CN117430709B - Nanometer antibody and application thereof - Google Patents

Nanometer antibody and application thereof Download PDF

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Publication number
CN117430709B
CN117430709B CN202311771263.8A CN202311771263A CN117430709B CN 117430709 B CN117430709 B CN 117430709B CN 202311771263 A CN202311771263 A CN 202311771263A CN 117430709 B CN117430709 B CN 117430709B
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seq
sequence
nanobody
nucleic acid
bcma
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CN117430709A (en
Inventor
庄文超
黎勇
李福胜
郭立君
张婉盈
冼俊杰
任永峰
崔颖杰
林兆新
关小莺
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Kangzhong Guangdong Biopharmaceutical Co ltd
Kangweizhonghe Zhongshan Bio Pharmaceutical Co ltd
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Kangzhong Guangdong Biopharmaceutical Co ltd
Kangweizhonghe Zhongshan Bio Pharmaceutical Co ltd
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • 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
    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C07K2317/50—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
    • C07K2317/567—Framework region [FR]
    • 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®
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a nano antibody and application thereof; the nanobody includes a heavy chain variable region including CDR1, CDR2, and CDR3. The anti-BCMA nano antibody provided by the scheme of the invention can be obviously combined with human multiple myeloma cells, has high affinity, and provides an alternative antibody for the subsequent treatment of human multiple myeloma; meanwhile, the nano antibody prepared by the invention can specifically identify and bind to BCMA, and has potential effect of treating diseases related to BCMA; according to the scheme, the prepared anti-BCMA nano antibody and a pseudomonas exotoxin (PE 38) sequence are prepared to obtain the fusion protein, so that the survival of human multiple myeloma cells can be effectively reduced, the fusion protein is used for treating human multiple myeloma, and a new direction is provided for developing medicaments for treating human multiple myeloma.

Description

Nanometer antibody and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a nano antibody and application thereof.
Background
Multiple Myeloma (MM) is a hematological neoplastic disease characterized by abnormal proliferation of Plasma Cells (PCs) while causing the production of large amounts of pathological immunoglobulins in the body, the tumor cells of which originate from plasma cells in the bone marrow. The incidence of MM in hematological tumors is next to lymphoma. In the field of immunotherapy, research shows that CD38, CD47, CD138, SLAMF7, GPRC5D, fcRH, BCMA and the like are highly expressed on the surface of malignant plasma cells, and monoclonal antibodies or antibody-coupled medicaments thereof show good anti-tumor effect. Nevertheless, MM patients develop resistance after standardized treatment due to their high clonal heterogeneity and complex interactions with the bone marrow environment, ultimately leading to relapse in most patients. Therefore, in order to solve the problems of reduced curative effect in the treatment process, the development of safer, more effective and diverse new drugs is needed.
BCMA (B-cell maturation antigen; CD269; TNFRSF 17) belongs to a member of the tumor necrosis factor receptor superfamily. In normal human tissues, BCMA mRNA and protein are almost exclusively found on plasma cells and selectively overexpressed during malignant transformation of plasma cells, promoting tumor cell growth, survival and the development of drug resistance. The uniformity and uniqueness of BCMA on the surface of MM cells, whether cell lines or patient samples, makes BCMA an attractive target for MM drug discovery and development. In addition, studies have found that BCMA is expressed at similar levels at different stages of multiple myeloma (from untreated to relapsed), suggesting that BCMA may be an effective therapeutic target throughout the disease process.
Currently, BCMA-targeted therapies mainly include three major lineups of Antibody Drug Conjugates (ADC), bispecific antibodies (bispecific antibody, bsAb) and chimeric antigen receptor T cell immunotherapy (chimeric antigen receptor T-cells, CAR-T), and BCMA-targeted immunotherapy has significant efficacy in preclinical and clinical studies, especially CAR-T technology, and can specifically recognize tumor antigens in a non-major histocompatibility complex-dependent manner to exert a powerful antitumor immune effect. However, adverse reactions such as CRS and off-target effects in the treatment process still need to be solved, but when the antibody is used for targeting MM, a drug with too large molecular weight cannot penetrate through a blood vessel to reach a focus, the treatment effect does not reach a particularly good curative effect, and simultaneously, FC fragments on monoclonal antibodies of the related technology have nonspecific binding with other normal cells, so that the drug cannot specifically target the target cells, the curative effect is weakened, and the side effect is large.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. For this purpose, the invention provides a nanobody.
The invention also provides a nucleic acid molecule for encoding the nano antibody.
The invention also provides a biological material related to the nucleic acid molecules.
The invention also provides application of the nano antibody, the nucleic acid molecule and the biological material.
The invention also provides a fusion protein.
The invention also provides application of the fusion protein.
The invention also provides a medicine for treating diseases related to BCMA.
The invention also provides a BCMA detection agent.
The invention also provides a screening method of the nano antibody.
According to a first aspect of the present invention there is provided a nanobody wherein the CDRs of the VHH chain complementarity determining regions are selected from one or more of the following (1) - (3):
(1) CDR1 shown in SEQ ID NO. 11, CDR2 shown in SEQ ID NO. 13, CDR3 shown in SEQ ID NO. 15;
(2) CDR1 shown in SEQ ID NO. 18, CDR2 shown in SEQ ID NO. 20, CDR3 shown in SEQ ID NO. 22;
(3) CDR1 shown in SEQ ID NO. 25, CDR2 shown in SEQ ID NO. 27, CDR3 shown in SEQ ID NO. 29.
In some embodiments of the invention, the VHH chain of the nanobody further comprises a framework region sequence FR.
In some embodiments of the invention, the framework region sequence FR is selected from at least one of the following 1) -3):
1) FR1 shown in SEQ ID NO. 10, FR2 shown in SEQ ID NO. 12, FR3 shown in SEQ ID NO. 14 and FR4 shown in SEQ ID NO. 16;
2) FR1 shown in SEQ ID NO. 17, FR2 shown in SEQ ID NO. 19, FR3 shown in SEQ ID NO. 21 and FR4 shown in SEQ ID NO. 23;
3) FR1 shown in SEQ ID NO. 24, FR2 shown in SEQ ID NO. 26, FR3 shown in SEQ ID NO. 28 and FR4 shown in SEQ ID NO. 30.
In some embodiments of the invention, the VHH chain of the nanobody is selected from at least one of A1) -A3):
a1 A sequence shown as SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6;
a2 Amino acid sequence with the same function as the protein shown in SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6 by substitution and/or deletion and/or addition of one or more amino acids;
a3 Amino acid sequence with 80%, 85% or more than 90% homology with the sequence shown in SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6 and the same function with the protein shown in SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6.
According to a second aspect of the present invention, a nucleic acid molecule is presented, which encodes a nanobody against BCMA as described above.
In some embodiments of the invention, the sequence of the nucleic acid molecule comprises:
b1 A nucleotide sequence shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3; or (b)
B2 A nucleotide sequence shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO.3 is subjected to substitution and/or deletion and/or addition of one or more nucleotides, and the nucleotide sequence is the same as the nucleotide sequence shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO.3 for encoding the same protein; or (b)
B3 Nucleotide sequence which has 80%, 85% or more than 90% homology with SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO.3 and codes the same protein with the nucleic acid molecule shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.
According to a third aspect of the present invention, there is provided a biological material associated with the nucleic acid molecule, the biological material being any one of (1) to (3);
(1) An expression cassette comprising the nucleic acid molecule described above;
(2) A recombinant vector comprising the nucleic acid molecule described above or the expression cassette of (1);
(3) Recombinant cells comprising the nucleic acid molecules described above, the expression cassette of (1) or the recombinant vector of (2).
According to a fourth aspect of the present invention, there is provided the use of the nanobody, nucleic acid molecule, biomaterial described above for the manufacture of a medicament for the treatment of BCMA-related diseases.
In some embodiments of the invention, the disease comprises hematological malignancy.
In some embodiments of the invention, the disease hematological malignancy includes multiple myeloma.
In some embodiments of the invention, the use is in the preparation of a detection reagent for BCMA.
According to a fifth aspect of the present invention, there is provided a fusion protein comprising the nanobody and pseudomonas exotoxin protein described above.
In some embodiments of the invention, the pseudomonas exotoxin sequence is a PE38 sequence.
In some embodiments of the invention, the sequence of the pseudomonas exotoxin protein is selected from at least one of SEQ ID No. 7, SEQ ID No. 8, and SEQ ID No. 9.
In some embodiments of the invention, the fusion protein is obtained by ligating the nanobody described above and the pseudomonas exotoxin protein via a linker sequence.
In some embodiments of the invention, the linking sequence comprises (GGGS) 2 。
According to a sixth aspect of the present invention, there is provided the use of the fusion protein described above for the manufacture of a medicament for the treatment of BCMA-related diseases.
In some embodiments of the invention, the BCMA-related disease comprises hematological malignancy.
In some embodiments of the invention, the hematological malignancy comprises multiple myeloma.
According to a seventh aspect of the present invention, a medicament for the treatment of BCMA-related diseases is presented, said medicament comprising the nanobody, nucleic acid molecule, biomaterial or fusion protein as described above.
In some embodiments of the invention, the medicament further comprises pseudomonas exotoxin (PE 38).
In some embodiments of the invention, the pseudomonas exotoxin has a sequence selected from at least one of SEQ ID No. 7, SEQ ID No. 8, and SEQ ID No. 9.
In some embodiments of the invention, the medicament further comprises a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable adjuvant.
In some embodiments of the invention, the pharmaceutically acceptable carrier comprises at least one of diluents, excipients, fillers, binders, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, sweeteners, and flavoring agents.
In some embodiments of the invention, the BCMA-related disease comprises hematological malignancy.
In some embodiments of the invention, the hematological malignancy comprises multiple myeloma.
According to an eighth aspect of the present invention, there is provided a BCMA detector comprising the above nanobody, nucleic acid molecule, biomaterial.
According to a ninth aspect of the present invention, there is provided a screening method of the above nanobody or antigen-binding fragment thereof, comprising the steps of:
s1, using an immune antigen to immunize a target animal;
s2, separating peripheral blood lymphocytes of the target animal to extract RNA, and obtaining cDNA through reverse transcription;
s3, carrying out two-round PCR amplification by taking the cDNA as a template to obtain a nanobody fragment, and connecting the nanobody fragment with a display carrier to construct a nanobody phage display library;
s4, screening BCMA nanobodies based on the nanobody phage display library.
In some embodiments of the invention, the immune antigen comprises BCMA protein.
In some embodiments of the invention, the animal of interest comprises alpaca.
In some embodiments of the invention, the step of immunizing the subject animal comprises introducing the immunizing antigen into the subject animal at a dose of 0.2-0.4 mg/dose for 3-5 total immunizations at a 12-15d interval.
In some embodiments of the invention, the primers used for obtaining the nano antibody fragment by two PCR amplification with the cDNA as a template are SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35 and SEQ ID NO. 36 respectively.
According to some embodiments of the invention, at least the following benefits are provided: the anti-BCMA nano antibody provided by the scheme of the invention can be obviously combined with human multiple myeloma cells, has high affinity, and provides an alternative antibody for the subsequent treatment of human multiple myeloma; meanwhile, the nano antibody prepared by the invention can specifically identify and bind to BCMA, and has potential effect of treating diseases related to BCMA; according to the scheme, the prepared anti-BCMA nano antibody and a pseudomonas exotoxin (PE 38) sequence are prepared to obtain the fusion protein, so that the survival of human multiple myeloma cells can be effectively reduced, the fusion protein is used for treating human multiple myeloma, and a new direction is provided for developing medicaments for treating human multiple myeloma.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a graph showing the results of animal immunotiter tests in example 1 of the present invention;
FIG. 2 is a diagram showing the detection results of the second round PCR products in example 1 of the present invention, wherein M is a marker;
FIG. 3 is a graph showing ELISA detection results of 165 BCMA antibodies of different sequences in example 3 of the present invention;
FIG. 4 is a graph showing the flow-type binding results of candidate molecules NbBCMA-5 and RPMI8226 cells in example 3, wherein A is a graph showing the flow-type binding results of blank and RPMI8226 cells, B is a graph showing the flow-type binding results of candidate molecules NbBCMA-5 and RPMI8226 cells, C is a graph showing the flow-type binding results of candidate molecules NbBCMA-7 and RPMI8226 cells, and D is a graph showing the flow-type binding results of candidate molecules NbBCMA-8 and RPMI8226 cells;
FIG. 5 is a graph showing the results of flow-through binding between candidate molecules and U266 in example 3 of the present invention, wherein A is a graph showing the results of flow-through binding between a blank and U266, B is a graph showing the results of flow-through binding between candidate molecules NbBCMA-5 and U266, C is a graph showing the results of flow-through binding between candidate molecules NbBCMA-7 and U266, and D is a graph showing the results of flow-through binding between candidate molecules NbBCMA-8 and U266;
FIG. 6 is a diagram showing the SDS-PAGE detection result in embodiment 3 of the present invention, wherein M is a marker,1 is a NbBCMA-5 candidate molecule, 2 is a NbBCMA-7 candidate molecule, and 3 is a NbBCMA-8 candidate molecule;
FIG. 7 is a graph showing the results of measurement of protein concentration and activity in example 4 of the present invention, wherein M is a marker;
FIG. 8 is a graph showing the results of measurement of protein concentration and activity in example 4 of the present invention;
FIG. 9 is a graph showing the results of measurement of protein concentration and activity in example 4 of the present invention;
FIG. 10 is a graph showing the detection result of killing effect of the nano-antibody fusion expression drug prepared in examples 4-6 of the test example on the multiple myeloma cell line U266;
FIG. 11 is a graph showing the detection result of killing effect of the nanobody fusion expression drug prepared in examples 7 to 9 of the test example on the multiple myeloma cell line U266;
FIG. 12 is a graph showing the detection result of killing effect of the nanobody fusion expression drug prepared in examples 10-12 of the test example on multiple myeloma cell line U266;
FIG. 13 is a graph showing the results of detection of the killing effect of Pseudomonas exotoxin sequences on multiple myeloma cell line U266 in the test examples of the present invention.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
EXAMPLE 1 construction of phage library
1. llama immunity
Adult male healthy llama alpaca were subjected to subcutaneous multipoint injection immunization. 0.25mg BCMA protein (purchased from offshore protein, cat No. cs 79) was injected every 14 days for a total of 3 injections. The titers were measured by taking 2mL of blood 14 days after the third immunization.
The titer detection utilizes an ELISA method, the target antigen is BCMA protein with HIS label, the primary antibody is anti-HIS-HRP, TMB color development, and the O.D value is detected at 450 nm). In the detection, serum is diluted by PBS buffer solution with pH of 7.4, wherein the dilution times of the serum are 0.1k times, 1k times, 2k times, 4k times, 8k times, 16k times, 32k times, 64k times, 128k times, 256k times, 512k times and 1024k times, the detection result is shown in figure 1, and the immune titer of the invention reaches 1024k times of serum dilution and is far beyond the common industry value.
2. Acquisition of alpaca Peripheral Blood Mononuclear Cell (PBMC) total RNA and cDNA
And (3) carrying out a large amount of blood collection on alpaca meeting the titer requirement, separating PBMC, and extracting total RNA. The method for extracting total RNA is a TRZOL portable operation method, and comprises the following specific operations: taking out the separated PBMC cells from the liquid nitrogen tank, and rapidly thawing; centrifuging at 800g for 5min, discarding supernatant, adding 1mL TRIZOL solution into the precipitate, and lightly blowing back and forth with a gun for several times to completely lyse the cells; 200. Mu.L of chloroform was added and the mixture was shaken vigorously by hand for 30S, left stand for 5min at 13500r/min, centrifuged for 10min, and the supernatant was separated into layers, and the supernatant was pipetted into another new RNase-free 1.5mL tube, and an equal volume of isopropanol was added. Mixing, and precipitating at-20deg.C for 20 min. The pellet was centrifuged at 13500rpm for 10 minutes and the supernatant was discarded. Washing with 75% ice ethanol twice, blow-drying with a super clean bench, and re-dissolving with 60 μl RNase-free water to obtain a total RNA solution of Peripheral Blood Mononuclear Cells (PBMC) of alpaca for later use.
After the total RNA is reversely transcribed into cDNA by using a Thermo company reverse transcription kit (see the Thermo company reverse transcription kit for specific scheme), quantitative split charging is carried out, and the total RNA is preserved at-80 ℃ for standby.
3. Phage library construction
(1) The target fragment was obtained by performing two-step PCR amplification.
1) First round PCR
A first round of PCR was performed using Kz-001 and Kz-002 as primers (sequences shown in Table 1) and amplified using TAKARA high-fidelity PCR polymerase as follows: the reaction was performed at 94℃for 3 minutes, followed by 18 cycles of denaturation at 94℃for 30 seconds, annealing at 53℃for 30 seconds and elongation at 72℃for 40 seconds, elongation at 72℃for 10 minutes and cooling at 4℃for 1 minute. The 750bp band was recovered. The reaction system is shown in the specification of TAKARA high-fidelity PCR polymerase, and the template is cDNA of alpaca peripheral blood PBMA cells.
TABLE 1
2) Second round PCR
A second round of PCR was performed using Kz-003, kz-004, kz-005 and Kz-006 as primers (sequences shown in Table 1) and amplified using TAKARA high-fidelity PCR polymerase as follows: pre-denaturation at 94℃for 3 min, (denaturation at 94℃for 30 sec, annealing at 53℃for 30 sec, extension at 72℃for 30 sec), 20 cycles, extension at 72℃for 10min, cooling at 4℃for 1 min. The 450bp band was recovered. The reaction system is referred to the instruction of TAKARA high-fidelity PCR polymerase, and the template DNA is the DNA product obtained by the PCR in the step 1.
The agarose gel electrophoresis detection result of the second round of PCR products is shown in figure 2, and a specific target band with the size of 450bp can be seen from the figure, and the band is the nano antibody fragment. These bands were collected and quantified using an agarose gel collection kit.
(2) Digestion, ligation and electrotransformation of fragments and vectors to obtain phage libraries
The nanobody fragment obtained in the step (1) is subjected to enzyme digestion by adopting restriction enzymes, and two restriction enzyme digestion sites are sfiI and NotI (the sfiI and NotI enzymes are purchased from Shanghai Bioengineering Co., ltd., enzyme digestion system and conditions are carried out according to the instruction book), and the nanobody fragment is recovered and quantified by using a gel recovery kit (purchased from Shanghai Bioengineering Co., ltd.); the invention uses a commercial vector pcantab5e (purchased from the biological technology Co., ltd., chengdu, cat# trans 12-4), and the vector is also subjected to restriction enzyme digestion by the two types of restriction enzyme digestion, and is recovered by a gel recovery kit and then quantified.
All the digested carriers and digested nano antibody fragments are subjected to a molar ratio of 1:3 overnight ligation followed by shock transformation into TG1 competent cells, dilution plating for clonal enumerationThe storage capacity was 9.9X10 7 The method comprises the steps of carrying out a first treatment on the surface of the Randomly picking 20 clones, sequencing, positive rate 100%, packaging to phage titer 2.14X10 13 pfu/mL。
Example 2 screening of phage libraries
The phage library is screened by adopting a solid phase screening method, and the specific screening method is as follows:
target molecules were coated on 96-well surfaces, unadsorbed target molecules were washed off, then blocked, and then bound by adding a background-subtracted phage antibody library to the wells. Unbound phage were washed away and eluted with 0.2M glycine-HCl to give affinity phage. Each round of phage with high affinity was obtained by decreasing the concentration of coated target molecules and increasing the wash power. Each round of panning amplification test will allow the nanobody bound to the target molecule to be enriched in phage libraries. After 2 rounds of screening, monoclonal verification is carried out, the result is shown in table 2, and enrichment after screening reaches more than 1000 times.
TABLE 2 anti-BCMA nanobody phage library 2 round screening results
EXAMPLE 3 screening of nanobody molecules
(1) The specific clones of the target antigen were identified by ELISA as follows:
752 monoclonal antibodies were selected from round 2 of the screening product of example 2 for ELISA identification as follows: the target protein was diluted with a pH9.6 sodium bicarbonate coating solution, and the solution was allowed to stand at 37℃for 1h for coating, followed by washing with PBS 3 times. Adding a sealing liquid, sealing, standing at 37 ℃ for 1 hour, and throwing out the redundant sealing liquid PBS for 3 times. The amplified product was diluted 10 times with 1%M-PBS and mixed to 50. Mu.L/well and allowed to stand at 37℃for 1 hour. An antibody: the samples were taken with 1%M-PBS at 1: the 1000 dilution rabbit anti-M13, 50. Mu.L/well, is left to stand at 37℃for 1.0h. And (2) secondary antibody: the samples were taken with 1%M-PBS at 1: the HRP-goat anti-rabbit was diluted at 3000. Mu.L/well and allowed to stand at 37℃for 1.0h. Color development: taking 0.2M/L Na 2 HPO 4 4.5mL of +0.1M/L citric acid was added with a small amount of OPD, 60. Mu. L H 2 O was mixed well at 50. Mu.L/well. The 2M sulfuric acid was stopped at 50. Mu.L/well. 490nAnd (3) detecting m. The Phage ELISA was repeated at least once. Positive clone preservation: mixing 0.5mL Phage+0.3mL 50% glycerol. Preserving at-80 ℃.
Sequencing all positive sequences in ELISA to obtain 165 different nanometer antibody sequences, wherein ELISA results of the sequences are shown in figure 3; as can be seen from fig. 3, all the sequences screened had significantly greater absorbance at 490nm than the blank, indicating that BCMA nanobody bound to BCMA protein with positive results.
(2) Candidate nanobody molecule and cell binding Activity assay
Cloning and constructing 165 candidate sequences obtained in the step (1) into an expression vector PET30a (+) (which is obtained from biological (Anhui) Co., ltd.) prokaryotic expression vector, carrying out 37 ℃ and 220rpm/min induction expression of 1mM IPTG overnight, taking 3mL of overnight induction expression bacteria, carrying out ultrasonic disruption and 13500rpm/min, centrifuging for 10 minutes, taking the supernatant, carrying out a flow cell binding experiment, and carrying out experiment to select two multiple myeloma cell lines, one RPMI8226 strain with low expression of BCMA protein and one U266 cell line with high expression of BCMA protein. And respectively detecting the binding condition of 165 candidate sequences and the two cell lines, and screening to obtain 3 nanometer antibody candidate molecules (NbBCMA-5, nbBCMA-7 and NbBCMA-8), wherein the binding of the 3 candidate sequences and the RPMI8226 cell line with low BCMA expression is shown in figure 4. The cell line was then replaced, and the BCMA-expressing cell line U266 was used to detect the flow binding of the candidate molecules to the cell line, and as a result, as shown in fig. 5, the 3 candidate molecules were also bound simultaneously to the BCMA-expressing cell line U266.
The flow cytometry detection method specifically comprises the following steps: collecting cells, preparing single cell suspension, and counting; the supernatant was centrifuged off and resuspended in 1.5mL FACS BUFF (2% FBS-PBS); adding 60 μl of blocking regenant (invitrogen 14-9161-73), mixing, and incubating on ice for 10min; cells were added to a 1.5mL EP tube (300. Mu.L/sample) containing the sample and incubated on ice for 15min; setting one blank control and antibody only control; flow buffer was washed 2 times, the supernatant was discarded, the raffinate (100. Mu.L) was added with the flow antibody anti-his-FITC, incubated on ice for 20min, mixed well across the cell screen, and tested with a flow meter (Beckmann cytoFlex).
(3) Antigen-antibody affinity detection of candidate nanobody molecules
TABLE 3 Table 3
3 candidate molecules are respectively fused with FC labels and expressed in a pCDNA3.1 (+) eukaryotic vector, 293 cells are transiently rotated to form a 30mL system, then Protein A columns are used for purification, purified proteins are obtained, protein samples are subjected to reduction treatment and run out of SDS-PAGE gel, as shown in FIG. 6, and antigen-antibody affinity detection is carried out, wherein the specific detection method is as follows: the immobilized BCMA non-tagged protein antigen, combined with self-purified NBBCMA-FC protein gradient dilutions (100 nM,50nM,25nM,12.5nM,6.25 nM), using anti-humanFC probe, affinity results are shown in Table 3, where it can be seen that the affinity of the NbBCMA-8 molecule, nbBCMA-5 molecule is nanomolar and the NbBCMA-7 molecule reaches picomolar.
FC tag sequence:
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK（SEQ ID NO:40）。
(4) Epitope detection of candidate molecules
According to the invention, an anti-humanFC probe is adopted, the protein is purified by NBBCMA-FC at a sample position, and epitopes of NbBCMA-5, nbBCMA-7 and NbBCMA-8,3 candidate molecules are detected by a sandwich method. The results are shown in Table 4: the epitopes of NbBCMA-5 (5#) and NbBCMA-7 (7#) are different, and NbBCMA-7 and NbBCMA-8 have the same epitope.
TABLE 4 Table 4
The CDR region sequences of the 3 nano antibody molecules obtained by the invention are shown in Table 5.
TABLE 5
The protein sequence of the obtained nano antibody molecule is as follows:
Seq5:QVQLVETGGGLVQAGGSLRLSCAASGTIFSVYNMGWFRQAPGKQRELVAAITKNGSTSYKDSVKGRFTISTDNAKNTVDLRMSSLKLGDTAVYFCAAWDWDKNQAYWGQGTQVTVSS（SEQ ID NO:4）。
Seq7:QVQLVETGEGLGQVGGPLTFSWACSESIFMTYNMGGFRQAPGQAPQVGPCSYYRGDHTLYLLCERAILPLQRQRPEHDRSANEQPETCWTQPSITVPPGIVDKDQAYWIQETHVTVSS（SEQ ID NO:5）。
Seq8:QVQLVESGGGLVQAGGSLRLSCAASTRTFSSYVMAWFRQVPGKEREFVASRACSGGTPYYADSVKGRFFISRDDAKNTVYLRMNSLKPEDTAVYYCAAASIGATQYDYWGQGTQVIVSS（SEQ ID NO:6）。
the nucleic acid sequences of the candidate molecules obtained by the invention are as follows:
Seq5:
CAGGTGCAGCTGGTGGAAACTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAACGATCTTCAGCGTCTATAACATGGGCTGGTTCCGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCAGCTATAACTAAGAATGGTAGCACAAGCTATAAAGACTCCGTGAAGGGCCGATTCACCATCTCCACAGACAACGCCAAGAACACGGTGGATCTGCGAATGAGCAGCCTGAAACTTGGGGACACAGCCGTCTATTTCTGTGCCGCCTGGGATTGGGACAAAAATCAGGCCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA（SEQ ID NO:1）。
Seq7:
CAGGTGCAGCTGGTGGAAACTGGGGAAGGTTTGGGGCAGGTTGGGGGGCCTCTGACATTCTCCTGGGCATGCTCTGAATCGATCTTCATGACCTATAACATGGGCGGGTTCCGCCAGGCTCCAGGACAAGCACCGCAAGTAGGCCCCTGCTCTTACTATAGGGGTGATCACACGCTATATTTACTCTGCGAAAGGGCGATTCTCCCTCTCCAGAGACAACGCCCAGAACACGATAGATCTGCTAATGAACAGCCTGAAACTTGCTGGACACAGCCGTCTATTACTGTGCCGCCTGGGATTGTGGACAAAGACCAGGCCTACTGGATACAGGAGACCCACGTCACCGTCTCCTCA（SEQ ID NO:2）。
Seq8:
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCGGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTACACGCACCTTCAGTAGCTATGTCATGGCCTGGTTCCGCCAGGTTCCAGGAAAGGAGCGTGAGTTTGTAGCTTCTCGTGCCTGCAGTGGTGGTACCCCTTACTATGCAGACTCCGTGAAGGGCCGATTCTTCATCTCCAGAGACGACGCCAAGAACACGGTGTATCTGCGAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGCCTCCATTGGGGCCACTCAGTATGACTACTGGGGCCAGGGGACCCAGGTCATCGTCTCCTCA（SEQ ID NO:3）。
example 4A drug against multiple myeloma
The present example provides a drug against multiple myeloma, which is prepared by passing a sequence of Seq5 (sequence shown as SEQ ID NO: 1) and a sequence of Seq9 (sequence shown as SEQ ID NO: 7) of Pseudomonas exotoxin (PE 38) through linker (GGGS) as prepared in example 3 2 The medicine obtained by fusion expression is prepared by the following specific method:
pseudomonas exotoxin (PE 38) sequence Seq9:
PEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK（SEQ ID NO:7）。
gene synthesis seq9 linker (GGGS) 2 The recombinant vector was obtained by inserting two restriction enzyme sites BamHI/XhoI into the vector PET30 (a) and was kept ready for use. The primers SEQ1-F, SEQ-R shown in Table 6 are used, the nucleotide sequence shown in SEQ ID NO. 1 is used as a template to amplify the SEQ5, ndeI/BamHI restriction sites are used after the amplification, the fragment of the SEQ5 purpose is inserted into the recombinant vector, the sequence is carried out, and the correct sequencing reserve is reserved.
TABLE 6
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Clones sequenced correctly above were shake-cultured using 2YT medium at 22℃and 220 r/min. The expression was induced by adding IPTG at a final concentration of 1mg/mL for 6 hours at OD 0.7. 12000 The cells were collected by centrifugation at r/min for 5min, 50mmol/L Tris-HCl was added, and after ultrasonic disruption in ice bath, the pellet was collected by centrifugation at 12000r/min for 30 min. Adding inclusion body washing liquid into inclusion bodies: 50mmol/L Tris-HCl,1mmol/L EDTA,50mmol/L NaCl, w=0.5% Triton X-100, pH8.0; washing for 1-2 h at 37 ℃ in an oscillating way, centrifuging for 15min at 8000r/min, and collecting precipitate. Adding a proper amount of inclusion body (inclusion body dissolving solution) after washing: 6mol/L urea, 50mmol/L Tris-HCl,1mmol/L EDTA,50mmol/L NaCl,10mmol/L beta-ME, pH8.0, stirring overnight on a magnetic stirrer, centrifuging at 1000r/min for 30min, and collecting supernatant to obtain inclusion body solution. The 8mol/L urea dissolved sample is put into a dialysis bag, sealed and placed in renaturation solution I (0.2 mol/L Tris-HCl;0.5mol/L NaCl;5% glycerol; 5 mu mol/L EDTA; pH8.5), dialyzed for 12 hours at 4 ℃, then transferred into renaturation solution II (0.2 mol/L Tris-HCl;0.5mol/L NaCl; 4 ℃) and dialyzed for 12 hours at 12000r/min, and the supernatant is collected for protein concentration and activity measurement.
The results of the protein concentration detection are shown in fig. 7-9, and it can be seen from the figures that fusion expression sequences are successfully prepared, and the positions of the fusion expression sequences are consistent with the expectations.
Example 5A drug against multiple myeloma
The present example provides a drug against multiple myeloma, which is prepared by passing a sequence of Seq5 (sequence shown as SEQ ID NO: 1) and a sequence of Seq10 (sequence shown as SEQ ID NO: 8) of Pseudomonas exotoxin (PE 38) through linker (GGGS) as prepared in example 3 2 The drug obtained by fusion expression differs from example 4 only in that the pseudomonas exotoxin (PE 38) sequence Seq9 is replaced by a pseudomonas exotoxin (PE 38) sequence Seq10.
Pseudomonas exotoxin (PE 38) sequence Seq10:
PEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPKDEL（SEQ ID NO:8）。
example 6A drug against multiple myeloma
The present example provides a drug against multiple myeloma, which is prepared by passing a sequence of Seq5 (sequence shown as SEQ ID NO: 1) and a sequence of Seq11 (sequence shown as SEQ ID NO: 9) of Pseudomonas exotoxin (PE 38) through linker (GGGS) as prepared in example 3 2 The drug obtained by fusion expression differs from example 4 only in that the pseudomonas exotoxin (PE 38) sequence Seq9 is replaced by a pseudomonas exotoxin (PE 38) sequence Seq11.
Pseudomonas exotoxin (PE 38) sequence Seq11:
ASGGPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPKDEL（SEQ ID NO:9）。
example 7A drug against multiple myeloma
The present example provides a drug against multiple myeloma, which is prepared by passing a sequence of the nanometer antibody obtained in example 3, seq7 (the sequence shown as SEQ ID NO: 2), and a sequence of the pseudomonas exotoxin (PE 38) Seq9 (the sequence shown as SEQ ID NO: 7) through linker (GGGS) 2 And (5) fusion expressing the obtained medicine.
The preparation method comprises the following steps: gene synthesis seq9 linker (GGGS) 2 The recombinant vector was obtained by inserting two restriction enzyme sites BamHI/XhoI into the vector PET30 (a) and was kept ready for use. The primers SEQ1-F, seq-R shown in Table 6 are used, the nucleotide sequence shown in SEQ ID NO. 2 is used as a template to amplify the SEQ7, ndeI/BamHI restriction sites are used after the amplification, the fragment of the SEQ7 purpose is inserted into the recombinant vector, the sequence is carried out, and the reserve of correct sequencing is reserved.
Clones sequenced correctly above were shake-cultured using 2YT medium at 22℃and 220 r/min. The expression was induced by adding IPTG at a final concentration of 1mg/mL for 6 hours at OD 0.7. 12000 The cells were collected by centrifugation at r/min for 5min, 50mmol/L Tris-HCl was added, and after ultrasonic disruption in ice bath, the pellet was collected by centrifugation at 12000r/min for 30 min. Adding inclusion body washing liquid into inclusion bodies: 50mmol/L Tris-HCl,1mmol/L EDTA,50mmol/L NaCl, w=0.5% Triton X-100, pH8.0; washing for 1-2 h at 37 ℃ in an oscillating way, centrifuging for 15min at 8000r/min, and collecting precipitate. Adding a proper amount of inclusion body (inclusion body dissolving solution) after washing: 6mol/L urea, 50mmol/L Tris-HCl,1mmol/L EDTA,50mmol/L NaCl,10mmol/L beta-ME, pH8.0, stirring overnight on a magnetic stirrer, centrifuging at 1000r/min for 30min, and collecting supernatant to obtain inclusion body solution. The 8mol/L urea dissolved sample is put into a dialysis bag, sealed and placed in renaturation solution I (0.2 mol/L Tris-HCl;0.5mol/L NaCl;5% glycerol; 5 mu mol/L EDTA; pH8.5), dialyzed for 12 hours at 4 ℃, then transferred into renaturation solution II (0.2 mol/L Tris-HCl;0.5mol/L NaCl; 4 ℃) and dialyzed for 12 hours at 12000r/min, and the supernatant is collected for protein concentration and activity measurement.
The results of the protein concentration detection are shown in fig. 7-9, and it can be seen from the figures that fusion expression sequences are successfully prepared, and the positions of the fusion expression sequences are consistent with the expectations.
Example 8A drug against multiple myeloma
The present example provides a drug against multiple myeloma, which is prepared by passing a sequence of the nanometer antibody obtained in example 3, seq7 (the sequence shown as SEQ ID NO: 2) and a sequence of the pseudomonas exotoxin (PE 38) Seq10 (the sequence shown as SEQ ID NO: 8) through a linker (GGGS) 2 The drug obtained by fusion expression was identical to example 7 except that the Pseudomonas exotoxin (PE 38) sequence Seq9 was replaced with the Pseudomonas exotoxin (PE 38) sequence Seq10.
Example 9A drug against multiple myeloma
The present example provides a drug against multiple myeloma, which is prepared by passing the sequence of the nanometer antibody obtained in the example 3 (the sequence shown as SEQ ID NO: 2) and the sequence of the pseudomonas exotoxin (PE 38) sequence Seq11 (the sequence shown as SEQ ID NO: 9) through a linker (GGGS) 2 The specific preparation method of the drug obtained by fusion expression is the same as that of example 7,the only difference is that the Pseudomonas exotoxin (PE 38) sequence Seq9 is replaced by the Pseudomonas exotoxin (PE 38) sequence Seq11.
Example 10A drug against multiple myeloma
The present example provides a drug against multiple myeloma, which is prepared by passing a sequence of the nanometer antibody obtained in example 3, seq8 (sequence shown as SEQ ID NO: 3) and a sequence of the pseudomonas exotoxin (PE 38) Seq9 (sequence shown as SEQ ID NO: 7) through linker (GGGS) 2 And (5) fusion expressing the obtained medicine.
The preparation method comprises the following steps: gene synthesis seq9 linker (GGGS) 2 The recombinant vector was obtained by inserting two restriction enzyme sites BamHI/XhoI into the vector PET30 (a) and was kept ready for use. The primers SEQ1-F, SEQ-R shown in Table 6 are used, the nucleotide sequence shown in SEQ ID NO.3 is used as a template to amplify the SEQ8, ndeI/BamHI two restriction enzyme sites are used after the amplification, the fragment of the SEQ8 purpose is inserted into the recombinant vector, the sequence is carried out, and the correct sequence is reserved for standby.
Clones sequenced correctly above were shake-cultured using 2YT medium at 22℃and 220 r/min. The expression was induced by adding IPTG at a final concentration of 1mg/mL for 6 hours at OD 0.7. 12000 The cells were collected by centrifugation at r/min for 5min, 50mmol/L Tris-HCl was added, and after ultrasonic disruption in ice bath, the pellet was collected by centrifugation at 12000r/min for 30 min. Adding inclusion body washing liquid into inclusion bodies: 50mmol/L Tris-HCl,1mmol/L EDTA,50mmol/L NaCl, w=0.5% Triton X-100, pH8.0; washing for 1-2 h at 37 ℃ in an oscillating way, centrifuging for 15min at 8000r/min, and collecting precipitate. Adding a proper amount of inclusion body (inclusion body dissolving solution) after washing: 6mol/L urea, 50mmol/L Tris-HCl,1mmol/L EDTA,50mmol/L NaCl,10mmol/L beta-ME, pH8.0, stirring overnight on a magnetic stirrer, centrifuging at 1000r/min for 30min, and collecting supernatant to obtain inclusion body solution. The 8mol/L urea dissolved sample is put into a dialysis bag, sealed and placed in renaturation solution I (0.2 mol/L Tris-HCl;0.5mol/L NaCl;5% glycerol; 5 mu mol/L EDTA; pH8.5), dialyzed for 12 hours at 4 ℃, then transferred into renaturation solution II (0.2 mol/L Tris-HCl;0.5mol/L NaCl; 4 ℃) and dialyzed for 12 hours at 12000r/min, and the supernatant is collected for protein concentration and activity measurement.
The results of the protein concentration detection are shown in fig. 7-9, and it can be seen from the figures that fusion expression sequences are successfully prepared, and the positions of the fusion expression sequences are consistent with the expectations.
Example 11A drug against multiple myeloma
The present example provides a drug against multiple myeloma, which is prepared by passing the sequence Seq8 (sequence shown as SEQ ID NO: 3) of the nanobody prepared in example 3 and the sequence Seq10 (sequence shown as SEQ ID NO: 8) of the pseudomonas exotoxin (PE 38) through linker (GGGS) 2 The drug obtained by fusion expression was identical to example 10 except that the Pseudomonas exotoxin (PE 38) sequence Seq9 was replaced with the Pseudomonas exotoxin (PE 38) sequence Seq10.
Example 12A drug against multiple myeloma
The present example provides a drug against multiple myeloma, which is prepared by passing the sequence Seq8 (sequence shown as SEQ ID NO: 3) of the nanobody prepared in example 3 and the sequence Seq11 (sequence shown as SEQ ID NO: 9) of the pseudomonas exotoxin (PE 38) through linker (GGGS) 2 The drug obtained by fusion expression was identical to example 10 except that the Pseudomonas exotoxin (PE 38) sequence Seq9 was replaced with the Pseudomonas exotoxin (PE 38) sequence Seq10.
Test examples
This test example tests the killing effect of the anti-multiple myeloma drugs prepared in examples 4-12 on multiple myeloma cell line U266 which expresses BCMA.
The invention adopts CCK-8 kit to detect cell proliferation and cytotoxicity. The working principle is as follows: in the presence of an electron coupling reagent, WST-8 can be reduced by intramitochondrial dehydrogenases to produce a highly water-soluble orange-yellow formazan product (formazan) whose color shade is proportional to cell proliferation, inversely proportional to cytotoxicity, and linearly related to the same cells, color shade and cell number. The OD value measured at a wavelength of 450nm using an enzyme-labeled instrument can indirectly reflect the number of living cells.
The specific test steps are as follows:
1. seed plate: 10000U 266 cells (available from Shanghai Biotechnology Co., ltd.) were seeded in 96-well plates, 4 duplicate wells were set per group, and a blank group was set. The cells were then exposed to 37℃5% CO 2 Culturing in a cell culture box for 18h (12-24 h).
2. Adding the medicine: the culture medium was aspirated from each well, the experimental group was added with medium containing different concentrations of drug (concentration 20000ng/mL, 10000ng/mL, 5000ng/mL, 2500ng/mL, 1250ng/mL, 625ng/mL, 312.5ng/mL, 156.25ng/mL, 78.125ng/mL, 39.0625ng/mL, 19.53125ng/mL, 9.765625ng/mL, respectively) and the drug-free medium (drug group against multiple myeloma prepared in examples 4-12), the negative control group was replaced with U266 cells by the jarset cell line, the pseudomonas exotoxin control group was added at concentrations 9000ng/mL, 2500ng/mL, 1250ng/mL, 625ng/mL, 312.5ng/mL, 156.25ng/mL, 78.125ng/mL, 39.0625ng/mL, 19.53125ng/mL, 9.765625ng/mL, and the pseudomonas exotoxin (PE 38) protein Seq9, seq10, seq11, respectively; the 96-well plate was then incubated at 37℃with 5% CO 2 Incubate in an air cell incubator for 24h.
3. Adding CCK-8: in both methods, 10. Mu.L of CCK-8 solution was added directly to each well. Placing the CCK-8-added culture plate into 37 ℃ and 5% CO 2 Incubating for 4 hours in an incubator, taking out the 96-well plate, detecting the OD value of each well at the wavelength of 450nm by using an enzyme label instrument, analyzing and processing data, and drawing a proliferation curve.
4. Analysis of results:
cell viability: OD values of each duplicate wells were averaged ± SD by subtracting the background OD value from the OD value of each test well (blank). The cell viability in the test was calculated according to the cell viability calculation formula on the cck8 specification.
Cell viability = [ (As-Ab)/(Ac-Ab) ]x100%;
as: experimental hole absorbance (cell, medium, CCK-8 solution and drug solution);
ac: control well absorbance (with cells, medium, CCK-8 solution, without drug);
ab: blank well absorbance (medium, CCK-8 containing solution, no cells, drug).
As shown in FIGS. 10-13, it can be seen that the sequences seq5, seq7 and seq8 of the nano-antibody prepared by the invention are respectively linked with the sequences seq9, seq10 and seq11 of the pseudomonas exotoxin (PE 38) through linker (GGGS) 2 The fusion expressed antitumor drug molecule has obvious cell killing effect on the multiple myeloma cell line U266 and also has obvious specificity.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Claims (10)

1. A nanobody, wherein the CDRs of the VHH chain complementarity determining regions in the nanobody are selected from one or more of the following (1) - (3):
(1) CDR1 shown in SEQ ID NO. 11, CDR2 shown in SEQ ID NO. 13, CDR3 shown in SEQ ID NO. 15;
(2) CDR1 shown in SEQ ID NO. 18, CDR2 shown in SEQ ID NO. 20, CDR3 shown in SEQ ID NO. 22;
(3) CDR1 shown in SEQ ID NO. 25, CDR2 shown in SEQ ID NO. 27, CDR3 shown in SEQ ID NO. 29;
the nano antibody is anti-BCMA nano antibody.
2. The nanobody of claim 1, wherein the VHH chain of the nanobody further comprises a framework region sequence FR; the framework region sequence FR is selected from at least one of the following 1) -3):
1) FR1 shown in SEQ ID NO. 10, FR2 shown in SEQ ID NO. 12, FR3 shown in SEQ ID NO. 14 and FR4 shown in SEQ ID NO. 16;
2) FR1 shown in SEQ ID NO. 17, FR2 shown in SEQ ID NO. 19, FR3 shown in SEQ ID NO. 21 and FR4 shown in SEQ ID NO. 23;
3) FR1 shown in SEQ ID NO. 24, FR2 shown in SEQ ID NO. 26, FR3 shown in SEQ ID NO. 28 and FR4 shown in SEQ ID NO. 30.
3. The nanobody of claim 1, wherein the VHH chain of the nanobody is selected from at least one of A1) -A3):
a1 A sequence shown as SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6;
a2 Amino acid sequence with the same function as the protein shown in SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6 by substitution and/or deletion and/or addition of one or more amino acids;
a3 Amino acid sequence with 80%, 85% or more than 90% homology with the sequence shown in SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6 and the same function with the protein shown in SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6.
4. A nucleic acid molecule encoding the nanobody of any of claims 1-3; the sequence of the nucleic acid molecule comprises:
b1 A nucleotide sequence shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3; or (b)
B2 A nucleotide sequence shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO.3 is subjected to substitution and/or deletion and/or addition of one or more nucleotides, and the nucleotide sequence is the same as the nucleotide sequence shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO.3 for encoding the same protein; or (b)
B3 Nucleotide sequence which has 80%, 85% or more than 90% homology with SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO.3 and codes the same protein with the nucleic acid molecule shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.
5. A biological material associated with the nucleic acid molecule of claim 4, which is any one of (1) to (3);
(1) An expression cassette comprising the nucleic acid molecule of claim 4;
(2) A recombinant vector comprising the nucleic acid molecule of claim 4 or the expression cassette of (1);
(3) A recombinant cell comprising the nucleic acid molecule according to claim 4, the expression cassette according to (1) or the recombinant vector according to (2).
6. Use of at least one of the nanobody of any of claims 1-3, the nucleic acid molecule of claim 4 and the biological material of claim 5 in any of the following (1) - (2):
(1) Preparing a medicament for treating multiple myeloma;
(2) Preparing detection reagent of BCMA.
7. A fusion protein comprising the nanobody of any one of claims 1-3 and a pseudomonas exotoxin protein; the sequence of the pseudomonas exotoxin protein is selected from at least one of SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9.
8. Use of the fusion protein of claim 7 in the manufacture of a medicament for the treatment of multiple myeloma.
9. A medicament for the treatment of multiple myeloma, characterized in that it comprises a nanobody according to any one of claims 1-3, a nucleic acid molecule according to claim 4, a biological material according to claim 5 and/or a fusion protein according to claim 7.
10. A BCMA detector comprising the nanobody of any one of claims 1-3, the nucleic acid molecule of claim 4, and/or the biological material of claim 5.
CN202311771263.8A 2023-12-21 2023-12-21 Nanometer antibody and application thereof Active CN117430709B (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109678961A (en) * 2019-01-15 2019-04-26 深圳市南科生物工程有限公司 A kind of construction method of the Chimeric antigen receptor based on BMCA nano antibody sequence and its application
CN109694413A (en) * 2019-01-17 2019-04-30 深圳市前海精准生物科技有限公司 A kind of Chimeric antigen receptor and its application based on BMCA nano antibody sequence
CN111848798A (en) * 2020-07-27 2020-10-30 南京安锐生物科技有限公司 Nano antibody capable of being combined with BCMA (brain cell activating antigen) and application thereof
CN112851814A (en) * 2020-03-17 2021-05-28 西安宇繁生物科技有限责任公司 BCMA-targeted fully human single-chain antibody and preparation method and application thereof
CN116396385A (en) * 2022-09-23 2023-07-07 四川大学 anti-BCMA nano antibody and preparation method and application thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109678961A (en) * 2019-01-15 2019-04-26 深圳市南科生物工程有限公司 A kind of construction method of the Chimeric antigen receptor based on BMCA nano antibody sequence and its application
CN109694413A (en) * 2019-01-17 2019-04-30 深圳市前海精准生物科技有限公司 A kind of Chimeric antigen receptor and its application based on BMCA nano antibody sequence
CN112851814A (en) * 2020-03-17 2021-05-28 西安宇繁生物科技有限责任公司 BCMA-targeted fully human single-chain antibody and preparation method and application thereof
CN111848798A (en) * 2020-07-27 2020-10-30 南京安锐生物科技有限公司 Nano antibody capable of being combined with BCMA (brain cell activating antigen) and application thereof
CN116396385A (en) * 2022-09-23 2023-07-07 四川大学 anti-BCMA nano antibody and preparation method and application thereof

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