达宿主细胞中，培养，进行上述抗BCMA单链抗体scFv的大量表达和纯化。
优选地，上述表达载体是pComb3XSS载体，上述宿主细胞是大肠杆菌XL1-Blue和TG1菌株。
根据第七方面，一种实施例中提供第一方面的抗BCMA单链抗体scFv在制备BCMA靶向药物中的用途，或在非疾病诊断治疗目的免疫学检测BCMA中的用途。
本发明结合定点突变和随机突变技术人工合成了靶向BCMA的噬菌体展示库，结合噬菌 体展示技术，获得了3株靶向BCMA抗原的新型scFv株，可作为靶向BCMA的抗体开展深入的研究开发。
附图说明
图1为本发明实施例1中定点突变的二级文库和随机文库电泳检测结果图；
图2为本发明实施例1中克隆PCR验证构建的噬菌体展示库中scFv片段的重组效率结果图，其中克隆1-36为二级库的克隆，克隆37-72为随机库的克隆；
图3为本发明实施例2中ELISA检测4轮亲和淘洗后的噬菌体库靶向BCMA的亲和力结果图；
图4为本发明实施例2中靶向BCMA的scFv单克隆PCR筛选结果图，其中克隆1-36为随机库筛出的克隆，克隆37-56为二级库筛出的克隆，箭头表示序列发生突变的克隆；
图5为本发明实施例2中ELISA验证scFv候选株靶向BCMA的亲和力结果图。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。在以下的实施方式中，很多细节描述是为了使得本发明能被更好的理解。然而，本领域技术人员可以毫不费力的认识到，其中部分特征在不同情况下是可以省略的，或者可以由其他元件、材料、方法所替代。
另外，说明书中所描述的特点、操作或者特征可以以任意适当的方式结合形成各种实施方式。同时，方法描述中的各步骤或者动作也可以按照本领域技术人员所能显而易见的方式进行顺序调换或调整。因此，说明书和附图中的各种顺序只是为了清楚描述某一个实施例，并不意味着是必须的顺序，除非另有说明其中某个顺序是必须遵循的。
本发明在抗BCMA的人源化scFv序列的基础上，对其重链和轻链的CDR3区域进行定点突变和随机突变，人工合成抗BCMA的scFv的噬菌体库；之后结合噬菌体展示技术，对BCMA抗原进行多轮亲和淘洗和筛选，最后对筛选获得抗体单克隆株进行序列分析和亲和力检测，已获得新型的抗BCMA的scFv株可作为BCMA靶向药物的抗体，用于下游免疫疗法的治疗药物的开发。
本发明以人源化的scFv序列为骨架，仅对其CDR3区域进行了人工改造，可以极大的降低后期对抗体人源化改造难度，同时减少改造对于抗体靶向抗原亲和力的影响；同时，本发 明结合使用噬菌体展示技术，能够比较快速直观的获得抗体亲和信息，在较短时间内获得多株新型的高亲和力的scFv，为开发以BCMA为靶点的免疫疗法提供了更多的途径，具有继续开发价值。
本发明在BCMA的scFv序列基础上，对其重链和轻链的CDR3去进行了突变，并结合噬菌体展示技术，开发出了3株靶向BCMA的新型scFv抗体株。
上述获得的3株抗BCMA的scFv抗体的氨基酸和核苷酸序列信息如表1所示：
表1抗BCMA的阳性克隆株scFv_20、scFv_43和scFv_46的核苷酸和氨基酸序列信息
Figure PCTCN2018109564-appb-000007
Figure PCTCN2018109564-appb-000008
Figure PCTCN2018109564-appb-000009
上述获得的3株scFv的轻链(L chain)和重链(H chain)的框架区(FR)和互补结构区(CDR)的氨基酸序列如表2所示：
表2抗BCMA的阳性克隆株scFv_20、scFv_43和scFv_23的轻链和重链的框架区(FR1-FR4)和互补决定区(CDR1-CDR3)氨基酸序列信息
Figure PCTCN2018109564-appb-000010
Figure PCTCN2018109564-appb-000011
Figure PCTCN2018109564-appb-000012
本发明获得的scFv抗体能高效的靶向结合BCMA抗原，应用于靶向BCMA诊断或者治疗的用途中。
需要说明的是，众所周知，抗体的特异性结合特性由互补决定区决定。因此，本发明一个技术方案要求保护一种抗BCMA单链抗体scFv，包括轻链可变区(VL)和重链可变区(VH)，轻链可变区(VL)包括框架区(FR)和互补结构区(CDR)，其中互补结构区(CDR)包括CDR1，其序列为QDISNY(SEQ ID NO：14)，CDR2，其序列为YTS(SEQ ID NO：16)，CDR3，其序列为QQYRKLAWT(SEQ ID NO：18)；重链可变区(VH)包括框架区(FR)和互补结构区(CDR)，其中互补结构区(CDR)包括CDR1，其序列为GGTFSNYW(SEQ ID NO：21)，CDR2，其序列为TYRGHSDT(SEQ ID NO：23)，CDR3，其序列为ARGAIYNGYDVLDN(SEQ ID NO：25)；或者轻链可变区(VL)包括框架区(FR)和互补结构区(CDR)，其中互补结构区(CDR)包括CDR1，其序列为QDISNY(SEQ ID NO：28)，CDR2，其序列为YTS(SEQ ID NO：30)，CDR3，其序列为QQALVVTPFT(SEQ ID NO：32)；重链可变区(VH)包括框架区(FR)和互补结构区(CDR)，其中互补结构区(CDR)包括CDR1，其序列为GGTFSNYW(SEQ ID NO：35)，CDR2，其序列为TYRGHSDT(SEQ ID NO：37)，CDR3，其序列为ARFGMLDN(SEQ ID NO：39)；或者轻链可变区(VL)包括框架区(FR)和互补结构区(CDR)，其中互补结构区(CDR)包括CDR1，其序列为QDISNY(SEQ ID NO：42)，CDR2，其序列为YTS(SEQ ID NO：44)，CDR3，其序列为QQRLTPSPFT(SEQ ID NO：46)；重链可变区(VH)包括框架区(FR)和互补结构区(CDR)，其中互补结构区(CDR)包括CDR1，其序列为GGTFSNYW(SEQ ID NO：49)，CDR2，其序列为TYRGHSDT(SEQ ID NO：51)，CDR3，其序列为ARNTALLDN(SEQ ID NO：53)。
在优选的技术方案中，一种抗BCMA单链抗体scFv，包括轻链可变区(VL)和重链可变区(VH)，其中，轻链可变区(VL)序列为SEQ ID NO：4；重链可变区(VH)序列为SEQ ID NO：3；或者轻链可变区(VL)序列为SEQ ID NO：8；重链可变区(VH)序列为SEQ ID NO：7；或者轻链可变区(VL)序列为SEQ ID NO：12；重链可变区(VH)序列为SEQ ID NO：11。
在更优选的技术方案中，一种抗BCMA单链抗体scFv的序列为SEQ ID NO：1；或者SEQ ID NO：5；或者SEQ ID NO：9。
考虑到编码基因的简并性，并同时考虑到抗体的特异性结合特性由互补决定区决定。因 此，本发明一个技术方案要求保护一种编码本发明的抗BCMA单链抗体scFv的多核苷酸序列，包括编码轻链可变区(VL)的互补结构区序列SEQ ID NO：14、SEQ ID NO：16和SEQ ID NO：18的核苷酸序列；以及编码重链可变区(VH)的互补结构区序列SEQ ID NO：21、SEQ ID NO：23和SEQ ID NO：25的核苷酸序列；或者多核苷酸序列包括编码轻链可变区(VL)的互补结构区序列SEQ ID NO：28、SEQ ID NO：30和SEQ ID NO：32的核苷酸序列；以及编码重链可变区(VH)的互补结构区序列SEQ ID NO：35、SEQ ID NO：37和SEQ ID NO：39的核苷酸序列；或者多核苷酸序列包括编码轻链可变区(VL)的互补结构区序列SEQ ID NO：42、SEQ ID NO：44和SEQ ID NO：46的核苷酸序列；以及编码重链可变区(VH)的互补结构区序列SEQ ID NO：49、SEQ ID NO：51和SEQ ID NO：53的核苷酸序列。这样的多核苷酸序列，由于编码基因的简并性，碱基序列可以变化，只要是能够编码各自对应的互补决定区即可。
在优选的技术方案中，多核苷酸序列包括编码轻链可变区(VL)序列SEQ ID NO：4的核苷酸序列；以及编码重链可变区(VH)序列SEQ ID NO：3的核苷酸序列；或者多核苷酸序列包括编码轻链可变区(VL)序列SEQ ID NO：8的核苷酸序列；以及编码重链可变区(VH)序列SEQ ID NO：7的核苷酸序列；或者多核苷酸序列包括编码轻链可变区(VL)序列SEQ ID NO：12的核苷酸序列；以及编码重链可变区(VH)序列SEQ ID NO：11的核苷酸序列。
在更优选的技术方案中，多核苷酸序列为：SEQ ID NO：2；或者SEQ ID NO：6；或者SEQ ID NO：10。
在本发明一个实施例中，提供一种噬菌粒表达载体，含有本发明的多核苷酸序列。本领域技术人员知晓，在本发明精神下，众多载体，如pComb3XSS，pComb3XTT，pComb3HSS等均可作为本发明的多核苷酸序列的表达载体。在一个优选实施例中，表达载体是噬菌体展示载体pComb3XSS载体(购自武汉淼灵生物科技有限公司)。
在本发明一个实施例中，提供一种宿主细胞，含有本发明的表达载体，能够在噬菌体表面表达出抗BCMA单链抗体scFv。本领域技术人员知晓，在本发明精神下，众多细胞如ER2738，SS320，TG1，XL1-Blue等均可作为本发明的表达载体的宿主细胞。在一个优选实施例中，宿主细胞是大肠杆菌XL1-Blue菌株(购自TAKARA)和TG1菌株(购自Lucigen，美国)。
在本发明一个实施例中，提供一种药物组合物，包含本发明的抗BCMA单链抗体scFv，以及药学上可接受的载体、稀释剂或赋形剂。
本发明的药物组合物可通过本领域众所周知的方法来制备(例如，Remington:The Science and Practice of Pharmacy,19th ed.(1995),A.Gennaro等人,Mack Publishing Co.)，且包含如本发明所公开的抗BCMA单链抗体scFv和一种或多种药学上可接受的载体、稀释剂或赋形剂。
在本发明一个实施例中，提供一种制备本发明的抗BCMA单链抗体scFv的方法，包括：将包含本发明的多核苷酸序列的表达载体转化至表达宿主细胞中，培养，进行抗BCMA单链抗体scFv的大量表达和纯化。在一个优选实施例中，表达载体是噬菌体展示载体pComb3XSS载体，宿主细胞是大肠杆菌XL1-Blue菌株和TG1菌株。
本发明的抗BCMA单链抗体scFv能够用于制备抗BCMA蛋白单抗类药物，还可用于免疫学检测BCMA。因此，在本发明一个实施例中，提供本发明的抗BCMA单链抗体scFv在制备BCMA靶向药物中的用途，或在非疾病诊断治疗目的免疫学检测BCMA中的用途。
以下通过实施例详细说明本发明的技术方案，应当理解，实施例仅是示例性的，不能理解为对本发明保护范围的限制。
实施例1随机合成单链抗体的噬菌体展示库构建
(1)CDR3区突变设计
分别以抗BCMA的人源化的scFv的重链和轻链序列为基础，设计突变引物，分别在重链和轻链的CDR3区引入突变，构建人工合成的二级库(用于定点突变)和随机库(用于随机突变)，突变引物序列如表3。
表3抗BCMA抗体scFv突变使用的引物信息
Figure PCTCN2018109564-appb-000013
Figure PCTCN2018109564-appb-000014
(注：兼并碱基K＝G/T，M＝A/C，R＝A/G，S＝G/C，W＝A/T，Y＝C/T)
(2)模板制备
合成BCMA抗体的重链和轻链可变区基因，从位点Sfi I插入到pComb3XSS载体(购自武汉淼灵生物科技有限公司)中，抗体片段扩增的引物信息见表4。
表4 BCMA抗体片段扩增使用的引物信息
Figure PCTCN2018109564-appb-000015
取1μL构建好的模板质粒，转化XL1-Blue感受态细胞(购自TAKARA)，37℃复苏1h，取200μL涂布平板，37℃过夜培养。
挑取单克隆至含有氨苄霉素(Amp +)M13KO7(辅助噬菌体)(实验室制备)的LB培养基，37℃过夜培养。
菌液离心，收集上清，加入1/5体积的PEG/NaCl，混匀，冰上孵育30min，沉淀噬菌体颗粒，12000rpm离心30min，弃上清，用PBS重悬噬菌体颗粒，再次12000rpm离心5min，收集上清加入到培养好的CJ236菌液(购自TAKARA)中，用于形成dU-ssDNA(含尿嘧啶的单链模板DNA，uracil-containing single-stranded template DNA)，37℃培养30min，加入M13KO7，继续培养1h，菌液转接至300ml含有Amp +、卡那霉素(Kan +)以及尿嘧啶(Uridine)的培养基，37℃过夜培养。
菌液离心，收集上清，加入1/4体积的PEG/NaCl，混匀，冰上孵育30min，沉淀噬菌体颗粒，12000rpm离心25min，弃上清，收集噬菌体颗粒，使用DNA提取试剂盒，提取dU-ssDNA，并定量。
(3)文库构建
各引物干粉配制成100μM工作液，随机库引物混合成重链引物H3-211(H3-2、H3-4、H3-9、H3-11各5μL)和轻链引物L3-459(L3-4、L3-5、L3-9各5μL)。
对二级库和随机库各引物分别进行磷酸化反应，每个库2个反应，每个反应体系20μL，如下：引物(100μM)2μL，10X T4 Buffer 2μL，T4 polynucleotide kinase(T4多核苷酸激酶)2μL，去离子水14μL。37℃水浴1.5h。
将二级库和随机库相应的磷酸化突变引物分别混合后与dU-ssDNA模板进行退火反应，每个库1个反应，每个反应体系250μL，如下：引物(每个库包括轻链和重链2个引物)40μL，10X T4 Buffer 25μL，dU-ssDNA模板20μL，去离子水补齐至250μL。
在PCR仪中设定退火程序：90℃，5min；75℃，45s；70℃，1min；65℃，1min；60℃，1min；55℃，5min；50℃，5min；45℃，30s；37℃，10min；30℃，45s；25℃，45s；22℃，90s；20℃，5min；1个循环。
在每个退火反应中加入：10X T4 Buffer 10μL，dNTP(每种10mM)25μL，DTT(100mM)5μL，T7 DNA polymerase(T7 DNA聚合酶)3μL。37℃延伸反应4h。
每个体系中加入5μL T4连接酶，室温连接反应2h。
连接产物经DNA电泳验证，如图1所示，条带与模板相比有明显位移，表明scFv成功构建到载体上。
(4)噬菌体展示库的构建
向二级文库和随机文库中分别加入15μL 3M NaAc(pH 5.5)调节pH，连接产物经PCR Purification Kit(QIAGEN)纯化后，并用Nanodrop并对纯化产物定量；分别取1μL纯化后 的二级文库和随机文库转化高效TG感受态细胞(Lucigen，美国)，37℃复苏1h，梯度稀释至10 6，分别取300μL涂布平板，37℃，过夜培养；次日计数平板上的克隆数，根据公式：
库容＝克隆数*稀释度*1000/30
计算获得的二级库和随机库的库容大于10 8/ml。将所有克隆用LB洗脱下，5,000g，离心5min，沉淀用2ml LB悬浮，加入等体积的30％甘油，-80冻存。
(5)噬菌体展示库的多样性检测
分别随机挑取步骤(4)的二级库和随机库的36个克隆，作为模板，进行克隆(clone)PCR，用1.5％琼脂糖凝胶电泳检测PCR产物，如图2所示，二级库(克隆1-36)和随机库(克隆37-72)的重组率为94％和100％。随机挑选56个克隆送Sanger测序，分析噬菌体展示库的多样性；分析scFv序列的结果显示，二级库中26条序列11条有突变，随机库中25条序列中14条有突变，多样性分别达到42％和56％，可用于下一步新型抗体的筛选。
(6)噬菌体扩增和拯救
对上述得到的二级库和随机库的噬菌体展示进行增殖和噬菌体拯救，将步骤(4)中保存的噬菌体库1ml分别接入100ml培养基中培养至对数生长期，加入MOI(multiplicity of infection，感染复数)为20的辅助噬菌体M13K01，室温，静置30min，低速离心后，沉淀用培养基悬起，接入300ml培养基中，培养过夜。次日，3,000离心30min，收集上清，加入PEG沉淀噬菌体，冰上静置30min，3,000离心30min，沉淀即为携带BCMA的scFv的噬菌体库，用PBS悬浮沉淀后，测定其滴度，二级库为2.6X 10 12pfu/ml，随机库为3.3X 10 12pfu/ml。
实施例2用噬菌体展示技术筛选BCMA的高亲和抗体
(1)噬菌体亲和淘洗
分别取100ng BCMA-his抗原包被ELISA板作为样品孔，100μL Na 2CO 3作为阴性对照孔，4℃，过夜孵育。次日，分别加入实施例1中步骤(6)拯救出的二级库和随机库的噬菌体库，室温，孵育2h；PBST洗孔10次，加入100μL三乙胺，室温，孵育30min，收集的噬菌体即亲和淘洗获得的抗BCMA的scFv的噬菌体库；取10μL感染TG细胞并涂布平板，次日观察亲和噬菌体的淘洗结果，剩余的噬菌体的用于扩增和拯救。
(2)淘洗后噬菌体的扩增和拯救
扩增和拯救方法同实施例1中步骤(6)，获得的PBS悬浮液即扩增的第一轮淘洗后的噬菌体，置于4℃保存，并用于下一轮的筛选；按实施例2中步骤(1)的淘洗步骤，每轮逐次递减抗原量，淘洗3-4轮。
(3)ELISA评价特异性抗体的富集程度
取100ng BCMA-his抗原，4℃，过夜；次日加2％的BSA室温封闭1h；实验组分别加入每轮淘洗后扩增的噬菌体，对照组加入等量野生型的噬菌体，室温，孵育2h；PBST洗10次，以去除没有结合的噬菌体；加入HRP标记的抗M13抗体，室温孵育1h；加入显色液，避光反应10-30min，测吸光值OD450，ELISA结果如图3所示，吸光值在第一轮到第三轮淘洗时趋于稳定，表明有新型的特异性的抗体得到了富集。
(4)鉴定靶向BCMA高亲和的scFv阳性克隆
取第二轮和第三轮淘洗获得的噬菌体涂布的平板，分别从随机库和二级库中挑选36和20个克隆进行克隆(clone)PCR；结果如图4所示，挑其中条带有位移的克隆进行序列测定；分析测序结果，发现5条(图中箭头指示)在CDR3区域有突变的scFv单克隆株。
ELISA板包被100ng BCMA-Fc抗原，4℃孵育过夜；挑取这5个单克隆于1ml培养基中，37℃，培养至对数期，加入1mM IPTG诱导过夜；次日，离心收集菌沉，破碎后，5,000g离心15min，收集上清；同时取ELISA板，加2％的BSA室温封闭1h；实验组每孔加入单克隆破碎上清，对照组加入空白TG破碎上清，室温，孵育2h；PBST洗10次，加入鼠抗HA标签的抗体，室温1h；PBST洗3-5次，加入AP标记的抗鼠IgG抗体，室温1h；加入底物，反应10-20min，在酶标仪上读取吸光值；当吸光值与对照孔比值大于2.1(基准线)时，判定为阳性克隆；ELISA验证结果显示，获得3个阳性克隆，分别为克隆20，克隆43和克隆46(图5)。
(5)阳性克隆序列信息
将步骤(4)中获得的3株阳性克隆与原始序列相比，发现它们在重链CDR3区域和轻链的CDR3区域有显著突变。这3株scFv单克隆分别命名为scFv_20，scFv_43，和scFv_46，其核苷酸和氨基酸序列如下所示：
scFv_20核苷酸序列：
Figure PCTCN2018109564-appb-000016
Figure PCTCN2018109564-appb-000017
scFv_20氨基酸序列：
Figure PCTCN2018109564-appb-000018
scFv_43核苷酸序列：
Figure PCTCN2018109564-appb-000019
scFv_43氨基酸序列：
Figure PCTCN2018109564-appb-000020
scFv_46核苷酸序列：
Figure PCTCN2018109564-appb-000021
scFv_46氨基酸序列：
Figure PCTCN2018109564-appb-000022
以上应用了具体个例对本发明进行阐述，只是用于帮助理解本发明，并不用以限制本发明。对于本发明所属技术领域的技术人员，依据本发明的思想，还可以做出若干简单推演、变形或替换。

Claims (12)

  1. 一种抗BCMA单链抗体scFv，其特征在于，所述单链抗体scFv包括轻链可变区(VL)和重链可变区(VH)，
    所述轻链可变区(VL)包括框架区(FR)和互补结构区(CDR)，其中互补结构区(CDR)包括CDR1，其序列为QDISNY(SEQ ID NO：14)，CDR2，其序列为YTS(SEQ ID NO：16)，CDR3，其序列为QQYRKLAWT(SEQ ID NO：18)；所述重链可变区(VH)包括框架区(FR)和互补结构区(CDR)，其中互补结构区(CDR)包括CDR1，其序列为GGTFSNYW(SEQ ID NO：21)，CDR2，其序列为TYRGHSDT(SEQ ID NO：23)，CDR3，其序列为ARGAIYNGYDVLDN(SEQ ID NO：25)；或者
    所述轻链可变区(VL)包括框架区(FR)和互补结构区(CDR)，其中互补结构区(CDR)包括CDR1，其序列为QDISNY(SEQ ID NO：28)，CDR2，其序列为YTS(SEQ ID NO：30)，CDR3，其序列为QQALVVTPFT(SEQ ID NO：32)；所述重链可变区(VH)包括框架区(FR)和互补结构区(CDR)，其中互补结构区(CDR)包括CDR1，其序列为GGTFSNYW(SEQ ID NO：35)，CDR2，其序列为TYRGHSDT(SEQ ID NO：37)，CDR3，其序列为ARFGMLDN(SEQ ID NO：39)；或者
    所述轻链可变区(VL)包括框架区(FR)和互补结构区(CDR)，其中互补结构区(CDR)包括CDR1，其序列为QDISNY(SEQ ID NO：42)，CDR2，其序列为YTS(SEQ ID NO：44)，CDR3，其序列为QQRLTPSPFT(SEQ ID NO：46)；所述重链可变区(VH)包括框架区(FR)和互补结构区(CDR)，其中互补结构区(CDR)包括CDR1，其序列为GGTFSNYW(SEQ ID NO：49)，CDR2，其序列为TYRGHSDT(SEQ ID NO：51)，CDR3，其序列为ARNTALLDN(SEQ ID NO：53)。
  2. 根据权利要求1所述的单链抗体scFv，其特征在于，所述轻链可变区(VL)序列为SEQ ID NO：4；所述重链可变区(VH)序列为SEQ ID NO：3；或者
    所述轻链可变区(VL)序列为SEQ ID NO：8；所述重链可变区(VH)序列为SEQ ID NO：7；或者
    所述轻链可变区(VL)序列为SEQ ID NO：12；所述重链可变区(VH)序列为SEQ ID NO：11。
  3. 根据权利要求1所述的单链抗体scFv，其特征在于，所述单链抗体scFv的序列为SEQ ID NO：1；或者SEQ ID NO：5；或者SEQ ID NO：9。
  4. 一种编码权利要求1-3任一项所述的抗BCMA单链抗体scFv的多核苷酸序列，其特 征在于，所述多核苷酸序列包括编码所述轻链可变区(VL)的互补结构区序列SEQ ID NO：14、SEQ ID NO：16和SEQ ID NO：18的核苷酸序列；以及编码所述重链可变区(VH)的互补结构区序列SEQ ID NO：21、SEQ ID NO：23和SEQ ID NO：25的核苷酸序列；或者所述多核苷酸序列包括编码所述轻链可变区(VL)的互补结构区序列SEQ ID NO：28、SEQ ID NO：30和SEQ ID NO：32的核苷酸序列；以及编码所述重链可变区(VH)的互补结构区序列SEQ ID NO：35、SEQ ID NO：37和SEQ ID NO：39的核苷酸序列；或者
    所述多核苷酸序列包括编码所述轻链可变区(VL)的互补结构区序列SEQ ID NO：42、SEQ ID NO：44和SEQ ID NO：46的核苷酸序列；以及编码所述重链可变区(VH)的互补结构区序列SEQ ID NO：49、SEQ ID NO：51和SEQ ID NO：53的核苷酸序列。
  5. 根据权利要求4所述的多核苷酸序列，其特征在于，所述多核苷酸序列包括编码所述轻链可变区(VL)序列SEQ ID NO：4的核苷酸序列；以及编码所述重链可变区(VH)序列SEQ ID NO：3的核苷酸序列；或者
    所述多核苷酸序列包括编码所述轻链可变区(VL)序列SEQ ID NO：8的核苷酸序列；以及编码所述重链可变区(VH)序列SEQ ID NO：7的核苷酸序列；或者
    所述多核苷酸序列包括编码所述轻链可变区(VL)序列SEQ ID NO：12的核苷酸序列；以及编码所述重链可变区(VH)序列SEQ ID NO：11的核苷酸序列。
  6. 根据权利要求4所述的多核苷酸序列，其特征在于，所述多核苷酸序列为：SEQ ID NO：2；或者SEQ ID NO：6；或者SEQ ID NO：10。
  7. 一种表达载体，其特征在于，所述表达载体含有权利要求3-6任一项所述的多核苷酸序列。
  8. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求7所述的表达载体，能够表达出抗BCMA单链抗体scFv。
  9. 一种药物组合物，其特征在于，所述药物组合物包含权利要求1-3任一项所述的抗BCMA单链抗体scFv，以及药学上可接受的载体、稀释剂或赋形剂。
  10. 一种制备权利要求1-3任一项所述的抗BCMA单链抗体scFv的方法，其特征在于，所述方法包括：将包含权利要求3-6任一项所述的多核苷酸序列的表达载体转化至表达宿主细胞中，培养，进行所述抗BCMA单链抗体scFv的大量表达和纯化。
  11. 根据权利要求10所述的方法，其特征在于，所述表达载体是pComb3XSS载体，所 述宿主细胞是大肠杆菌XL1-Blue和TG1菌株。
  12. 权利要求1-3任一项所述的抗BCMA单链抗体scFv在制备BCMA靶向药物中的用途，或在非疾病诊断治疗目的免疫学检测BCMA中的用途。
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