CN112538115B

CN112538115B - Anti-human BCMA nano antibody and preparation method and application thereof

Info

Publication number: CN112538115B
Application number: CN202011424935.4A
Authority: CN
Inventors: 陈明久; 马志清
Original assignee: Boaoxin Biotechnology Nanjing Co ltd
Current assignee: Boaoxin Biotechnology Nanjing Co ltd
Priority date: 2020-12-08
Filing date: 2020-12-08
Publication date: 2022-05-03
Anticipated expiration: 2040-12-08
Also published as: CN112538115A

Abstract

The invention belongs to the technical field of biology, and particularly relates to an anti-human BCMA nano antibody consisting of a variable region of a heavy chain antibody, a preparation method and application thereof, and provides a coding nucleic acid molecule, an expression vector, a host cell and the like of the antibody. The anti-human BCMA nano antibody prepared by the invention has high affinity binding to human BCMA antigen, strong antibody endocytosis activity and novel sequence, and is a novel anti-BCMA nano antibody.

Description

Anti-human BCMA nano antibody and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an anti-human BCMA nano antibody consisting of a variable region of a heavy chain antibody, and a preparation method and application thereof.

Background

B Cell Maturation Antigen (BCMA), also known as tumor necrosis factor receptor superfamily member 17(TNFRS17), is a transmembrane protein rich in the extracellular domain of cysteine, which is involved in B cell maturation, growth and survival (Madry C et al, "The characterization of The human BCMA gene definitions as a new member of The molecular characterization factor reporter subunit. Int Immunol.1998,10(11): 1693-. BCMA is a receptor for two ligands of the TNF superfamily: APRIL (proliferation-inducing ligands, also known as TNFSF13, TALL-2 and TRDL-1; high affinity ligands for BCMA), and B-cell activating factor BAFF (also known as BLyS, TALL-1, THANK, zTNF4, TNFSF20 and D8Ertd387 e; low affinity ligands for BCMA). APRIL and BAFF are growth factors that bind BCMA and promote plasma cell survival. The negative regulator TACI also binds BAFF and APRIL. Synergistic binding of APRIL and BAFF to BCMA and/or TACI activates the transcription factor NF- κ B and increases expression of pro-survival (pro-survival) Bcl-2 family members (e.g., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, a1) and down-regulation of pro-apoptotic factors (e.g., Bid, Bad, Bik, Bim, etc.), thereby inhibiting programmed cell death and promoting survival. This combination promotes B cell differentiation, proliferation, survival and antibody production (reviewed in Rickert RC et al, "signalling by the promoter factor receptor super family in B-cell biology and disease". Immunol Rev.2011,244(1): 115-133).

In 1993, a novel natural antibody derived from camelidae was found. The antibody naturally lacks a light chain and consists of only a heavy chain comprising two constant regions (CH2 and CH3), one hinge region and one heavy chain variable region (i.e., antigen binding site) with a relative molecular mass of about 15kD, which is only 1/10 of conventional antibodies, and has a molecular height and diameter on the nanometer scale, which is the smallest functional antibody fragment currently available, and thus is called nanomumab (Nb), i.e., heavy chain single domain antibody VHH (variable domain of heavy-chain antibody). The single domain antibody which is obtained by cloning the variable region and only consists of one heavy chain variable region is the smallest unit which can be obtained at present and has complete functions and stable ability to combine with antigen. The single domain antibody has the characteristics of high stability, good water solubility, simple humanization, high targeting property, strong penetrability and the like, and plays an extremely imaginable huge function in immune experiments, diagnosis and treatment. Single domain antibodies are becoming an emerging force in the diagnosis and treatment of new generation antibodies.

Therefore, there is a need in the art to develop an anti-BCMA nanobody, particularly an anti-BCMA nanobody having a good BCMA antigen binding ability and a strong functional activity.

Disclosure of Invention

The invention aims to provide a nano antibody specifically aiming at human BCMA, and simultaneously provides a coding sequence, a preparation method and application of the nano antibody. Meanwhile, the invention provides a research and development basis for researches based on BCMA targets, including development of antibody coupling drugs and multispecific antibody drugs.

In order to achieve the purpose, the invention provides the following technical scheme:

an anti-human BCMA nanobody comprising a variable region of a heavy chain antibody;

the variable region of the heavy chain antibody comprises the Complementarity-determining region (CDR);

the amino acid sequence of CDR1 of the anti-human BCMA nano antibody is SEQ ID NO: 1 is shown in the specification;

the amino acid sequence of CDR2 of the anti-human BCMA nano antibody is SEQ ID NO: 2 is shown in the specification;

the amino acid sequence of CDR3 of the anti-human BCMA nano antibody is SEQ ID NO: 3, respectively.

The heavy chain variable region amino acid sequence of the nano antibody is SEQ ID NO: 4, respectively.

A nucleotide molecule encoding said anti-human BCMA nanobody;

the sequence of the nucleotide molecule is selected from SEQ ID NO: 5;

sequence SEQ ID NO: 5 encoding the heavy chain variable region of the nanobody.

An expression vector containing the nucleotide molecule.

A host cell comprising the above expression vector.

A preparation method of an anti-human BCMA nano antibody comprises the following steps:

step 1: preparing an expression vector containing a nucleotide molecule for expressing the antihuman BCMA nano antibody;

step 2: transfecting a eukaryotic or prokaryotic host cell with the expression vector of step 1;

and step 3: culturing the eukaryotic or prokaryotic host cell transfected in the step 2;

and 4, step 4: separating and purifying to obtain the antibody.

Further, the expression vector comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5.

The invention also relates to the development of an antibody conjugate drug and a multispecific antibody drug or drug composition containing the anti-human BCMA nano antibody.

The invention also provides application of the anti-human BCMA nano antibody in preparation of anti-tumor drugs, infectious diseases or other drugs of pathological conditions mediated by BCMA.

Compared with the prior art, the invention has the following advantages: the invention can specifically identify the nanometer antibody of human BCMA, the binding affinity of the antibody and human BCMA is stronger than that of the existing anti-human BCMA monoclonal antibody, and the sequence is novel.

Drawings

FIG. 1 is a first round PCR electrophoresis of antihuman BCMA nano antibody, M is molecular weight standard (the size of the band is 2000, 1500, 1000, 750, 500, 250, 100bp in sequence), "1" represents the PCR product amplified by the first pair of primers, "2" represents the PCR product amplified by the second pair of primers, the size of the PCR product band is about 700 bp;

FIG. 2 is a second round of PCR electrophoresis of anti-human BCMA nanobody, wherein M is molecular weight standard (same size of band), "1" represents PCR product amplified by first pair of primers, "3" represents PCR product amplified by second pair of primers, "5" represents PCR product amplified by third pair of primers, "7" represents PCR product amplified by fourth pair of primers, "9" represents PCR product amplified by fifth pair of primers, "11" represents PCR product amplified by sixth pair of primers, numbers "2", "4", "6", "8", "10" and "12" are negative control wells, and the size of PCR product band is about 400 bp;

FIG. 3 shows the positive cloning efficiency of VHH fragment identified by colony PCR method, wherein M is molecular weight standard (band size same as above), 94 single clones (numbered 1-94) are randomly picked, number "95" is positive control strain (PCR band size of bacterial liquid about 750bp), and the positive cloning efficiency of VHH fragment is about 93.6%;

FIG. 4 is a SDS-PAGE (SDS-PAGE) of purified anti-human BCMA nanobody, wherein lane M shows molecular weight standard (bands are 250, 130, 100, 70, 55, 35, 25, 15, 10kDa in sequence), lane 1 shows positive control Herceptin, lane 2 shows purified anti-human BCMA nanobody, and the protein molecular weight is about 35 kDa;

FIG. 5 is a graph of indirect ELISA to determine the binding capacity of nanobodies to human BCMA protein;

FIG. 6 shows the detection of the endocytic activity of anti-human BCMA nanobody.

Detailed Description

The invention will be further illustrated by means of the following examples, without however restricting its scope to these examples.

Example 1 construction of alpaca phage VHH display library

1.1 alpaca immunization

1mg of human BCMA-Fc (Acro biosystems, Cat # BC7-H5254) protein and Freund's complete adjuvant 1:1 are mixed in equal volume and fully emulsified, and a healthy adult alpaca is immunized for the first time in a subcutaneous multipoint injection mode; thereafter, 3 boosts with 0.5mg BCMA protein 1:1 equal volume of mixed incomplete Freund's adjuvant were performed 4 times in total, with 20 days intervals for a single time. After 7 days of each immunization, a small amount of alpaca peripheral blood was drawn for titer detection. After the last serum titer test, 100ml of peripheral blood was collected, and peripheral blood mononuclear cells were obtained from the blood.

1.2 extraction of Total RNA and cDNA from Peripheral Blood Mononuclear Cells (PBMC) of alpaca

Total RNA was extracted from alpaca PBMC stored in TRIzol using the FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme, Cat # RC 101). The procedure is briefly described as follows, chloroform is added in a proportion of 200. mu.l chloroform per 1ml TRIzol, the EP tube cap is closed, shaking vigorously for 15s, and standing at 4 ℃ for 5 min. The samples were centrifuged at 13,000g for 10min at 4 ℃ whereupon the samples were separated into three layers: the red organic phase, the middle layer and the upper colorless aqueous phase, and the RNA is mainly in the upper aqueous phase. To the resulting aqueous solution was added 1.6 times the volume of Buffer RL2, and the mixture was gently mixed. The mixture was transferred to RNAPure Columns, centrifuged at 13,000g for 1min at room temperature, and the waste liquid was discarded. To RNAcure Columns was added 500. mu.l Buffer RW1, and 13,000g was centrifuged at room temperature for 1min, and the waste liquid was discarded. Add 700. mu.l Buffer RW2, centrifuge at 13,000g for 1min at room temperature, discard the waste. Then 700. mu.l of Buffer RW2 was added, and the mixture was centrifuged at 13,000g at room temperature for 1min, and the waste liquid was discarded.

Centrifugation at 13,000g for 2min was performed to completely remove Buffer RW2 remaining in RNAcure Columns. The column was transferred to a new RNase-free Collection Tubes 1.5ml centrifuge tube, and 50. mu.l of RNase-free ddH2O was suspended and dropped into the center of the column. The mixture was left at room temperature for 2min, and centrifuged at 13,000g at room temperature for 1min to elute RNA.

The steps for reverse transcription of total RNA into cDNA using Hiscript II 1st Strand cDNA Synthesis kit (+ gDNA wrapper) (Vazyme, Cat # R212-01) were as follows: RNA template denaturation: preparing the following reaction system in an RNase-free centrifuge tube: 11 μ l Total RNA +1 μ l Oligo (dT)₂₃VN (50. mu.M) (12. mu.l total). Preparing a system, instantly centrifuging a PCR tube, heating a sample at 65 ℃ for 5min, rapidly placing the sample on ice for quenching, and standing for 2 min; b. genomic DNA removal: adding 4 mu l of 4 XgDNA wiper mix in the step a, uniformly mixing, and then, carrying out temperature control at 42 ℃ for 2 min; c. preparing a first strand cDNA synthesis reaction solution: adding 2. mu.l of 10 XRT Mix + 2. mu.l of Hiscript II Enzyme Mix (20. mu.l of the system) in the step b, and uniformly mixing; d. the first strand cDNA synthesis reaction was performed under the following conditions: 50 ℃ for 45 min; 85 ℃ for 2 min. The obtained cDNA was purified using the MiniElute Reaction Cleanup Kit (50) (QIAGEN, Cat #28204) Kit.

1.3 obtaining variable region fragment of heavy chain antibody by nested PCR amplification using cDNA as template

1.3.1 first round PCR

Using the cDNA obtained above as a template, first PCR amplification was performed using the primers shown in Table 1 below.

Table 1: construction of first round PCR primers for alpaca phage VHH display library

First round PCR reaction System: mu.l cDNA + 25. mu.l 2 XTAQA Master Plus Mix + 0.2. mu.l upstream primer + 0.2. mu.l downstream primer + 24.42. mu.l ddH₂O (total 50. mu.l system). And (3) circulating amplification conditions: 95 ℃ for 5 min; 94 ℃ 1min, 62 ℃ 1min, 72 ℃ 1min 10s, 30 cycles; 72 ℃ 10 min.

The obtained PCR product was then subjected to agarose gel electrophoresis, as shown in FIG. 1, and the result showed that the size of the amplified VHH-CH2 fragment was about 700bp, i.e., the nanobody gene electrophoresis band was about 700 bp.

1.3.2 second round PCR

The first round of PCR products were used as templates for the second round of PCR amplification using the primers shown in table 2 below.

Table 2: construction of second round PCR primers for alpaca phage VHH display library

Second round PCR reaction System: 0.71. mu.l VHH-CH2+ 25. mu.l 2 XTAQ Master Plus Mix + 0.2. mu.l upstream primer + 0.2. mu.l downstream primer + 23.89. mu.l ddH₂O (total 50. mu.l system). And (3) circulating amplification conditions: 95 ℃ for 5 min; 94 ℃ 1min, 56 ℃ 1min, 72 ℃ 30s, 30 cycles; 72 ℃ 10 min.

And then, carrying out agarose gel electrophoresis on the obtained PCR product, wherein the result is shown in figure 2, and the size of the amplified VHH fragment is about 400bp, thus obtaining the heavy chain variable region fragment of the antibody of the natural deleted light chain in the peripheral blood of the alpaca.

1.4 cleavage, ligation and electrotransformation of the fragment with the vector

Second round of PCR products after double digestion and purification with restriction enzymes Pst I and Not I (from NEB)The phage vector pMECS was digested with restriction enzymes Pst I, Xba I and Not I (from NEB) in a triple-restriction enzyme, which was used for the restriction of VHH, and the products of both digestions were ligated with T4 DNA ligase (NEB, Cat # M0202L) at a molar ratio of 1: 5. The specific enzyme digestion system and the connection system are as follows. And (3) digesting a VHH system of a second round PCR product: 3.58 μ l VHH (2ng) +5 μ l CutSmart Buffer +1 μ l Pst I +1 μ l Not I +39.42 μ l ddH₂O (50. mu.l total system); enzyme digestion of the phage vector pMECS System: 4.7 μ l pMECS +5 μ l CutSmart Buffer +1 μ l Pst I +1 μ l Not I +1 μ l Xba I +37.3 μ l ddH₂O (total 50. mu.l system). Enzyme cutting conditions are as follows: the cleavage was carried out at 37 ℃ overnight. After completion of the digestion, the fragment and the vector were purified using a MiniElute Reaction clean Kit (50) (QIAGEN, Cat #28204), respectively, and then ligated. The ligation reaction system is as follows: mu.l of digested VHH + 0.81. mu.l of digested pMECS + 1. mu. l T4 DNA Ligase + 2. mu. l T4 DNA Ligase Buffer (10X) + 15.19. mu.l ddH₂O (20. mu.l total system). Connection conditions are as follows: ligation was performed overnight at 16 ℃ and then at 65 ℃ for 15 min.

1.5 preparation of a Camel phage VHH display library

The ligation products were electroporated into electroporation competent cells TG1 (available from Lucigen Corporation), under the electroporation conditions: 1.8KV, 200 M.OMEGA.and 25. mu.F. Shake culturing the electro-transformed bacteria liquid in SOC liquid culture medium at 37 deg.C for 1 hr, and diluting with 100 μ l of 10⁵、10⁶、10⁷、10⁸After doubling, 100. mu.l of each of the bacterial suspension was applied to the surface of 2 XYT solid culture dish for library capacity measurement, and the other bacterial suspension was applied to the surface of 2 XYT solid culture dish and cultured overnight in a 37 ℃ incubator, and all the colonies were collected the next day as VHH antibody libraries. The positive cloning efficiency (i.e. the VHH fragment insertion rate) of the VHH fragment was identified by colony PCR using the primers: MP 57: TTATGCTTCCGGCTCGTATG, respectively; GIII: CCACAGACAGCCCTCATAG, the results in FIG. 3 show that the positive cloning efficiency of VHH fragments is around 93.6%, demonstrating that a library of VHH antibodies is obtained with a library size of 9.0X 10⁸。

The helper phage rescue procedure was as follows: 1) collecting 73 μ l library, inoculating in 60ml 2 XYT/Amp/Glucose (2 YTAG) culture medium, culturing at 37 deg.C and 220rpm with shaking to logarithmThe OD600 is about 0.3 to 0.5; 2) 10ml of the culture medium after completion of the culture was taken out, and 1X 10 cells were added¹⁰pfu helper phage M13KO7(NEB, Cat # N3015S), gently mixing, and standing at 37 deg.C for 30min for infection; 3) placing the centrifuge tube at 45 ° in an incubator, culturing at 220rpm and 37 deg.C for about 1.5 h; 4) centrifuging the culture solution at 2800g for 10min at room temperature, discarding supernatant, precipitating thallus, resuspending with 50ml 2 XYT/Amp/Kana culture solution at 30 deg.C and 200rpm, and culturing overnight; 5) centrifuging the culture solution at 4 deg.C and 8000g for 20min, collecting supernatant, adding 1/4 volume of PEG6000/2.5M NaCl, and standing in ice bath for 2 hr; 6) centrifuging at 12000g at 4 deg.C for 15min, discarding supernatant, and resuspending the precipitate with about 6ml of ice-bath DPBS; 7) centrifuging at 12000g at 4 deg.C for 5min, collecting supernatant about 6ml, adding 1/4 volume of PEG6000/2.5M NaCl, mixing, and standing in ice bath for 2 hr; 8) at 4 ℃, 8000g, centrifuge for 20min, discard the supernatant, precipitate with ice bath DPBS 20% Glycerol (0.7ml) heavy suspension. The recombinant phage antibody library was obtained and dispensed into 1.5ml EP tubes and stored at 4 ℃. Purification Phage final titer determination procedure: preparing a clean 1.5ml EP tube, taking 20 mu l of Phage solution, adding to 80 mu l of DPBS 20% Glycerol, mixing uniformly, taking 20 mu l of Phage solution from the mixed solution, adding to 80 mu l of DPBS 20% Glycerol, mixing uniformly, and so on, carrying out 5 times of gradient dilution, measuring OD260 value (namely RNA concentration reading value) after dilution, and calculating Phage titer: [ Dilution X (reading of RNA concentration)]×22.14×10¹⁰pfu/ml, finally obtaining Phage titer of 1.1X 10¹⁴pfu/ml。

Example 2: screening of positive clone strain expressing nano antibody BCMA-VHH

2.1 affinity panning

Preparation of solid phase protein ELISA plates: human BCMA-His antigen (purchased from ACRO, Cat # BCA-H522y) was prepared with 1 Xcarbonate/bicarbonate buffer to a final concentration of 1. mu.g/ml, and applied to a 96-well plate (divided into A, B, C three zones with 4 wells in each zone) at 100. mu.l/well overnight at 4 ℃. After coating was completed, the plate was washed 1 time with 0.05% Tween 20 in PBS (i.e., 1xPBST), 200. mu.l/well of 2% BSA in PBST was added, and the plate was blocked at 37 ℃ for 2 hours. Phage blocking: mu.l of purified Phage, 16. mu.l of human immunoglobulin IgG for intravenous injection (pH4) (purchased from Tabang, Guizhou) was added to 374. mu.l of PBST buffer containing 2% BSA, mixed well and suspended using a rotary suspension apparatus at the lowest rotation speed for 1 hour at room temperature. Phage solid phase panning: after the blocking of the 96-well microplate, the plate was washed, 100. mu.l of the blocked Phage solution was added to each well of the A-zone, and 100. mu.l of the PBST solution was added to each well of the B, C-zone labeled plate, and the plate was incubated at room temperature for about 1 hour. After that, the liquid in the B region was discarded, and the supernatant of each well in the A region was transferred to the corresponding well in the B region (100. mu.l/well), and incubated at room temperature for about 1 hour. The liquid in zone C was discarded, and the supernatant from each well in zone B was transferred to the corresponding well in zone C (100. mu.l/well), and incubated at room temperature for about 1 hour. Washing Phage: after incubation, the labeled wells in zone B, C were washed repeatedly 22 times with 200. mu.l/well PBST solution and 3 times with 200. mu.l/well PBS. Eluting and neutralizing Phage: after the completion of washing, 100. mu.l of 0.1M Glycine (pH 2.2) was added to each of the labeled wells at B, C for elution, and the eluted products from B, C wells were combined with each other in a new 1.5ml centrifuge tube, mixed well, and neutralized with 1M Tris & HCl (pH 9.1). And adding the B area neutralizing solution into 3ml of TG1(OD600 is 0.48) in a logarithmic growth phase, generating and purifying phage for the next round of screening, and continuously enriching positive clones through 2 rounds of screening, thereby achieving the purpose of screening BCMA specific antibodies in an antibody library by using a phage display technology.

2.2 Indirect Phage ELISA Primary screening of antigen-positive Nanobodies

2.2.1 preparation of monoclonal supernatants of VHH phages

A VHH phage monoclonal supernatant was first prepared: 2 rounds of panning were performed, and the single colonies growing on 2YTAG plates were spread and inoculated into 2 96-well plates, labeled Master Plate, cultured at 37 ℃ and 220rpm until OD600 reached 0.5-0.6, and transferred to 2 new 96-well plates containing 2 XYT. Amp, labeled P1 Plate, cultured at 37 ℃ and 220rpm until OD600 reached 0.6-0.8. Adding 1mM IPTG (QIAGEN, Cat # RT108-01) to the final concentration, and inducing overnight at 220rpm at 30 ℃; and after the culture is finished, centrifuging at 4000rpm for 15min at room temperature, and obtaining the supernatant, namely the monoclonal supernatant of the VHH phage.

2.2.2 Indirect ELISA screening of antibodies binding to human BCMA antigen

Human BCMA-His antigen was prepared at a final concentration of 1. mu.g/ml using a1 Xcarbonate/bicarbonate buffer, and 100. mu.l/well was applied to a 96-well plate and coated overnight at 4 ℃. After coating, the plates were washed 1 time with PBST, 200. mu.l/well of PBST solution containing 5% skim milk powder was added and blocked at 37 ℃ for 2 hours. Plates were washed again and 100. mu.l/well of VHH phage monoclonal supernatant and the biotin-labeled reference antibody human BAFF-Fc (purchased from Acro biosystems, Cat # BAF-H4268) were added. Incubate at 37 ℃ for 40min, then wash the plate 4 times. Sample wells were loaded with 100 μ l/well 1: horseradish peroxidase-labelled anti-HA Tag (GenScript, Cat # a01296) antibody diluted 2000, control wells 100 μ Ι/well added 1: 5000 dilution of horseradish peroxidase-labeled streptavidin (Jackson Immuno Research, Cat # 016-. Incubate at 37 ℃ for 40min, then wash the plate 4 times and pat dry. 100. mu.l/well of an ELISA chromogenic substrate TMB (Innoreagents, Cat # TMB-S-002) was added, color development was carried out at room temperature, and then color development was stopped with a 1M sulfuric acid solution, and the absorbance at 450nm was measured for each well. Positive clones were considered when the OD450 value of the sample well was at least 10-fold greater than the negative control, and the results are shown in table 3. The clone BCMA-VHH-1F7 was finally selected for subsequent experiments.

TABLE 3 anti-human BCMA Nanobody supernatant detection ELISA data

Example 3: in vitro analysis method for determining functional activity of anti-human BCMA nano antibody

3.1 Indirect ELISA for determining the binding Capacity of antibodies

The clone BCMA-VHH-1F7 was extracted and transferred to Expi293F cells using PEI (biosciences, Cat #24765-1) transfection reagent. Cell supernatant expressed anti-human BCMA nanobody (containing Fc tag) was purified using Protein a or G column purification standard protocols. The purified protein obtained was examined by SDS-PAGE, and the results are shown in FIG. 4.

Human BCMA-His antigen was prepared with 1 Xcarbonate/bicarbonate buffer to a final concentration of 2. mu.g/ml, and 100. mu.l/well was applied to a 96-well plate and coated at 37 ℃ for 2 hours. After the coating was completed, the plate was washed 4 times with 1xPBST, and 200. mu.l/well of PBST solution containing 5% skim milk powder was added and blocked at 37 ℃ for 2 hours. The plate was washed again, and after adding 100. mu.l/well of the gradient diluted antibody solution and BCMA Benchmark1 (i.e., BCMA-BM1 whose sequence was selected from the antibody sequence of antibody conjugate GSK2857916 available from Glaxo Smith Kline, Inc., the antibody was synthetically expressed in Booxcin, whose heavy chain amino acid was QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK and light chain amino acid was DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC), the plate was incubated at 37 ℃ for 40min, and then washed 4 times. Add 1: a5000 dilution of horseradish peroxidase-labeled goat anti-human Fc γ specific fragment (Jackson ImmunoResearch Laboratories, Inc., Cat # 109-. 100. mu.l/well of an ELISA chromogenic substrate TMB was added, development was carried out at room temperature for 3 to 10min, and then the development was stopped with a 1M sulfuric acid solution, and the absorbance of each well at 450nm was measured. Data processing was performed using GraphPad Prism software, and the results are shown in fig. 5. As can be seen from the figure, the BCMA-VHH-1F7 nano antibody can recognize human BCMA protein, and the binding activity is slightly better than that of Benchmark 1.

3.2 detection of endocytic activity of anti-human BCMA Nanobody

The DT3C protein can automatically bind to the Fc region of an antibody and further undergo antibody-mediated endocytosis to serve as a mechanism for killing target cells (Miki Yamaguchi, et al, "Development of a reactive screening method for selecting monoclonal antibodies to a target intracellular cells". Biochemical and Biophysical researcharch communications, 2014,454(4):600-3), anti-human BCMA nanobody was quantitatively compared for endocytic activity with BCMA Benchmark1 by DT3C, by collecting human multiple myeloma U266 cells (purchased from ATCC) at logarithmic growth phase, centrifuging, discarding supernatant, resuspending and counting with 1640 containing 15% FBS (i.e., Assay medium) and adding to the cell plate at 100 μ l/well such that 1.5 × 10 per well⁴Single cell at 5% CO₂And then, the culture was allowed to stand at 37 ℃ for 24 hours. The next day, nanobody and toxin (DT3C-His) were diluted with 1640 (i.e., Assay buffer) to working concentrations of 10. mu.g/ml each. Mixing the prepared antibody and toxin according to a ratio of 1:1, and incubating for 30min at room temperature. The incubated mixture was diluted in a gradient and added to the cell plate at 100. mu.l/well in 5% CO₂And then, the culture was allowed to stand at 37 ℃ for 3 days. After the completion of the culture, the Cell plate was removed, 50. mu.l/well of CellCount-Lite 2.0 luminescence Cell vitality Assay (Vazyme, Cat # DD1101-02) thawed to room temperature was added thereto, and the plate was left to stand at room temperature in the dark for 3 to 5min, and the fluorescence signal value was detected by Tecan i-control. Data processing was performed using GraphPad Prism software, and the results are shown in fig. 6. As can be seen from the figure, the nano-antibody of the invention has good endocytosis effect, and the endocytosis activity is superior to that of Benchmark 1.

Example 4: variable region sequencing of anti-human BCMA Nanobodies

Firstly, cloning and expanding culture of the expression nano antibody: taking out BCMA-VHH-1F7, and carrying out reaction according to the weight ratio of 1: 100 was added 2 XYT medium and incubated at 37 ℃ and 200rpm overnight. The next day, the cultured bacterial solution was taken out and used in PureYield^TMPlasmid Miniprep System kit (Promega, Cat # A1223) was used for Plasmid extraction. The method comprises the following steps: 1) taking 600 mu l of bacterial liquid to a 1.5ml centrifugal tube; 2) add 100. mu.l cell lysis buffer, reverse up and down 6 times; 3) adding 350 μ l of the neutralized solution stored at 4 deg.C, and mixing; 4) centrifuging at 12,000rpm at room temperature for 3 min; 5) the supernatant was removed and transferred to PureYield^TMMinicolumn, and put into PureYield^TMCollecting Tube; 6) centrifugation at 12,000rpm for 15s at room temperature; 7) discarding the supernatant, adding 200. mu.l endotoxin removing solution, centrifuging at 12,000rpm at room temperature for 15 s; 8) adding 400 μ l of column cleaning solution, centrifuging at 12,000rpm at room temperature for 30 s; 9) transfer Minicolumn to a new 1.5ml EP tube, add 3Mu.l of Elution Buffer, left to stand at room temperature for 1min, at 12,000rpm, and centrifuged at room temperature for 15s to obtain plasmid DNA. The extracted plasmids were sent out for sequencing to obtain the variable region sequences of nanobodies, as shown in Table 4.

TABLE 4 amino acid and nucleotide sequences of anti-human BCMA Nanobodies

The nucleotide sequence of the anti-human BCMA nano antibody is as follows:

nucleotide sequence of BCMA-VHH-1F7 nanobody:

5’-GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAGGCACCTTCAATAATGATAACGTGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGTTATTAGTTGGAGGGGTGATGGCACGGATTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACTACGCCAAGAAGATGGTGTATCTGCAAATGAACAGCCTGCAACCTAAGGACACGGCCGTTTATTACTGTGCAGTAGGCGATGGTAGCAGATGGCACGGCGCTTATGCCTACTGGGGCCAGGGGACCCGGGTCACCGTCTCCTCA-3’(SEQ ID No.:5)

in conclusion, the anti-BCMA nano antibody (BCMA-VHH-1F7 nano antibody) prepared by the invention has high affinity binding to human BCMA antigen, strong antibody endocytosis activity and novel sequence, and is a novel anti-human BCMA nano antibody.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.

Sequence listing

<110> Boaoxin Biotechnology (Nanjing) Ltd

<120> anti-human BCMA nano antibody and preparation method and application thereof

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Gly Gly Thr Phe Asn Asn Asp Asn

1 5

<210> 2

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Ile Ser Trp Arg Gly Asp Gly Thr

1 5

<210> 3

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Ala Val Gly Asp Gly Ser Arg Trp His Gly Ala Tyr Ala Tyr

1 5 10

<210> 4

<211> 121

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Asn Asn Asp

20 25 30

Asn Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Val Ile Ser Trp Arg Gly Asp Gly Thr Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Tyr Ala Lys Lys Met Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Gln Pro Lys Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Val Gly Asp Gly Ser Arg Trp His Gly Ala Tyr Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Arg Val Thr Val Ser Ser

115 120

<210> 5

<211> 363

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gaggtgcagc tggtggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60

tcctgtgcag cctctggagg caccttcaat aatgataacg tgggctggtt ccgccaggct 120

ccagggaagg agcgtgagtt tgtagcagtt attagttgga ggggtgatgg cacggattat 180

gcagactccg tgaagggccg attcaccatc tccagagact acgccaagaa gatggtgtat 240

ctgcaaatga acagcctgca acctaaggac acggccgttt attactgtgc agtaggcgat 300

ggtagcagat ggcacggcgc ttatgcctac tggggccagg ggacccgggt caccgtctcc 360

tca 363

Claims

1. An anti-human BCMA nanobody, comprising a heavy chain variable region;

the heavy chain variable region comprises a complementarity determining region;

2. The anti-human BCMA nanobody according to claim 1, wherein the heavy chain variable region amino acid sequence of said nanobody is SEQ ID NO: 4, respectively.

3. A nucleotide molecule encoding the anti-human BCMA nanobody according to claim 1 or 2.

4. The nucleotide molecule of claim 3, wherein the sequence of the nucleotide molecule is selected from the group consisting of SEQ ID NO: 5;

sequence SEQ ID NO: 5 encoding the heavy chain variable region of the nanobody.

5. An expression vector comprising the nucleotide molecule of claim 3.

6. A host cell comprising the expression vector of claim 5.

7. A preparation method of an anti-human BCMA nano antibody is characterized by comprising the following steps:

step 1: preparing an expression vector containing a nucleotide molecule for expressing the anti-human BCMA nanobody of claim 1 or 2;

and 4, step 4: separating and purifying to obtain the antibody.

8. The method of claim 7, wherein the expression vector comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 5.

9. An antibody-conjugated drug, a multispecific antibody drug, a chimeric antigen receptor or a pharmaceutical composition comprising the anti-human BCMA nanobody of claim 1 or 2.

10. The use of the anti-human BCMA nanobody of claim 1 or 2 for the preparation of a medicament for treating multiple myeloma.