WO2020038147A1 - 抗bcma的单域抗体及其应用 - Google Patents

抗bcma的单域抗体及其应用 Download PDF

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WO2020038147A1
WO2020038147A1 PCT/CN2019/095507 CN2019095507W WO2020038147A1 WO 2020038147 A1 WO2020038147 A1 WO 2020038147A1 CN 2019095507 W CN2019095507 W CN 2019095507W WO 2020038147 A1 WO2020038147 A1 WO 2020038147A1
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bcma
domain antibody
cell
domain antibodies
domain
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PCT/CN2019/095507
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English (en)
French (fr)
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张继帅
栗红建
包朝乐萌
蔡清华
李莹莹
宋宗培
丁怡瑾
蔡志波
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深圳普瑞金生物药业有限公司
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Priority to CA3110262A priority Critical patent/CA3110262C/en
Priority to JP2021534412A priority patent/JP7289562B2/ja
Priority to US17/270,788 priority patent/US20220251226A1/en
Priority to EA202190608A priority patent/EA202190608A1/ru
Priority to BR112021003410-2A priority patent/BR112021003410A2/pt
Priority to AU2019323389A priority patent/AU2019323389B2/en
Priority to CN201980006291.6A priority patent/CN111542343B/zh
Priority to KR1020217008611A priority patent/KR20210050535A/ko
Priority to SG11202101675UA priority patent/SG11202101675UA/en
Priority to EP19852395.3A priority patent/EP3845244A4/en
Publication of WO2020038147A1 publication Critical patent/WO2020038147A1/zh

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Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to a group of single-domain antibodies against B-cell maturation antigen (BCMA) and applications thereof.
  • BCMA B-cell maturation antigen
  • BCMA Breast Cellular Antigen
  • TNFR tumor necrosis factor receptor
  • BAFF B cell-activating factor
  • B lymphocyte stimulation factor B lymphocyte stimulation factor
  • APRIL proliferation-inducing ligand
  • BCMA one candidate target antigen for immunotherapy of MM.
  • MM treatment can induce remission, but almost all patients will eventually relapse and die.
  • Some monoclonal antibody candidate drugs have shown promise in the treatment of MM in preclinical studies and early clinical trials, but have not been consistently recognized and no monoclonal antibody drugs have been marketed.
  • new immunotherapy for MM is highly needed, and the development of effective antigen-specific adoptive T cell therapy for this disease will be a major research advance.
  • Single domain antibody (sdAb) or nanobody is a heavy chain antibody with missing light chain found in alpaca blood. It was developed by Belgian scientists using molecular biology technology combined with the concept of nanoparticle science. The latest and the smallest molecular antibody fragment that can bind to the antigen. It has a series of advantages such as simple structure, strong penetrability, easy expression and purification, high affinity and stability, non-toxic side reactions, etc. Using single domain antibody platform The technology studies single-domain antibodies of various target antigens and is applied in the field of biomedicine.
  • the present invention aims to develop a set of anti-BCMA single domain antibodies that can be applied to therapeutic antibody candidate drugs and chimeric antigen receptor T cells targeting BCMA.
  • the technical problem to be solved by the present invention is to provide a set of novel anti-BCMA single domain antibodies with good effects.
  • Another technical problem to be solved by the present invention is the development of various applications of anti-BCMA single domain antibodies.
  • the present invention provides the following technical solutions:
  • the present invention provides a set of single-domain antibodies against BCMA.
  • the set of single-domain antibodies is composed of a framework region and a complementarity determining region, and the amino acid sequence of the complementarity determining region is selected from SEQ ID NO: 1-66 (Schedule 1 : Screened BCMA-sdAbs complementarity determining region amino acid sequence).
  • the amino acid sequence of the complementarity determining region is greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% with the amino acid sequence shown in SEQ ID NO: 1-66.
  • the differential amino acids are conservative substitutions.
  • the group of single domain antibodies has an amino acid sequence selected from the group consisting of SEQ ID NO: 67-132, or the amino acid sequence thereof is SEQ ID NO: 67-132 (Schedule 2: Screened BCMA-sdAbs amino acids sequence).
  • the amino acid sequence is greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% in identity with the amino acid sequence shown in SEQ ID NOs: 67-132.
  • the differential amino acids are conservative substitutions, and more preferably, one or more conservative substitutions.
  • the invention provides a set of single-domain antibody genes against BCMA, said set of single-domain antibody genes having a nucleotide sequence selected from SEQ ID NO: 133-198 (Annex Table 3: Screened BCMA-sdAbs nucleotides Sequence), or its nucleotide sequence is the nucleotide sequence of SEQ ID NO: 133-198, or the nucleotide sequence encoding the single-domain antibody described above.
  • the nucleotide sequence is greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% in identity with the nucleotide sequence shown in SEQ ID NOs: 133-198.
  • the different bases are conservative substitutions, and more preferably, one or more conservative substitutions.
  • the present invention provides a polypeptide having one or more single domain antibodies selected from the single domain antibody set described above.
  • the multiple single domain antibodies are the same or different.
  • the present invention provides an expression vector comprising one or more genes selected from the single domain antibody genome described above.
  • the expression vector is a prokaryotic cell expression vector, a eukaryotic cell expression vector, or another cell expression vector.
  • the present invention provides a host cell comprising the expression vector described above.
  • the host cell is a prokaryotic expression cell, a eukaryotic expression cell, a fungal cell or a yeast cell, and the prokaryotic expression cell is preferably E. coli.
  • the present invention provides a chimeric antigen receptor having one or more single domain antibodies selected from the single domain antibody set described above.
  • the plurality of single domain antibodies are the same or different.
  • the present invention provides a chimeric antigen receptor-modified T cell, which is modified by the chimeric antigen receptor described above.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising, as an active ingredient, one or more single-domain antibodies selected from the single-domain antibody group described above.
  • the present invention provides a humanized form of an anti-BCMA single domain antibody obtained by humanizing a single domain antibody selected from the single domain antibody group described above.
  • the invention provides the application of the single-domain antibody in the single-domain antibody group described above in the detection of BCMA.
  • the invention provides the use of the single-domain antibody in the single-domain antibody group described above in blocking the interaction between BAFF and BCMA.
  • the single domain antibody is linked to one or more of a cytotoxic agent, an enzyme phase, a radioisotope, a fluorescent compound, or a chemiluminescent compound.
  • the invention provides the application of the single-domain antibody in the single-domain antibody group described above in the preparation of a medicament for treating a disease associated with abnormal expression of BCMA.
  • the disease associated with abnormal BCMA expression is a multiple myeloma disease.
  • the present invention screens a set of single-domain antibodies against BCMA.
  • the single-domain antibodies of each group in this group have high activity and strong neutralization or binding ability.
  • This group of single-domain antibodies can specifically bind to human BCMA antigen and tumor cell lines expressing BCMA on the cell surface, effectively blocking the binding of BAFF antigen to BCMA and generating the corresponding signal cascade effect.
  • This group of single-domain antibodies can be used for detection and / Or treat a variety of diseases associated with abnormal BCMA expression.
  • Figure 1 shows the amplification of common heavy chain antibody and single chain antibody genes in the first round of PCR.
  • Reference (Marker) (1500BP, 1000BP, 800BP, 500BP, 250BP, and 100BP) 1.
  • PCR amplification products there are common antibody heavy chain gene amplified fragments larger than 800BP and heavy chain antibody gene amplified fragments smaller than 800BP.
  • 2 and 3 are PCR amplification products, which are only about 500BP of the heavy chain antibody gene amplified fragment.
  • Figure 2 shows the second round of PCR amplification to obtain the VHH target gene fragment.
  • Marker (1500BP, 1200BP, 1000BP, 800BP, 700BP, 600BP, 500BP, 250BP, and 100BP).
  • 1 to 12 are PCR amplification products, and about 500BP of the heavy chain antibody VHH gene amplified fragment.
  • Figure 3 is an example of SDS-PAGE before purification of expressed BCMA-sdAbs.
  • FIG. 4 is an example of an SDS-PAGE chart of the expressed BCMA-sdAbs purified by a nickel column.
  • Figure 5 is a concentration gradient (ELISA) of the binding of purified BCMA single domain antibody to BCMA protein.
  • FIG. 6 shows that BCMA single domain antibodies can compete to inhibit the binding of BAFF protein to BCMA protein.
  • Figure 7 shows the killing efficiency of BCMA CART on tumor cells.
  • a set of single-domain antibodies against BCMA are obtained through a series of steps. They have high activity and potential for high neutralization or binding ability. These single-domain antibodies have similar structures (consisting of framework regions and complementarity determining regions). , And similar functional effects, it can be seen as a group of anti-BCMA single domain antibodies with a common structure and common performance effects.
  • BCMA tumor necrosis factor receptor
  • TNFR tumor necrosis factor receptor
  • APRIL proliferation-inducing ligand
  • MM Multiple myeloma
  • BCMA RNA is commonly detected in MM cells, and BCMA protein can be detected on the plasma cell surface of patients with multiple myeloma.
  • MM multiple myeloma
  • MM treatment can induce remission, but almost all patients will eventually relapse and die.
  • monoclonal antibodies have shown promise in treating MM in preclinical studies and early clinical trials, they have not been consistently recognized. Obviously, new antibodies to MM and new immunotherapy are clearly needed.
  • New antibodies against BCMA are the object of development and ultimately the object of protection in this article.
  • the scope of this article relates to the resulting anti-BCMA antibodies, their various forms (e.g., single domain antibodies), and substances using the antibody as a component (such as , Pharmaceutical compositions, kits, vectors, chimeric antigen receptors, chimeric antigen receptor-modified T cells, etc.), applications (eg, diagnostic applications, therapeutic applications, manufacturing applications, etc.), however, techniques in the art Personnel should understand that the object of protection in this article is not limited to these examples.
  • single-domain antibody refers to a fragment containing a single variable domain in an antibody, also known as a Nanobody. Like intact antibodies, it can selectively bind to specific antigens. Compared to the 150-160kDa mass of the intact antibody, the single domain antibody appears to be much smaller, only about 12-15kDa.
  • the first single-domain antibody was artificially engineered from the alpaca heavy chain antibody and is called the "VHH segment".
  • single-domain antibodies for alpaca are used in the present invention.
  • the single domain antibody of the invention is a single domain antibody against BCMA.
  • Framework region that is, the framework region, has approximately 110 amino acid sequences at the near N-terminus of the H and L chains of the immunoglobulin, and the amino acid sequences of other parts are relatively constant.
  • Light and heavy chains are distinguished into variable regions (V) and constant regions (C).
  • the variable region contains a hypervariable region (HVR) or a complementaryarity-determining region (CDR) and a FR framework region.
  • FR is less variable than CDR.
  • CDR Complementarity Determining Region
  • constant region variable region
  • variable region A small part of the amino acid residues in the variable region are particularly strongly changed. The composition and arrangement of these amino acid residues are more prone to mutation.
  • the region is called a hypervariable region.
  • hypervariable regions There are three hypervariable regions (HVRs) in the V region of the L chain and the H chain. Because of their spatial complementarity with the epitopes, the hypervariable regions are also called complementarity determining regions.
  • identity of a sequence is used interchangeably with “identity” and refers to the degree of similarity between sequences determined by sequence alignment software such as BLAST. Methods and software for sequence alignment are well known to those skilled in the art. This can be done by performing one or several of the known sequences (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more Multiple) amino acid or base substitutions, deletions and / or additions to obtain a modified nucleotide sequence.
  • sequence alignment software such as BLAST.
  • amino acid or nucleotide sequence shown in one or more of the sequences of the present invention SEQ ID ID: 1-198 can be modified to obtain 85%, more than 90%, more than 95%, or more than 99% sequence identity, and have substantially the same performance, which are all within the protection scope of the present invention.
  • the present invention obtains sequence identity through conservative substitutions, but is not limited to conservative substitutions.
  • amino acid sequence refers to the sequence in which amino acids are connected to each other to form a peptide chain (or polypeptide), and the amino acid sequence can only be read in one direction.
  • amino acids There are more than 100 different types of amino acids, of which 20 are commonly used.
  • the present invention does not exclude modification of other substances on the amino acid chain, such as sugars, lipids, and the like, and the present invention is not limited to the 20 commonly used amino acids.
  • nucleotide sequence refers to the sequence of bases in DNA or RNA, that is, the sequence of A, T, G, and C in DNA, or the sequence of A, U, G, and C in mRNA. Sequences of bases in rRNA, tRNA, and mRNA are also included. It should be understood that, in addition to the DNA sequence, the antibody genes claimed in the present invention also cover RNA (rRNA, tRNA, mRNA) and their complementary sequences.
  • genes encoding the antibodies of the present invention are not equivalent to the sequences of the present invention SEQ ID NO: 133-198, these genes encoding the antibodies of the present invention but different from the nucleotides described in SEQ ID NO: 133-198 The sequences are also within the scope of the present invention.
  • the polypeptide, pharmaceutical composition, chimeric antigen receptor or CART of the present invention comprises a single domain antibody.
  • the present invention is not limited thereto, and the above-mentioned substance of the present invention may include two single domain antibodies. , 3 single-domain antibodies or more single-domain antibodies, these multiple single-domain antibodies are the same or different, and besides the single-domain antibody of the present invention, other antibodies or single-domain antibodies other than the present invention may also be included Domain antibodies do not go beyond the scope of the invention.
  • expression vectors refers to a vector that adds an expression element (such as a promoter, RBS, terminator, etc.) to the basic skeleton of a cloning vector so that the gene of interest can be expressed.
  • an expression element such as a promoter, RBS, terminator, etc.
  • target gene a promoter, RBS, terminator, etc.
  • marker gene a gene that is expressed by the expression vector.
  • the invention includes, but is not limited to, prokaryotic cell expression vectors, eukaryotic cell expression vectors, or other cell expression vectors.
  • CAR Chimeric Antigen Receptor
  • CART Chomeric Antigen Receptor T Cells
  • the chimeric antigen receptor or the chimeric antigen receptor T cell of the present invention may comprise one single domain antibody, two single domain antibodies or more single domain antibodies of the present invention, and they may It is the same or different.
  • the "pharmaceutical composition” of the present invention may include one single domain antibody, two single domain antibodies or more single domain antibodies of the present invention, which may be the same or different of.
  • humanized antibody refers to the constant region portions of the antibody (ie, the CH and CL regions) or the antibody is all encoded by the human antibody gene. Humanized antibodies can greatly reduce the immune side effects of heterologous antibodies on the human body. Humanized antibodies include chimeric antibodies, modified antibodies, and fully humanized antibodies. It should be understood that those skilled in the art can prepare suitable humanized forms of the single-domain antibodies of the present invention according to actual needs, which is within the scope of the present invention.
  • lentivirus as used herein is a genus of the retroviridae family, including 8 viruses capable of infecting humans and vertebrates.
  • the primary infected cells are mainly lymphocytes and macrophages, and the infected individuals eventually develop the disease. .
  • Lentivirus species such as human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), equine infectious anemia (EIA), feline immunodeficiency virus (FIV).
  • HAV human immunodeficiency virus
  • SIV simian immunodeficiency virus
  • EIA equine infectious anemia
  • FIV feline immunodeficiency virus
  • the research on lentiviral vectors has developed rapidly, and the research is also very deep. The vector can effectively integrate foreign genes into the host chromosome, thereby achieving persistent expression.
  • infectivity it can effectively infect many types of cells such as neuron cells, liver cells, cardiac muscle cells, tumor cells, endothelial cells, stem cells, etc., so as to achieve a good effect of gene therapy.
  • those skilled in the art can also choose other suitable vectors besides lentivirus, which are all within the protection scope of the present invention.
  • BCMA antigen Human TNFRSF17 / BCMA / CD269Protein, manufacturer Beijing Yiqiao Shenzhou, article number 10620-H15H
  • the interval of immunization is 7-15 days.
  • serum is collected to determine the immunological titer of the antigen. When the titer reaches more than 10,000 times (ELISA method), about 100 ml of whole blood is collected, and lymphocytes are separated, -80 Store at °C for future use.
  • Isolate the peripheral blood lymphocytes of alpaca apply QIAGEN kit (QIAamp RNA Blood Mini Kit (50), article number, 52304), follow the instructions, briefly: 1ml whole blood, add 5-10ml of erythrocyte lysate, mix well, Leave in the ice bath for 30 minutes. After the red blood cells are lysed, centrifuge at 2000 rpm for 10 minutes, remove the supernatant, add 1 to 2 ml of red blood cell lysate, mix well, and place in the ice bath for 10 minutes to lyse the residual red blood cells. Centrifuge at 2000 rpm for 10 minutes. Minute, remove the supernatant, add 0.3ml of lysate, mix the white blood cells, store at -80 ° C, and reserve.
  • QIAGEN kit QIAamp RNA Blood Mini Kit (50), article number, 52304
  • RNA purification using QIAGEN kit (QIAamp RNA Blood Mini Kit (50), article number, 52304), according to the instructions, briefly: take 0.3ml isolated alpaca lymphocytes, add 0.3ml buffer RLT, shake and mix uniform. The liquid mixed in the previous step was transferred to a collection tube (QIAshredderSpinColumn) that had been packed in an adsorption column, and centrifuged at 14,000 rpm for 2 minutes, and the filtrate in the collection tube was transferred to a new centrifuge tube. Add 0.5 ml of 70% ethanol to the filtrate and mix by inverting it upside down.
  • QIAGEN kit QIAamp RNA Blood Mini Kit (50), article number, 52304
  • YF-1 CGC CAT CAA GGT ACC AGT TGA (SEQ ID NO: 199)
  • YF-2 GGG, GTA, CCT, GTC, ATC, CAC, GGA, CCA, GCT, GA (SEQ ID NO: 200)
  • YBN CAG CCG GCC ATG GCC SMK GTR CAG CTG GTG GAK TCG GGG GGA G (SEQ ID NO: 201)
  • YV-BACK CAT, GTG, CATGGCCTA, GAC, TCG, CGG, CCCAGC, CGG, CCA, TGG CC (SEQ ID NO: 202)
  • YV-FOR CAT, GTG, TAG, CCT, GGC, CGG, CCT, GGC, CTG, AGG, AGA, CGG, TGA, CCT, GG (SEQ ID NO: 203)
  • the VHH fragment and the pHEN6 vector were used. Ligase ligation, electrotransformation into TG1 competent cells, spreading the plate, and verifying the antibody insertion rate by colony PCR. Recombinant gene cloning efficiency test: the electrolyzed bacterial liquid was coated on LB / Amp plates, cultured overnight at 32 ° C, and the ligation efficiency of antibodies was verified by colony PCR the next day. The electro-transformed bacteria solution was spread on an LB / Amp plate, and the culture was incubated at 32 ° C overnight, washed with 2YT medium, 15% glycerol was added, and stored at -80 ° C.
  • the antibody library was supplemented with helper phage M13K07 (Invitrogen) for rescue: the phage antibody library was prepared according to the conventional method and stored at -80 ° C until use.
  • helper phage M13K07 Invitrogen
  • Example 2 Obtaining of a single domain antibody to BCMA
  • the first round of BCMA protein concentration was 150 ⁇ g / ml, 150 ⁇ l / well, 1 microwell, and incubated overnight at 4 ° C.
  • the second round of BCMA protein concentration was 10-100 ⁇ g / ml, 150 ⁇ l / well, 5 microwells, and incubated at 4 ° C overnight.
  • the third round of BCMA protein concentration was 10-50 ⁇ g / ml, 150 ⁇ l / well, 5 microwells, and incubated at 4 ° C overnight.
  • Blocking 1% CPBS, 300 ⁇ l / well, 37 ° C, incubation for 2 hours.
  • a single colony was randomly picked from the agar plate of the third round of screening of growing colonies, inoculated in a 96-well culture plate containing Amp's 2YT liquid medium, and the phage antibody was induced by superinfection with a helper phage. The expression supernatant was harvested, and BCMA was used as an antigen for ELISA assay. BCMA positive wells were selected and DNA sequencing was performed to identify the gene sequence of the anti-BCMA single domain antibody clone. A series of single-domain antibody gene sequences including the sequence of the genes listed in Table 3 were obtained for further expression and screening of specific, highly active single-domain antibodies.
  • the specific BCMA single-domain antibody gene obtained in Example 3 was amplified by PCR to obtain PCR products with restriction enzymes BbsI and BamHI.
  • the PCR products and vectors were treated with the restriction enzymes BbsI and BamHI, respectively.
  • pSJF2 vector (kim Is. Biosic Biochem. 2002, 66 (5): 1148-51, Chinese patent ZL 201110280031), ligated by T4 ligase to recombine, and a plasmid sdAb-pSJF2, which can be efficiently expressed in E. coli,
  • the gene sequence was determined to determine the correctness of the sequence.
  • the strain containing the plasmid BCMAsdAb-pSJF2 described in Example 3 was inoculated onto an LB culture plate containing ampicillin, and incubated at 37 ° C overnight. A single colony was picked and inoculated in 15 ml of LB culture solution containing ampicillin, and cultured at 37 ° C with shaking overnight. 10ml of the overnight culture was transfected in 1L of 2YT broth containing ampicillin, and cultured at 37 ° C with a shaker at 240 rpm. When the OD value reached 0.4 to 0.6, 0.5 to 1.0 mM IPTG was added and the culture was continued overnight. Centrifuge and harvest.
  • FIG. 3 shows the expressed anti-BCMA single domain antibody protein
  • FIG. 4 shows the results of SDS-PAGE electrophoresis of the expressed BCMA-sdAbs purified by a nickel column.
  • Bio-BCMA was diluted to 10 ⁇ g / ml with 1 ⁇ dynamic buffer (1 ⁇ PBS, containing 0.05% Tween 20, 0.1% BSA, pH 7.2);
  • Single domain antibody Dilute to 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM with 1 ⁇ kinetic buffer.
  • the antigen to be tested was loaded by SA sensor, the antigen was diluted by 5 dilutions, and the affinity of all BCMA single domain antibodies was 50nm, 20nm, 10nm, 1nm, 0.1nm, 0.01nm.
  • BCMA-Fc antigen diluted to 1 ⁇ g / ml, 100 ⁇ l antigen-coated 96-well plates, 4 °C overnight. 300 ⁇ l 0.5% BSA-PBS blocked 96-well plate at 37 ° C for 2 hours. The purified BCMA single domain antibody was added at different dilution concentrations, and was added at 100 ⁇ l / well at 37 ° C. for 1 hour. Wash the plate three times with 0.05% PBST. Add 1: 5000-fold diluted mouse anti-His-HRP, add 100 ⁇ l / well, 37 ° C, 1 hour. Wash the plate three times with 0.05% PBST.
  • Figure 5 shows the concentration gradient (ELISA) of the binding of purified BCMA single domain antibody to BCMA protein. Except for B35 (13) and B92 (6-1), the two antibodies have lower binding ability to BCMA antigen, and the remaining 11 antibodies have lower binding ability. Both are high.
  • Example 7 Test of BCMA single domain antibody on competition inhibition of binding of BAFF to BCMA
  • BCMA single domain antibodies should be able to compete to inhibit the binding of BAFF to BCMA.
  • BAFF protein was coated on a removable microtiter plate at 1 ⁇ g / ml and 100 ⁇ l / well and incubated overnight at 4 ° C. Add 2% BSA to block, 300 ⁇ l / well, 37 ° C, and incubate for 2 hours. Dilute the BCMA single domain antibody to a final concentration of 10 ⁇ g / ml. Add 100 ⁇ l of BCMA (10 ⁇ g / ml) single domain antibody, add 2 ⁇ l of BAFF (5 ⁇ g / ml) protein to each well, and mix well.
  • Example 8 Study of BCMA single domain antibodies as recognition antibodies for specific BCMA target antigens on CART cells.
  • the BCMA single-domain antibody gene and the second-generation CAR structural gene were synthesized, and the two genes were spliced by overlapping PCR to obtain the BCMA CAR gene. After the synthetic gene is obtained, molecular cloning is performed. First, the PCR products of the two gene fragments are obtained, and then overlapping PCR is performed to obtain the BCMA CAR gene of the second generation CAR structure connected by the two fragments.
  • the lentiviral vector Pre-Lenti-EF1 and BCMA CAR, which correctly expressed BCMA and CAR, were obtained by ligation, transformation, selection, cloning, plasmid extraction and sequencing.
  • 293T cells were trypsinized and plated in 150 cm petri dishes. The cells were placed in a 5% CO 2 incubator and incubated for 8-24 hours. When the cells adhered to the cells, they reached 80% of the total area of the culture dish and began to transfect 293T cells.
  • PBMC peripheral blood mononuclear cells
  • T cells 2 ⁇ 10 6 T cells / well are planted in 6-well plates, containing IL-2 (1000 U / ml) in x-vivo15 medium was cultured and stimulated with anti-CD3 for 24 hours. After 24 hours of stimulation, BCMA virus solution was added and infected overnight. The next day was supplemented with 2 ml of medium. 6-7 days after infection, the expression of CAR molecules was evaluated by flow cytometry. Biotinylated BCMA was used to analyze the positive rate of anti-BCMA-CAR expression in transfected T cells by flow cytometry.
  • CART cells / T cells target cells were set with four gradients, 0.5: 1, 1: 1, 2: 1, 4: 1, and Daudi cells 3 ⁇ 10 4 / well, make up 200 ⁇ L of the system in all other wells with X-VIVO medium / 1640 medium.
  • the 96-well plate was placed in a 37 ° C, 5% CO2 incubator and cultured. After 17 hours, 20 ⁇ l of lysate was added to the well with the maximum release, and the cells were completely ruptured.
  • the 96-well plate was placed in a CO2 incubator and incubated for 2 hours. After 2 h, observe the wells with the greatest release.

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Abstract

一组抗BCMA的单域抗体以及该组内单域抗体的基因、包含该组内单域抗体的载体、嵌合抗原受体和嵌合抗原受体修饰的T细胞，该组内单域抗体的检测和治疗应用。该抗BCMA的单域抗体活性高、稳定性高、特异性高、结合能力强。

Description

抗BCMA的单域抗体及其应用 技术领域
本发明属于生物技术领域，具体地，涉及一组抗B细胞成熟抗原(BCMA)的单域抗体及其应用。
背景技术
BCMA(B cell maturation antigen，BCMA)是肿瘤坏死因子受体(TNFR)超家族的成员，可结合B细胞激活因子(B cell-activating factor(BAFF))或B淋巴细胞刺激因子(B lymphocyte stimulator(BLyS))和增殖诱导配体(APRIL)。据报道，在正常细胞中，BCMA主要由浆细胞和一部分成熟B细胞表达，而在大部分B细胞以及其它器官上都不表达。多发性骨髓瘤(MM)，是以克隆性浆细胞大量增生为特征的恶性肿瘤。BCMA的RNA在MM细胞中普遍检测到，多发性骨髓瘤患者的浆细胞表面可检测到BCMA蛋白。因此，MM的免疫治疗的一种候选靶抗原是BCMA。目前MM治疗可诱导缓解，但几乎所有患者最终仍会复发并死亡。一些单克隆抗体候选药物已经在临床前研究和早期临床试验中显示出治疗MM的希望，但并没有得到一致性认可，也没有单抗药物上市。显然，对MM的新的免疫治疗是非常需要的，并且为该疾病开发有效的抗原特异性过继性T细胞疗法将是重大研究进展。
单域抗体(single domain antibody(sdAb)或称纳米抗体(nanobody)是由羊驼血液中发现的缺失轻链的重链抗体，经比利时科学家应用分子生物学技术结合纳米粒子科学的概念，而研发出来的最新及分子量最小的能结合抗原的抗体分子片段。其具有结构简单，穿透力强，易于表达和纯化，亲和力及稳定性高，无毒副反应等一系列优点。运用单域抗体平台技术研究各种靶抗原的单域抗体，并应用于生物医药领域。
本发明旨在研究出有应用前景的一组抗BCMA的单域抗体，可应用于治疗性抗体候选药物和靶向BCMA的嵌合抗原受体T细胞。
发明内容
本发明要解决的技术问题是提供一组具有良好效果的新颖的抗BCMA单 域抗体。
本发明要解决的另一个技术问题是开发抗BCMA单域抗体的各种应用。
为实现上述目的，本发明提供以下技术方案：
本发明提供了一组抗BCMA的单域抗体，所述组的单域抗体由框架区和互补决定区组成，所述互补决定区的氨基酸序列选自SEQ ID NO:1-66(附表1：筛选的BCMA-sdAbs互补决定区氨基酸序列)。
在一些实施方案中，所述互补决定区的氨基酸序列与SEQ ID NO:1-66所示的氨基酸序列的同一性大于80％、大于85％、大于90％、大于95％或大于99％。
优选地，差异的氨基酸为保守替换。
在一些实施方案中，所述组的单域抗体具有选自SEQ ID NO:67-132的氨基酸序列，或其氨基酸序列为SEQ ID NO:67-132(附表2：筛选的BCMA-sdAbs氨基酸序列)。
在一些实施方案中，其氨基酸序列与SEQ ID NO:67-132所示氨基酸序列的同一性大于80％、大于85％、大于90％、大于95％或大于99％。
优选地，差异的氨基酸为保守替换，更优选地，一个或多个保守替换。
本发明提供了一组抗BCMA的单域抗体基因，所述组的单域抗体基因具有选自SEQ ID NO:133-198的核苷酸序列(附表3：筛选的BCMA-sdAbs核苷酸序列)，或其核苷酸序列为SEQ ID NO:133-198的核苷酸序列，或编码上述单域抗体的核苷酸序列。
在一些实施方案中，其核苷酸序列与SEQ ID NO:133-198所示核苷酸序列的同一性大于80％、大于85％、大于90％、大于95％或大于99％。
优选地，差异的碱基为保守替换，更优选地，一个或多个保守替换。
本发明提供了一种多肽，具有选自上面所述的单域抗体组中的一个或多个单域抗体。
优选地，所述的多个单域抗体相同或不同。
本发明提供了一种表达载体，其包含选自上面所述的单域抗体基因组的一个或多个基因。
优选地，所述表达载体为原核细胞表达载体、真核细胞表达载体或其它细胞表达载体。
本发明提供了一种宿主细胞，其包含上面所述的表达载体。
优选地，所述的宿主细胞为原核表达细胞、真核表达细胞，真菌细胞或酵母细胞，所述原核表达细胞优选大肠杆菌。
本发明提供了一种嵌合抗原受体，其具有选自上面所述的单域抗体组中的一个或多个单域抗体。
优选地，所述多个单域抗体相同或不同。
本发明提供了一种嵌合抗原受体修饰的T细胞，由上面所述的嵌合抗原受体进行修饰。
本发明提供了一种药用组合物，其包含选自上面所述的单域抗体组中的一个或多个单域抗体作为活性成份。
本发明提供了一种人源化形式的抗BCMA的单域抗体，其通过对选自上面所述的单域抗体组中的单域抗体进行人源化而获得。
本发明提供了上面所述的单域抗体组中的单域抗体在检测BCMA中的应用。
本发明提供了上面所述的单域抗体组中的单域抗体在阻断BAFF与BCMA之间相互作用中的应用。
在一些实施方案中，所述单域抗体与细胞毒性剂、酶相、放射性同位素、荧光化合物或化学发光化合物中的一种或多种相连。
本发明提供了上面所述的单域抗体组中的单域抗体在制备用于治疗BCMA表达异常相关疾病的药物的应用。
优选地，所述的BCMA表达异常相关疾病是多发性骨髓瘤性疾病。
本发明的有益技术效果：
本发明筛选得到的一组抗BCMA的单域抗体。与现有的抗体相比，该组的各个抗BCMA的单域抗体活性高、具有强的中和或结合能力。该组单域抗体能特异性结合人BCMA抗原、结合细胞表面表达BCMA的肿瘤细胞株，有 效阻断BAFF抗原与BCMA结合以及产生相应的信号级联效应，该组单域抗体可用于检测和/或治疗多种与BCMA表达异常相关疾病。
以下结合附图和具体实施方式详细说明本发明，并不以此限定本发明的实施范围。
附图说明
图1为第一轮PCR普通重链抗体及单链抗体基因的扩增。参照(Marker)(1500BP、1000BP、800BP、500BP、250BP和100BP)1.PCR扩增物，有大于800BP的普通抗体重链基因扩增片段和小于800BP的重链抗体基因扩增片段。2和3为PCR扩增物，仅为约500BP的重链抗体基因扩增片段。
图2为第二轮PCR扩增得到VHH目的基因片段。Marker(1500BP、1200BP、1000BP、800BP、700BP、600BP、500BP、250BP和100BP)。1～12为PCR扩增物，约500BP的重链抗体VHH基因扩增片段。
图3为表达的BCMA-sdAbs纯化前SDS-PAGE图例。
图4为表达的BCMA-sdAbs经镍柱纯化后SDS-PAGE图例。
图5为纯化的BCMA单域抗体与BCMA蛋白结合的浓度梯度(ELISA)。
图6为BCMA单域抗体能够竞争抑制BAFF蛋白与BCMA蛋白的结合。
图7为BCMA CART对肿瘤细胞的杀伤效率。
具体实施方式
本发明通过一系列的步骤筛选得到了一组抗BCMA的单域抗体，它们具有高活性和高中和或结合能力的潜在，这些单域抗体具有相似的结构(由框架区和互补决定区组成)，以及相似功能效果，因此可以看作是具有共同结构和共同性能效果的一组抗BCMA的单域抗体。
本文所用的术语“BCMA”，是肿瘤坏死因子受体(TNFR)超家族的成员，可结合B细胞激活因子或B淋巴细胞刺激因子和增殖诱导配体(APRIL)。多发性骨髓瘤(MM)，是以克隆性浆细胞大量增生为特征的恶性肿瘤。BCMA的RNA在MM细胞中普遍检测到，多发性骨髓瘤患者的浆细胞表面可检测到 BCMA蛋白。
本文所用的术语“多发性骨髓瘤(MM)”是指以克隆性浆细胞大量增生为特征的恶性肿瘤。目前MM治疗可诱导缓解，但几乎所有患者最终仍会复发并死亡。尽管一些单克隆抗体已经在临床前研究和早期临床试验中显示出治疗MM的希望，但并没有得到一致性认可。显然，对MM的新的抗体以及新的免疫治疗显然是非常需要的。
针对BCMA的新抗体是本文的开发对象，最终也是本文的保护对象，本文的范围涉及所得到的抗BCMA抗体、其各种形式(如，单域抗体)、以该抗体为成分的物质(如，药物组合物、试剂盒、载体、嵌合抗原受体、嵌合抗原受体修饰的T细胞等等)、应用(如，诊断应用、治疗应用、制备应用等等)，然而，本领域技术人员应该理解，本文的保护对象并不限于举例的这些内容。
本文所用的术语“单域抗体(sdAb)”是指包含了抗体中单个可变域的片段，也称为纳体(Nanobody)。和完整的抗体一样，它可以选择性的和特定抗原结合。与完整抗体的150－160kDa的质量相比，单域抗体则显得小得多，大约只有12－15kDa。第一个单域抗体是从羊驼的重链抗体中人造工程制作出来的，称为“VHH区段”。在优选的实施方式中，本发明使用了羊驼的单域抗体，然而，本领域技术人员应该理解，来自其他物种的单域抗体也在本发明的保护范围之内。非限制性的，本发明的单域抗体是抗BCMA的单域抗体。
术语“框架区(Framework region)”，即骨架区，在免疫球蛋白的H和L链的近N端约有110个氨基酸序列的变化很大，其他部分的氨基酸序列相对恒定，据此可将轻链和重链区分为可变区(V)和恒定区(C)。可变区内包含超变区HVR(hypervariable region)或称互补决定区CDR(Complementarity-determining region)与FR骨架区。FR的可变性要低于CDR。FR分子共四个，分别为FRl、FR2、FR3和FR4。在识别抗体时，四个FR分子卷曲使CDR分子相互靠近。应该理解，本发明并不限于特定的框架区，本领域技术人员根据实际应用能够选择或取得合适的框架区，而不脱离本发明的保护范围。
术语“互补决定区(Complementarity determining region，CDR)”，整个 抗体分子可分为恒定区和可变区两部分。在可变区内有一小部分氨基酸残基变化特别强烈，这些氨基酸的残基组成和排列顺序更易发生变异区域称高变区。在L链、H链的V区中有三个高变区(hypervariable regions，HVR)，该部位因在空间结构上可与抗原决定簇形成精密的互补，故高变区又称互补性决定区。
本文所用的术语序列的“同一性(identity)”可以与“相同性”互换使用，指的是序列之间通过序列比对软件例如BLAST确定的相似程度。序列比对的方法和软件对于本领域技术人员是公知的。可以通过对已知序列进行一个或几个(例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18或更多个)氨基酸或碱基的取代、缺失和/或添加而获得经改造的核苷酸序列。例如，通过常规手段(例如保守取代等)，对本发明的序列SEQ ID NO：1-198中一个或多个所示的氨基酸或核苷酸序列进行改造，可以获得与这些具有大于80％、大于85％、大于90％、大于95％或大于99％的序列同一性，并且具有基本相同的性能，这都在本发明的保护范围之内。优选地，本发明通过保守取代获得序列同一性，但并不限于保守取代。
术语“氨基酸序列”是指氨基酸相互连接形成肽链(或多肽)的顺序，氨基酸序列只能按照一个方向读取。氨基酸有100多种不同类型，其中20种常用，本发明不排除氨基酸链上有其他物质，例如糖类、脂类等修饰，本发明也不限于20中常用的氨基酸。
术语“核苷酸序列”是指DNA或RNA中碱基的排列顺序，即在DNA中为A、T、G、C的排列顺序，或者在mRNA中A、U、G、C的排列顺序，也包括rRNA、tRNA、mRNA中碱基的排列顺序。应该理解，本发明请求保护的抗体基因除了DNA序列外，也涵盖RNA(rRNA、tRNA、mRNA)以及它们的互补序列。也应该理解，编码本发明的抗体的基因并不等同于本发明的序列SEQ ID NO:133-198，这些编码本发明的抗体的基因但不同于SEQ ID NO:133-198所述核苷酸的序列也在本发明的保护范围之内。
在一些实施方式中，本发明的多肽、药物组合物、嵌合抗原受体或CART包含一个单域抗体，应该理解，本发明并不限于此，本发明的上述物质可以包 含2个单域抗体、3个单域抗体或更多个单域抗体，这些多个单域抗体之间相同或不同，此外，除了包含本发明的单域抗体之外，还可以包含本发明以外的其他抗体或单域抗体，并没有超出本发明的范围。
术语“表达载体(Expression vectors)”是指在克隆载体基本骨架的基础上增加表达元件(如启动子、RBS、终止子等)，使目的基因能够表达的载体。表达载体四部分：目的基因、启动子、终止子、标记基因。本发明包括但不限于原核细胞表达载体、真核细胞表达载体或其它细胞表达载体。
“嵌合抗原受体(CAR)”是“嵌合抗原受体T细胞(CART)”的核心部件，赋予T细胞非依赖的方式识别肿瘤抗原的能力，这使得经过CAR改造的T细胞相较于天然T细胞表面受体能够识别更广泛的目标。CAR的基础设计中包括一个肿瘤相关抗原结合区，一个胞外铰链区，一个跨膜区和一个胞内信号区。
在本发明的实施方式中，本发明的嵌合抗原受体或嵌合抗原受体T细胞可以包含本发明的1个单域抗体、2个单域抗体或更多个单域抗体，它们可以是相同的，也可以是不同的。
在本发明的实施方式中，本发明的“药物组合物”可以包含本发明的1个单域抗体、2个单域抗体或更多个单域抗体，它们可以是相同的，也可以是不同的。
术语“人源化”抗体是指抗体的恒定区部分(即CH和CL区)或抗体所有全部由人类抗体基因所编码。人源化抗体可以大大减少异源抗体对人类机体造成的免疫副反应。人源化抗体包括嵌合抗体、改型抗体和全人源化抗体等几类。应该理解，本领域技术人员根据实际需要能够制备出本发明单域抗体的合适的人源化形式，这在本发明涵盖的范围之内。
本文所用的术语“慢病毒”是反转录病毒科下的一个属，包括8种能够感染人和脊椎动物的病毒，原发感染的细胞以淋巴细胞和巨噬细胞为主，感染个体最终发病。慢病毒种类例如人类免疫缺陷病毒(HIV)、猴免疫缺陷病毒(SIV)、马传染性贫血(EIA)、猫免疫缺陷病毒(FIV)。慢病毒载体的研究发展得很快，研究的也非常深入。该载体可以将外源基因有效地整合到宿主染色体上，从而达到持久性表达。在感染能力方面可有效地感染神经元细胞、肝细胞、心肌细 胞、肿瘤细胞、内皮细胞、干细胞等多种类型的细胞，从而达到良好的的基因治疗效果。此外，本领域技术人员也能选择除慢病毒之外其他合适的载体，这都在本发明的保护范围之内。
本发明通过下列实施例进行详细描述，然而本发明不限于这些实施例的特定细节，因为对于本领域的普通研究技术人员来说，其它的变化是已知的，或根据直接公开的内容和附属的权利要求是显而易见的。因此，凡基于本发明上述内容所实现的技术均属于本发明的范围。
下述实施例中所述实验方法，如无特殊说明，均为常规方法；所述试剂和生物材料，如无特殊说明，均可从商业途径获得。
实施例1：抗BCMA抗原特异性的单域抗体库构建
1)BCMA抗原免疫羊驼
按照常规免疫方法进行，简略介绍为，以购买的BCMA抗原(Human TNFRSF17/BCMA/CD269Protein，厂家北京义翘神州，货号10620-H15H)总量2mg左右，选择成年健康的羊驼，在颈背部皮下多点注射抗原，加入抗原和等体积福氏佐剂，分4-6次免疫，跟踪观察注射部位包块吸收情况，以确认免疫正确。免疫间隔时间为7-15天，第4次免疫后，采血清，测定抗原免疫效价，当效价达到1万倍以上时(ELISA方法)，采全血100ml左右，分离淋巴细胞，-80℃保存备用。
2)羊驼外周血淋巴细胞的分离和RNA的提取
分离羊驼外周血淋巴细胞，应用QIAGEN试剂盒(QIAamp RNA Blood Mini Kit(50)，货号，52304)，按照说明书进行，简述之：1ml全血，加入红细胞裂解液5～10ml，混匀，放冰浴中30分钟，待红细胞裂解后，2000rpm，离心10分钟，去上清，再加入红细胞裂解液1～2ml，混匀，放冰浴中10分钟，以裂解残余红细胞，2000rpm，离心10分钟，去净上清，加入0.3ml的裂解液，把白细胞混匀，-80℃保存，备用。
RNA纯化，应用QIAGEN试剂盒(QIAamp RNA Blood Mini Kit(50)，货号，52304)，按照说明书进行，简述之：取分离好的羊驼淋巴细胞0.3ml，加入0.3ml缓冲液RLT，振荡混匀。将上步混匀的液体转移到已装入吸附柱 的收集管(QIAshredderSpinColumn)中，14,000rpm离心2分钟，将收集管中的滤液转移到新的离心管中。向滤液中加入0.5ml的70％乙醇，上下颠倒混匀。10000rmp离心15秒，倒掉收集管中的废液，将吸附柱重新放回收集管中。将吸附柱转移到新的2ml收集管中，加入0.7ml缓冲液RW1，10000rmp离心15秒。将吸附柱转移到新的2ml收集管中，加入0.5ml缓冲液RPE，10000rmp离心15秒。加入0.5ml缓冲液RPE，14000rmp离心3分钟。将吸附柱转移到新的1.5ml离心管中，向吸附膜的中间部位悬空滴加入30-50μl RNase-free水，室温放置2-5分钟，12,000rpm离心1分钟，将质粒溶液收集到离心管中。测定得到的RNA浓度。
3)重链抗体可变区-VHH
合成cDNA第一链：用cDNA合成试剂盒(MiniBESTAgarose Gel DNA Extraction Kit Ver.4.0，TAKARA公司)，按照说明书进行。以此模板，分别用两套引物进行PCR扩增重链抗体VHH基因片段。采用巢式PCR方法，第一次PCR扩增中大于800bp的为普通重链基因片段，在800～500bp之间的为缺失轻链的重链抗体基因片段(参见图1)，切胶回收缺失轻链重链抗体基因片段，以此为模板用VHH特异性引物经PCR扩增得到VHH目的基因(～500bp)(参见图2)。
引物的合成：
第一轮PCRFd5’引物:
YF-1:CGC CAT CAA GGT ACC AGT TGA(SEQ ID NO:199)
YF-2:GGG GTA CCT GTC ATC CAC GGA CCA GCT GA(SEQ ID NO:200)
第一轮PCR Bd3’引物:
YBN:CAG CCG GCC ATG GCC SMK GTR CAG CTG GTG GAK TCT GGG GGA G(SEQ ID NO:201)
第二轮PCR引物：
YV-BACK：CAT GTG CATGGCCTA GAC TCG CGG CCCAGC CGG CCA TGG CC(SEQ ID NO:202)
YV-FOR：CAT GTG TAG ATT CCT GGC CGG CCT GGC CTG AGG AGA  CGG TGA CCT GG(SEQ ID NO:203)
4)VHH片段和噬菌体展示载体的连接及电转化TG1感受态。
SfI单酶切VHH片段和pHEN6载体质粒后，将VHH片段及pHEN6载体(Conrath,KEM other.Antimicrob Agents Chemother(Antimicrobial Chemotherapy)2001,45:(10)2807-12.，中国专利ZL20111028003.1)经连接酶连接，电转化至TG1感受态细胞中，涂布平板，经菌落PCR验证抗体***率。重组基因克隆效率检测：取电转化菌液涂布LB/Amp平板上，32℃，过夜培养，次日用菌落PCR的方法验证抗体的连接效率，噬菌体抗体库的连接效率在90％以上。将电转化菌液涂布LB/Amp平板上，32℃，过夜培养物，用2YT培养基洗下，加入15％甘油，-80℃保存。
5)VHH噬菌体抗体库的制备
抗体库加入辅助噬菌体M13K07(Invitrogen)进行拯救：按照常规方法进行噬菌体抗体库的制备，并保存于-80℃备用。
实施例2：BCMA的单域抗体的获得
针对BCMA特异性单域抗体的筛选
第一轮BCMA蛋白浓度150μg/ml，150μl/孔，1个微孔，4℃过夜孵育。
第二轮BCMA蛋白浓度10～100μg/ml，150μl/孔，5个微孔，4℃过夜孵育。
第三轮BCMA蛋白浓度10～50μg/ml，150μl/孔，5个微孔，4℃过夜孵育。
封闭：1％CPBS，300μl/孔，37℃，孵育2小时。
Figure PCTCN2019095507-appb-000001
Figure PCTCN2019095507-appb-000002
2.噬菌体ELISA方法挑选阳性克隆
从第3轮筛选生长菌落的琼脂平板上随机挑取单个菌落，接种在含有Amp的2YT液体培养基的96孔培养板中培养，用辅助噬菌体超感染诱导表达噬菌体抗体。收获表达上清，以BCMA为抗原进行ELISA测定，挑选出BCMA阳性孔，经DNA测序以鉴定抗BCMA单域抗体克隆的基因序列。得到包括序附表3基因序列在内的一系列单域抗体基因序列，用于进一步表达和筛选特异性、高活性的单域抗体。
实施例3：特异性BCMA单域抗体表达质粒的构建
PCR扩增实施例3所获得的特异性的BCMA单域抗体基因，而获得带有限制性内切酶BbsI和BamHI位点PCR产物，用限制性内切酶BbsI和BamHI分别处理PCR产物和载体(pSJF2载体)(kim Is.Biosic Biochem.2002,66(5):1148-51，中国专利ZL 201110280031)，经T4连接酶连接重组，而获得能在大肠杆菌中高效表达的质粒sdAb-pSJF2，并进行基因序列测定以确定其序列的正确性。
1)获得VHH目的基因的PCR扩增条件，50μl PCR体系的组成：
Figure PCTCN2019095507-appb-000003
PCR反应条件：
Figure PCTCN2019095507-appb-000004
Figure PCTCN2019095507-appb-000005
5’引物—GAA GAAGAA GAC AA CAG GCC SVK GTG MAG CTG GWG GAK TCT(SEQ ID NO:204)
3’引物——gaagatctccggatccTGAGGAGACGGTGACCTGGGT(SEQ ID NO:205)
2)目的基因和载体酶切，连接目的基因与载体，转化TG1，PCR鉴定含有目的片段的克隆，基因测序，获得基因序列正确的BCMA单域抗体表达质粒。
实施例4：抗BCMA单域抗体的表达和纯化
将实施例3所述的含有质粒BCMAsdAb-pSJF2的菌种接种在含氨基苄青霉素的LB培养板上，37℃过夜。挑选单个菌落接种于15ml的含氨基苄青霉素的LB培养液中，37℃，摇床培养过夜。转种10ml过夜培养物于1L含氨基苄青霉素的2YT培养液中，37℃摇床培养，240转/分，培养到OD值达0.4～0.6时，加入0.5～1.0mM IPTG，继续培养过夜。离心，收菌。加溶菌酶裂解细菌，离心，收上清中可溶性单域抗体蛋白。经Ni+离子亲和层析获得纯度达95％以上的蛋白。图3为表达的抗BCMA单域抗体蛋白，图4为表达的BCMA-sdAbs经镍柱纯化后SDS-PAGE电泳结果。
实验例5：BCMA单域抗体亲和力测定实验
1)样品制备
抗原：Bio-BCMA用1×动态缓冲液(1×PBS，含0.05％Tween 20、0.1％BSA，pH 7.2)稀释至10μg/ml；
单域抗体：用1×kinetic缓冲液依次稀释为400nM、200nM、100nM、50nM、25nM、12.5nM、6.25nM；
2)样品测试
待测抗原通过SA sensor进行加载，抗原稀释5个稀释度，所有BCMA单域抗体的亲和力在50nm、20nm、10nm、1nm、0.1nm、0.01nm。
实施例6：纯化的BCMA单域抗体与BCMA抗原结合试验(ELISA)
0.05M NaHCO 3(pH 9.5)稀释BCMA-Fc抗原至1μg/ml，100μl抗原包被96孔板，4℃过夜。300μl 0.5％BSA-PBS封闭96孔板，37℃，2小时。加入不同稀释浓度的纯化的BCMA单域抗体，按100μl/孔加入，37℃，1小时。用0.05％PBST洗板三次。加入1:5000倍稀释的鼠抗His-HRP，按100μl/孔加入，37℃，1小时。用0.05％PBST洗板三次。加TMB100μl，避光室温静置20分钟。加入1mol/LHCl 100μl终止反应。用酶标仪测定450nm波长下的样品OD值。图5为纯化的BCMA单域抗体与BCMA蛋白结合的浓度梯度(ELISA),除了B35(13)，B92(6-1)两个抗体结合BCMA抗原的结合力较低，其余11个抗体结合力都很高。
实施例7：BCMA单域抗体对BAFF与BCMA的结合竞争抑制作用试验
由于BCMA能够结合BAFF，有功能的BCMA单域抗体应该能够竞争抑制BAFF与BCMA的结合。按照1μg/ml，100μl/孔包被BAFF蛋白在可拆酶标板，4℃孵育过夜。加入2％BSA封闭，300μl/孔，37℃，孵育2小时。稀释BCMA单域抗体至终浓度为10μg/ml。加入100μl BCMA(10μg/ml)单域抗体，每孔加入BAFF(5μg/ml)蛋白2μl，混匀。稀释羊抗兔IgG-HRP(1:5000)，100μl/孔，37℃孵育1小时。加入TMB显色液，100μl/孔，避光反应10分钟。加入50μl/孔2M H 2SO 4终止反应。在450nm波长测OD值。图6显示BCMA单域抗体能够竞争抑制BAFF蛋白与BCMA蛋白的结合。不同BCMA单域抗体能够竞争抑制BAFF蛋白与BCMA蛋白的结合，抑制率从34～92％。
实施例8：BCMA单域抗体作为CART细胞上特异性BCMA靶抗原的识别抗体研究。
1)载体构建
合成BCMA单域抗体基因和2代CAR结构基因，两条基因通过重叠PCR进行拼接，得到BCMA CAR基因。得到合成基因后，进行分子克隆，首先得到两个基因片段的PCR产物，然后进行重叠PCR，得到两个片段连接在一起 的2代CAR结构的BCMA CAR基因，通过酶切Pre载体和BCMA CAR基因，连接，转化，挑克隆，提质粒，测序，得到序列正确的表达BCMA CAR的慢病毒载体Pre-Lenti-EF1 BCMA CAR。
2)慢病毒包装
包装病毒前一天，胰酶消化293T细胞，铺于150cm培养皿内。将细胞置于5％CO 2培养箱内，孵育8-24小时，当细胞贴壁后达到培养皿总面积的80％开始转染293T细胞。用脂质体(lipofectamine)2000共转染Pre-Lenti-EF1 BCMA CAR：psPAX2：pMD2G＝4：3：1，48小时收集病毒上清。4℃，1250rpm，5分钟，去除死亡的293T细胞及细胞碎片。然后将病毒上清过滤，浓缩，分装冻存-80℃冰箱。
3)CART细胞的制备
采取健康志愿者的新鲜血10ml，用淋巴细胞分离液分离外周血单核细胞(PBMC)后再经磁珠分离纯化T细胞，2×10 6个T细胞/孔种植在6孔板，用含IL-2(1000U/ml)的x-vivo15培养基培养并用抗-CD3刺激，刺激24小时。刺激24小时后，加入BCMA病毒液，感染过夜。第二天补加2ml培养基。感染6-7天后，流式细胞术评价CAR分子表达情况。用生物素化的BCMA经流式细胞术分析转染T细胞表达抗BCMA-CAR的阳性率。
4)杀伤活力测定
细胞杀伤实验采用LDH检测试剂盒(Promega)进行检测，CART细胞/T细胞：靶细胞均设置四个梯度，分别为0.5:1、1:1、2:1、4:1，Daudi细胞3×10 4/孔，用含X-VIVO培养基/1640培养基将其余所有孔的体系补足200μL。将96孔板放置于37℃，5％CO2培养箱中培养。17h后，在最大释放的孔内加入20μl的裂解液，混匀使细胞完全破裂，将96孔板放入CO2培养箱中孵育2h。2h后，观察最大释放的孔，靶细胞全部裂解后从每个孔吸出50μL上清液到平底96孔板中，再在各个孔中加入50μL底物液，避光显色30min。30min之后看颜色变化，最大释放MM.1S孔，和CART细胞孔颜色应该比较深。酶标仪进行测量，测量波长为490nm。杀伤结果参见图7，BCMA嵌合抗原受体修饰T细胞可以特异性杀伤BCMA阳性的细胞，对BCMA阴性的细 胞不具杀伤效应，并且杀伤活性很高，在20％以上。附表1
Figure PCTCN2019095507-appb-000006
Figure PCTCN2019095507-appb-000007
Figure PCTCN2019095507-appb-000008
附表2
Figure PCTCN2019095507-appb-000009
Figure PCTCN2019095507-appb-000010
Figure PCTCN2019095507-appb-000011
Figure PCTCN2019095507-appb-000012
Figure PCTCN2019095507-appb-000013
附表3
Figure PCTCN2019095507-appb-000014
Figure PCTCN2019095507-appb-000015
Figure PCTCN2019095507-appb-000016
Figure PCTCN2019095507-appb-000017
Figure PCTCN2019095507-appb-000018
Figure PCTCN2019095507-appb-000019
Figure PCTCN2019095507-appb-000020
Figure PCTCN2019095507-appb-000021
Figure PCTCN2019095507-appb-000022
Figure PCTCN2019095507-appb-000023
Figure PCTCN2019095507-appb-000024
Figure PCTCN2019095507-appb-000025
Figure PCTCN2019095507-appb-000026
Figure PCTCN2019095507-appb-000027
Figure PCTCN2019095507-appb-000028

Claims (18)

  1. 一组抗BCMA的单域抗体，所述组的单域抗体由框架区和互补决定区组成，所述互补决定区的氨基酸序列选自SEQ ID NO:1-66。
  2. 根据权利要求1所述的一组抗BCMA的单域抗体，所述互补决定区的氨基酸序列与SEQ ID NO:1-66所示的氨基酸序列的同一性大于80％、大于85％、大于90％、大于95％或大于99％，优选地，差异的氨基酸为保守替换。
  3. 根据权利要求1所述的一组抗BCMA的单域抗体，所述组的单域抗体具有选自SEQ ID NO:67-132的氨基酸序列，或其氨基酸序列为SEQ ID NO:67-132。
  4. 根据权利要求3所述的一组抗BCMA的单域抗体，其氨基酸序列与SEQ ID NO:67-132所示氨基酸序列的同一性大于80％、大于85％、大于90％、大于95％或大于99％，优选地，差异的氨基酸为保守替换。
  5. 一组抗BCMA的单域抗体基因，所述组的单域抗体基因具有选自SEQ ID NO:133-198的核苷酸序列，或其核苷酸序列为SEQ ID NO:133-198的核苷酸序列，或编码权利要求3或4所述单域抗体的核苷酸序列。
  6. 根据权利要求5所述的一组抗BCMA的单域抗体基因，其核苷酸序列与SEQ ID NO:133-198所示核苷酸序列的同一性大于80％、大于85％、大于90％、大于95％或大于99％，优选地，差异的碱基为保守替换。
  7. 一种多肽，具有选自权利要求1-4任一项所述的单域抗体组中的一个或多个单域抗体，优选地，所述的多个单域抗体相同或不同。
  8. 一种表达载体，其包含选自权利要求5或6所述的单域抗体基因组的一个或多个基因，优选地，所述表达载体为原核细胞表达载体、真核细胞表达载体或其它细胞表达载体，更优选地，慢病毒载体。
  9. 一种宿主细胞，其包含权利要求8所述表达载体，优选地，所述的宿主细胞为原核表达细胞、真核表达细胞，真菌细胞或酵母细胞，所述原核表达细胞优选大肠杆菌。
  10. 一种嵌合抗原受体，其具有选自权利要求1-4任一项所述的单域抗体组中的一个或多个单域抗体，优选地，所述多个单域抗体相同或不同。
  11. 一种嵌合抗原受体修饰的T细胞，由权利要求10所述的嵌合抗原受体进行修饰。
  12. 一种药用组合物，其包含选自权利要求1-4任一项所述的单域抗体组中的一个或多个单域抗体作为活性成份。
  13. 一种人源化形式的抗BCMA的单域抗体，其通过对选自权利要求1-4任一项所述的单域抗体组中的单域抗体进行人源化而获得。
  14. 权利要求1-4任一项所述的单域抗体组中的单域抗体在检测BCMA中的应用。
  15. 权利要求1-4任一项所述的单域抗体组中的单域抗体在阻断BAFF与BCMA之间相互作用中的应用。
  16. 根据权利要求16所述的应用，所述单域抗体与细胞毒性剂、酶相、放射性同位素、荧光化合物或化学发光化合物中的一种或多种相连。
  17. 权利要求1-4任一项所述的单域抗体组中的单域抗体在制备用于治疗BCMA表达异常相关疾病的药物的应用。
  18. 根据权利要求17所述的应用，所述的BCMA表达异常相关疾病是多发性骨髓瘤性疾病。
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