A nanoantibody targeting BCMA and its application

Abstract

The present invention relates to the field of biomedicine or biopharmaceutical technology, and specifically to a nanobody targeting BCMA and its application. Specifically, the present invention provides a nanobody targeting BCMA and its VHH chain. The present invention also provides a polynucleotide encoding the above-mentioned single-domain antibody or its VHH chain, a corresponding expression vector and a host cell capable of expressing the nanobody targeting BCMA, as well as a method for producing the nanobody targeting BCMA of the present invention. The nanobody provided by the present invention has both excellent specific BCMA binding ability and reduced immunogenicity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine or biopharmaceutical technology, and in particular to a nano antibody targeting BCMA and applications thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Human BCMA is a 184-amino acid transmembrane protein encoded by the TNFRSF17 gene (located at 16p13.13) with a theoretical molecular mass of 20 kDa. It is anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) motif. BCMA consists of three major domains: an extracellular segment (amino acids 1-54, linked by disulfide bonds at positions 8-21, 24-37, and 28-41), a transmembrane region (amino acids 55-77), and an intracellular segment (amino acids 78-184). BCMA is highly expressed in mature B lymphocytes and plasma cells and is selectively overexpressed during the malignant transformation of plasma cells. BCMA promotes tumor cell growth, survival, and drug resistance primarily by activating intracellular signaling cascades involving NFκB, AKT, phosphatidylinositol 3-kinase (PI3K), STAT3, and MAPK. Especially in the cancer cells of multiple myeloma patients, the high expression of BCMA makes it a very potential target in the treatment of multiple myeloma (MM). It can also be used as a biomarker for the diagnosis and prognosis of MM and as a possible predictor of treatment response.

[0004] Studies have found that BCMA controls a variety of physiological functions in the human body, such as the proliferation, differentiation, and survival of immune cells, as well as the production of antibodies. It mainly participates in the following physiological functions: (1) Affecting the proliferation and differentiation of lymphocytes. After BCMA binds to its ligand, it can activate lymphocytes, promote their proliferation and differentiation, and thus participate in the immune response. (2) Regulating the production of antibodies. BCMA can activate B cells and promote their differentiation into plasma cells, which in turn produce antibodies and participate in humoral immune responses. (3) Participating in the survival and proliferation of bone marrow cells. After BCMA binds to its ligand, it can promote the survival and proliferation of bone marrow cells, thereby participating in the hematopoietic process.

[0005] BCMA is considered to be the main target for the treatment of MM. Current anti-BCMA treatments are generally divided into the following types, including: antibody-drug conjugate (ADC), bispecific antibody (BsAb), CAR-T cell therapy (Chimeric antigen receptor T-Cell immunotherapy, CAR-T), CAR-NK cell therapy (Chimeric antigen receptor natural killer cell immunotherapy, CAR-NK), etc.

[0006] Whether used in atopic diseases or tumors, the immunogenicity of monoclonal antibodies has always been a top priority for researchers. Minimizing immunogenicity while ensuring efficacy is crucial. Consequently, many researchers have restructured and recombined monoclonal antibodies to reduce their molecular size, and then humanized them through methods such as amino acid mutations at key sites or affinity maturation. However, most humanization modifications reduce the affinity or stability of the antibody itself. Summary of the Invention

[0007] In order to address the deficiencies of the prior art, the present invention aims to provide a BCMA-targeted nanobody and its application, which has excellent specific BCMA binding ability and can reduce its own immunogenicity.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] In the first aspect, a VHH chain of a nanobody targeting BCMA, the variable region CDR of the VHH chain includes: a CDR1 as shown in the amino acid sequence of SEQ ID NO.1, a CDR2 as shown in the amino acid sequence of SEQ ID NO.2, and a CDR3 as shown in the amino acid sequence of SEQ ID NO.3.

[0010] Among them, any one of the above amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one amino acid and can retain BCMA binding affinity.

[0011] In some embodiments, the VHH chain further comprises a framework region FR. The framework region FR is used to separate the variable region CDRs, specifically, the framework region FR comprises: FR1 having an amino acid sequence as shown in SEQ ID NO. 4, FR2 having an amino acid sequence as shown in SEQ ID NO. 5, FR3 having an amino acid sequence as shown in SEQ ID NO. 6, and FR4 having an amino acid sequence as shown in SEQ ID NO. 7.

[0012] In some embodiments, the amino acid sequence of the VHH chain is as shown in SEQ ID NO.8; or, after adding, deleting or replacing one or several amino acids in the amino acid sequence shown in SEQ ID NO.8, the amino acid sequence has a sequence similarity of 80% and still exerts similar physiological activity.

[0013] In the second aspect, a nanobody targeting BCMA is provided, which is a single-domain antibody against the BCMA epitope and has the VHH chain described in the first aspect of the present invention.

[0014] The BCMA-targeting nanoantibodies of the present invention include monomers, bivalents (bivalent antibodies), and / or multivalents (multivalent antibodies).

[0015] In some embodiments, the Nanobody is Nanobody 1D7, and the amino acid sequence of 1D7 is shown in SEQ ID NO.8, wherein the amino acid sequence at positions 1 to 25 is FR1 (as shown in SEQ ID NO.4), the amino acid sequence at positions 26 to 33 is CDR1 (as shown in SEQ ID NO.1), the amino acid sequence at positions 34 to 49 is FR2 (as shown in SEQ ID NO.5), the amino acid sequence at positions 50 to 57 is CDR2 (as shown in SEQ ID NO.2), the amino acid sequence at positions 58 to 95 is FR3 (as shown in SEQ ID NO.6), the amino acid sequence at positions 96-111 is CDR3 (as shown in SEQ ID NO.3), and the amino acid sequence at positions 112 to 122 is FR4 (as shown in SEQ ID NO.7). In addition, the nanobody also includes a derivative polypeptide modified from the nanobody 1D7, wherein the modification method includes but is not limited to functional group modification or the addition of molecular labels such as polyethylene glycol, streptavidin, biotin, radioactive isotopes, fluorescent agents, etc.; further, the functional group modification includes modifying the FR region with a hydrophilic group or replacing the hydrophobic residues in the FR region.

[0016] In the third aspect, a polynucleotide encodes a protein selected from the group consisting of the VHH chain of the BCMA-targeting Nanobody according to the first aspect of the invention, or the BCMA-targeting Nanobody according to the second aspect of the invention.

[0017] The polynucleotides of the present invention include nucleic acids encoding the above-mentioned nanoantibodies that can be translated due to codon degeneracy, and the encoding nucleic acids are not limited to DNA or RNA; preferably, the encoding nucleic acid is DNA, including cDNA, genomic DNA or artificially synthesized DNA; the DNA may be single-stranded or double-stranded, and may be a coding strand or a non-coding strand.

[0018] In some embodiments, the nucleotide sequence of the polynucleotide is shown as SEQ ID NO.9.

[0019] A fourth aspect provides an expression vector comprising the polynucleotide according to the third aspect of the present invention.

[0020] The expression vector of the present invention is selected from the group consisting of DNA, RNA, viral vectors, bacteriophages, plasmids, transposons, other gene transfer systems, or combinations thereof. Specifically, the viral vector can be a plant cell virus, a mammalian cell virus, or the like, such as a lentivirus, adenovirus, AAV virus, a retrovirus, or combinations thereof. Specifically, the plasmid can be a bacterial plasmid, a yeast plasmid, or the like.

[0021] In a fifth aspect, a host cell comprises the expression vector according to the fourth aspect of the present invention, or the polynucleotide according to the third aspect of the present invention is integrated into its genome.

[0022] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell. Specifically, the prokaryotic cell is Escherichia coli, Bacillus subtilis, Lactobacillus, Streptomyces, Proteus mirabilis, or a combination thereof. Specifically, the eukaryotic cell is Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, or a combination thereof.

[0023] In some embodiments, the host cell is a plant cell, a mammalian cell, or a microbial cell.

[0024] In a sixth aspect, a method for producing a nanobody targeting BCMA comprises the following steps:

[0025] (a) culturing the host cell according to the fifth aspect of the invention under conditions suitable for the production of Nanobodies, thereby obtaining a culture containing the Nanobodies targeting BCMA; and

[0026] (b) isolating or recovering the BCMA-targeting Nanobody from the culture; and

[0027] (c) optionally, purifying the BCMA-targeting Nanobody obtained in step (b).

[0028] In some embodiments, the BCMA-targeting Nanobody contains the amino acid sequence shown in SEQ ID NO.8.

[0029] A seventh aspect, an immunoconjugate, comprising:

[0030] (a) the VHH chain of the Nanobody targeting BCMA as described in the first aspect of the invention, or the Nanobody targeting BCMA as described in the second aspect of the invention; and

[0031] (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.

[0032] In the eighth aspect, a VHH chain of the nanoantibody targeting BCMA as described in the first aspect of the present invention and the nanoantibody targeting BCMA as described in the second aspect of the present invention are used to prepare antibody-drug conjugates (ADCs), monoclonal antibody drugs, bispecific / multispecific antibody drugs, cell therapy-related drugs, etc. related to BCMA; or to prepare and develop biochemical detection reagents.

[0033] The biochemical detection reagent is specifically a BCMA detection antibody reagent, which is used in a detection method and kit based on antibody specific recognition.

[0034] The beneficial effects of the present invention are:

[0035] The BCMA-targeting Nanobodies provided by the present invention have specific recognition and binding capabilities for the BCMA antigen, with an affinity of up to 4.77E-10, demonstrating highly specific binding activity. Furthermore, the BCMA-targeting Nanobodies provided by the present invention have promising application prospects in the treatment of multiple myeloma. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0037] Figure 1 This is a diagram of the BCMA antigen protein in an embodiment of the present invention;

[0038] Figure 2 This is an electrophoresis identification diagram of the total RNA extracted in the embodiment of the present invention;

[0039] Figure 3 This is an electrophoresis identification diagram of the first round of PCR amplification of the antibody variable region gene in the embodiment of the present invention;

[0040] Figure 4 This is an electrophoresis identification diagram of the variable region gene of the antibody amplified by the second round of PCR in the embodiment of the present invention;

[0041] Figure 5 This is an electrophoresis identification diagram of transformants identified by colony PCR in an embodiment of the present invention;

[0042] Figure 6 This is an SDS-PAGE diagram of the purification of nanobodies in an embodiment of the present invention. DETAILED DESCRIPTION

[0043] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0044] Example 1. Screening of anti-BCMA nanobodies

[0045] 1.1 Expression of recombinant BCMA protein

[0046] According to the amino acid sequence of human BCMA on NCBI (NP_001183.2), Met1-Ala54 was synthesized, and an Fc tag was added to the C-terminus of the sequence and connected to the vector pCDNA3.1(+). After endotoxin-free plasmid extraction, HEK293 cells in logarithmic growth were used for transfection. After culturing the transfected cells for 36 hours, the cell culture medium was poured into a 50 mL centrifuge tube and centrifuged at 12000 g for 5 minutes. The supernatant was collected and filtered with a 0.22 μm filter membrane. The culture supernatant was purified by nickel column affinity chromatography. Protein expression was detected by SDS-PAGE. The results are shown in the table. Figure 1 ( Figure 1 : M is Thermo Fisher protein marker, product number 26616, and L is purified BCMA protein).

[0047] 1.2 Alpaca Immunity

[0048] A healthy adult alpaca was selected and immunized with a recombinant BCMA antigen mixed with Freund's adjuvant at a 1:1 ratio. 6-7 μg / kg was administered subcutaneously to the alpaca via multiple injections at the back for four immunizations, with a two-week interval. Subsequently, 10 mL of peripheral blood was collected from the alpaca for the construction of a phage display library.

[0049] 1.3 Isolation of alpaca lymphocytes

[0050] The alpaca peripheral blood was collected and lymphocytes were separated using the camel peripheral blood lymphocyte separation reagent kit (Tianjin Haoyang Company, product number LTS1076) according to the instructions. 7Add 1 mL of RNA isolation reagent to each living cell, take 1 mL for RNA extraction, and store the rest at -80℃.

[0051] 1.4 RNA extraction

[0052] Repeatedly pipette 1 mL of Tipure Isolation Reagent containing lymphocytes and let it sit for 5 minutes; add 200 μL of chloroform, vortex for 30 seconds, and let it sit for another 5 minutes; centrifuge at 12000 g at 4°C for 15 minutes, aspirate the aqueous phase and transfer it to a new EP tube; add an equal amount of isopropanol and let it sit for 10 minutes; centrifuge at 12000 g at 4°C for 10 minutes, discard the supernatant; wash with 1 mL of pre-cooled 70% ethanol, centrifuge at 7500 g at 4°C for 5 minutes, discard the supernatant and dry for 5 minutes; add 30 μL of RNase-free water to dissolve the precipitate, adjust the concentration to 1 μg / μL for gel electrophoresis detection, and the results are shown in the figure. Figure 2 .

[0053] 1.5 Reverse transcription and cDNA synthesis

[0054] According to the instructions of the reverse transcription kit (Roche's transcripor first stand cDNA synthesis KIT), reverse transcribe cDNA using the RNA obtained in step 1.4 as a template.

[0055] 1.6 Amplification of Antibody Variable Region Genes

[0056] The cDNA obtained by reverse transcription was used as a template for PCR reaction. Two rounds of amplification were performed. The primer sequences for the first round of PCR were as follows:

[0057] CALL001: GTCCTGGCTGCTCTTCTACAAGG, as shown in SEQ ID NO. 10;

[0058] CALL002: GGTACGTGCTGTTGAACTGTTCC, as shown in SEQ ID NO.11.

[0059] The PCR reaction conditions and program were as follows: 95°C for 5 minutes; 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 30 seconds, 30 cycles; 72°C for 7 minutes. An agarose gel recovery kit was used to recover a band of approximately 700 bp, and the final nucleic acid concentration was adjusted to 5 ng / μL with water ( Figure 3 : M is Trans 2K DNA Marker; 1 is the first-round PCR product).

[0060] The primer sequences for the second round of PCR are as follows:

[0061] VHH-Back: GATGTGCAGCTGCAGGAGTCTGGRGGAGG, as shown in SEQ ID NO. 12;

[0062] VHH-For: CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT, as shown in SEQ ID NO.13.

[0063] The PCR reaction conditions and procedures were as follows: 95°C for 5 minutes; 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, 15 cycles; 72°C for 7 minutes. The PCR product was purified using a PCR product recovery kit ( Figure 4 : M is Trans 2K DNA Marker; 1 is the second-round PCR product).

[0064] 1.7 Vector Construction

[0065] The pMES4 vector (purchased from Biovector) and the second PCR product were double-digested with PstI and BstEII, respectively. 1.5 μg of the digested vector and 450 ng of the second PCR product were added to 15 μL of T4 DNA ligase, supplemented with buffer and water to a total volume of 150 μL. The ligation reaction was incubated overnight at 16°C, and the ligated product was recovered. The product was recovered using a PCR product recovery kit and eluted with 20 μL of water.

[0066] 1.8 Electroporation and storage capacity determination

[0067] Take 10 μL of the purified ligation product and add it to a pre-cooled electroporation cuvette containing 50 μL of E. coli TG1 competent cells and place it in an electroporator (ECM630 electroporator from BTX, USA) for electroporation. Remove the electroporation cuvette, revive and culture the transformants. Randomly select clones and perform colony PCR identification ( Figure 5 M is DL5000 DNA Marker; N is negative control; 1–20 are randomly selected monoclonal PCR identification products. The colony PCR positive rate was 100%. The reservoir capacity was calculated based on the PCR positive rate (reservoir capacity = number of clones × dilution factor × PCR positive rate × 10). The primer sequences are as follows:

[0068] pMES-F: GCCGCTGGATTGTTATTACTC, as shown in SEQ ID NO. 14;

[0069] pMES-R: CTTTCAACAGTGGAACCGTAG, as shown in SEQ ID NO.15.

[0070] 1.9 Amplification of M13 phage

[0071] Take the recovered bacterial solution and inoculate it into YT-AG medium, and culture it at 37℃ and 200 rpm until the culture OD reaches 600 =0.5. Take out 10 mL of bacterial solution and add 4×10 10 VCSM13 cells were statically infected at 37°C for 30 min. Centrifuge at 4000 rpm for 10 min at room temperature, and the supernatant was discarded. Resuspend the cells in 2×YT-AK medium (containing ampicillin and kanamycin) and culture overnight at 37°C at 200 rpm. After centrifugation, 40 mL of the supernatant was transferred to a tube. 10 mL of 20% PEG / NaCl (2.5 M) solution was added and mixed thoroughly. The supernatant was discarded after centrifugation. The pellet was washed with 1 mL of ice-cold PBS and centrifuged. 250 μL of the supernatant was collected, mixed thoroughly, washed, and resuspended in pre-chilled PEG / NaCl.

[0072] Determine the phage titer: culture TG1 to OD 600 =0.4, gradient dilution of phage was performed with LB medium, and the serially diluted phage TG1 culture was mixed and cultured. The next day, the plaque formation in the culture plate was observed, and the plaque counts were performed on the dilution gradient plates with the number of plaques ranging from 30 to 300, and the phage titer (pfu) was calculated according to the following formula.

[0073] Phage titer (pfu / mL) = dilution factor × number of plaques × 100

[0074] 1.10 Phage Display of Nanobodies

[0075] Take 1 mL of the bacterial solution of the nanoantibody immune library and inoculate it into two 10 mL 2×YT-AG medium respectively, and culture at 37°C and 200 rpm until the OD 600 = 0.5, add 4 × 10 10 Infect with 100 pfu of helper phage at 37°C for 30 minutes. Centrifuge at 4000 rpm for 10 minutes at room temperature, discard the supernatant, and resuspend in 3 mL of 2×YT-AK medium. Finally, add the suspension to 100 mL of 2YT-AK medium and incubate overnight at 37°C at 200 rpm. The next day, concentrate and precipitate the displayed phage, and determine the titer.

[0076] Displayed phage titer (cfu / mL) = dilution factor × number of colonies × 100 × 2.

[0077] 1.11 Solid-phase panning of phage display libraries

[0078] All EP tubes used in this example were pre-filled with PBS buffer containing 1% BSA and allowed to stand at room temperature for 10 min to seal the tube walls to reduce the adsorption of trace proteins by the tube walls and the impact on screening results.

[0079] Dilute BCMA recombinant antigen to 10 μg / mL with CBS, coat the ELISA plate with 100 μL per well, incubate at 4°C overnight, and wash the plate 5 times with PBST; add 100 μL of 1% BSA to each well, block at 37°C for 1 hour, and wash the plate 5 times with PBST; add 100 μL of BCMA recombinant antigen diluted to 10 μg / mL to each well. 11 Incubate the cfu-displaying phage at 37°C for 2 hours, then wash the plate 15-25 times with PBST. After the final wash, add 100 μL of glycine solution to each well and incubate on a horizontal shaker for 15 minutes. Add the eluate from each well to an EP tube pre-filled with 15 μL of Tris solution, combine, and titer. Perform panning 3-4 times in total.

[0080] 1.12 Phage ELISA screening of positive clones

[0081] Screen positive clones by ELISA. Coat an ELISA plate with BCMA recombinant antigen, block with 5% BSA, and wash with PBST. Add 100 μL of phage supernatant to each well and incubate at 37°C for 1 hour. Discard the supernatant and add HRP-conjugated mouse anti-M13 secondary antibody. Incubate at 37°C for 1 hour. Discard the supernatant and add TMB solution. Incubate at room temperature for 5 hours. Add 2 M sulfuric acid stop buffer to each well and read at 450 nm using a microplate reader. Select clones with positive phage ELISA results and send for sequencing.

[0082] Example 2. Expression and purification of anti-BCMA nanobodies

[0083] 2.1 Amplification of the original strain TG1 of the nanobody and transformation of the recombinant plasmid of the nanobody into Escherichia coli BL21 (DE3)

[0084] Positive clones were selected and the original TG1 glycerol stock containing the nanobody nucleic acid was inoculated into 5 mL of fresh LB-A medium at a 1:1000 ratio and cultured overnight at 37°C at 200 rpm. The next day, plasmids were extracted using a Plasmid mini kit (OMEGA) according to the manufacturer's instructions. After verification, 1 μL of the plasmid was transformed into 100 μL of competent cells. The cells were gently mixed and placed on ice for 30 minutes. Heat-shocked in a 42°C water bath for 90 seconds, and then cooled in an ice bath for 3 minutes. 600 μL of LB medium was added to the centrifuge tube and incubated at 37°C with shaking for 60 minutes. 100 μL of the supernatant was spread onto an LB-A plate using a triangular spreader and incubated inverted at 37°C overnight.

[0085] 2.2 Inducible expression of nanobodies

[0086] Pick the positive monoclonal colony obtained in step 2.1 and culture it in LB-A medium at 37℃ overnight. The next day, take the bacterial solution and add 100 mL of fresh LB-A medium at a ratio of 1:100, and culture it at 37℃ for 3 hours until the bacterial solution OD reaches 0.600 = approximately 0.8, add IPTG to a final concentration of 1 mM, and induce overnight at 30°C. On the third day, harvest the cells by centrifugation at 8000 rpm for 10 minutes, and resuspend the pellet in 1.5 mL of pre-chilled TES buffer. Incubate on ice for 2 minutes, then gently shake for 30 seconds. Repeat this cycle six times. Add 3.0 mL of TES / 4 (TES diluted 4-fold with water), gently shake for 30 seconds, then incubate on ice for 2 minutes. Repeat the shaking and incubation steps six times. Centrifuge at 9000 rpm at 4°C for 10 minutes, and collect approximately 4.5 mL of supernatant (periplasmic extract).

[0087] 2.3 Purification and identification of nanobodies

[0088] After resuspending IMAC Sepharose (GE), 2 mL was added to a gravity column. The column was allowed to stand for 30 min, allowing the sepharose to naturally settle at the bottom of the column and draining the storage buffer. Two column volumes of 0.1 M nickel sulfate solution were added, and the nickel sulfate solution was drained at a flow rate of approximately 8 s / drop. The sepharose was equilibrated and washed with 10 column volumes of equilibration buffer, maintaining the flow rate constant. The sample was diluted 2-fold with equilibration buffer and added to the gravity column, adjusting the flow rate to 6 s / drop, and the flowthrough was collected. The sepharose was washed with 10 column volumes of wash buffer, maintaining the flow rate constant, and the wash was collected. Three column volumes of elution buffer were added, maintaining the flow rate at 6 s / drop, and the eluate containing the target protein was collected. Finally, the sepharose was washed with 10 column volumes of equilibration buffer, 10 column volumes of pure water, and 10 column volumes of 20% ethanol, leaving 4 mL of 20% ethanol to store the column. The samples collected above were subjected to SDS-PAGE detection ( Figure 6 :M is Thermo Fisher protein marker, product number 26616; lanes 1 to 3 are nanoantibodies 1E7, 1D7, and 1A4). The results are as follows Figure 6 As shown, all three nanobodies were expressed.

[0089] The nanobody screened in this example was named "1D7". The results of DNA sequencing are as follows:

[0090] The nucleic acid sequence of Nanobody 1D7 is:

[0091] 5’-CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTCCTTCTGCCATGACCTGGGCCCGCCAGGCACAAGGAAAGGGGCTCGAGTGGGTGGCCAGTATTTACGGTGATGGTGTCGCATACTACACAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACATGGTGTATCTGCAAATGAACAGCCTGATCGTTGAGGACACGGCCCTTTATCACTGTGCGATTGGGAATAGTAATGTTGTTGCCAGCGGGGGGGCCAACAAGTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA-3’, as shown in SEQ ID NO.9.

[0092] The amino acid sequence of nanobody 1D7 is:

[0093] 5’-QVQLQESGGGLVQPGGSLRLSCAASGFTFSPSAMTWARQAQGKGLEWVASIYGDGVAYYTDSVKGRFTISRDNAKNMVYLQMNSLIVEDTALYHCAIGNSNVVASGGANKYWGQGTQVTVSS-3’, as shown in SEQ ID NO.8.

[0094] In the amino acid sequence of Nanobody 1D7, the amino acid sequence of positions 1 to 25 is FR1, and its amino acid sequence is: 5'-QVQLQESGGGLVQPGGSLRLSCAAS-3', as shown in SEQ ID NO.4; the amino acid sequence of positions 26 to 33 is CDR1, and its amino acid sequence is: 5'-GFTFSPSA-3', as shown in SEQ ID NO.1; the amino acid sequence of positions 34 to 49 is FR2, and its amino acid sequence is: 5'-MTWARQAQGKGLEWVA-3', as shown in SEQ ID NO.5; the amino acid sequence of positions 50 to 57 is CDR2, and its amino acid sequence is: 5'-SIYGDGVA-3', as shown in SEQ ID NO.2; the amino acid sequence of positions 58 to 95 is FR3, and its amino acid sequence is: 5'-YYTDSVKGRFTISRDNAKNMVYLQMNSLIVEDTALYHC-3', as shown in SEQ ID NO.6; the amino acid sequence at positions 96 to 111 is CDR3, and its amino acid sequence is: 5'-AIGNSNVVASGGANKY-3', as shown in SEQ ID NO.3; the amino acid sequence at positions 112 to 122 is FR4, and its amino acid sequence is: 5'-WGQGTQVTVSS-3', as shown in SEQ ID NO.7.

[0095] Example 3 Determination of the affinity of nanobodies to antigens

[0096] 3.1 Chip Antigen Coupling

[0097] BCMA was prepared into a 50 μg / mL working solution in sodium acetate buffer at different pH values ​​(pH 5.5, pH 5.0, pH 4.5, and pH 4.0). A 50 mM NaOH regeneration solution was also prepared. Electrostatic binding between the antigen and the surface of a chip (GE Biosystems) was analyzed using the template method in the Biacore T100 Protein Interaction Analysis System at different pH values. The most neutral pH was selected as the coupling condition, with the antigen concentration adjusted as needed, based on a signal increase of 5-fold relative to the real-time (RL). The chip was coupled using the instrument's built-in template method: blank coupling mode was selected for channel 1, and target coupling mode was selected for channel 2, with the target set to the designed theoretical coupling amount. The coupling process took approximately 60 minutes.

[0098] 3.2 Exploration of analyte concentration setting conditions and optimization of regeneration conditions

[0099] Use manual injection mode, select 2-1 mode injection for channels 1 and 2, and set the flow rate to 30 μL / min. The injection conditions are all 120 s, 30 μL / min. The regeneration conditions are all 30 s, 30 μL / min. First, continue to empty the running buffer until all baselines are stable. Prepare nanoantibody solutions with a large concentration span and configure them with running buffer. It is recommended to set them to 200 μg / mL, 150 μg / mL, 100 μg / mL, 50 μg / mL, 20 μg / mL, 10 μg / mL, and 2 μg / mL. Prepare the regeneration solution and select the regeneration solution with four pH gradients of the glutamic acid hydrochloride system: 1.5, 2.0, 2.5, and 3.0. Manually inject 200 μg / mL of analyte sample, observe channel 2, and regenerate from the regeneration buffer with the most neutral pH until the response line of channel 2 after regeneration returns to the same height as the baseline. Manually inject another 200 μg / mL analyte sample, observe the signal change in channel 2-1, and record the binding amount. After regeneration with the regeneration solution used to return the response line to baseline in the previous step, manually inject another 200 μg / mL analyte sample, observe the signal change in channel 2-1, and record the binding amount. Compare the binding amount with the previous value. If the deviation is less than 5%, the regeneration solution at this pH is considered optimal. If the binding amount of the second injection is low, continue the experiment with a regeneration buffer at a lower pH. Use the selected optimal regeneration solution as the chip surface regeneration reagent after each injection. Inject samples at the analyte concentrations set above and analyze the binding amount at each concentration to ultimately determine the concentration gradient required for affinity testing.

[0100] 3.3 Affinity test

[0101] Following the optimized sample concentration gradient, the solution was regenerated and the affinity between the nanobody and the antigen was tested using the instrument's built-in template method (with injection conditions set to 60 s at 30 μL / min, dissociation time: 600 s, and regeneration conditions: 30 s at 30 μL / min). The signal in channel 2-1 was monitored continuously. The affinity test process took approximately 200 minutes.

[0102] 3.4 Results Analysis

[0103] Several appropriate concentration gradient binding and dissociation curves were selected and fitted using a 1:1 binding model. Finally, affinity values ​​and important parameters such as binding and dissociation constants were obtained. The results are shown in Table 1. The results showed that the 1D7 nanobody could specifically bind to the BCMA protein coupled to the chip with an affinity of 4.77E-10.

[0104] Table 1 Affinity values, binding constants and dissociation constants

[0105]

[0106] Example 4. In vitro experiments with nanobodies

[0107] 4.1 Cell killing assay

[0108] U266 cells were used to investigate the cell killing effects of 1D7 nanoantibodies expressed by Escherichia coli BL21, 1E5 nanoantibodies targeting CD36 that are not related to the BCMA target, and recombinant proteins NbBCMA-PE38-1D7 and NbCD36-PE38-1E5 linked to PE38 toxin, in order to verify the effect of nanoantibody immunotoxins on BCMA-high-expressing cells in vitro.

[0109] Test method:

[0110] 1) Subculture U266 cells, observe cell status, and count cells. Dilute with complete culture medium to contain 1x10 cells per 100 μL. 4 cells.

[0111] 2) Add cells to a 96-well plate, adding 1x10 4 Cells were plated (i.e., 100 μL of cell suspension was added) and incubated in a constant temperature incubator containing 5% CO2 for 24 h.

[0112] 3) The nanobody immunotoxin to be tested is serially diluted using complete culture medium.

[0113] 4) Add the serially diluted drugs to the U266 cells after incubation and continue incubation for 12 hours. Set up five replicates for each drug concentration.

[0114] 5) Add CCK8 solution to the 96-well plate to a final volume concentration of 10%. Since the amount of CCK8 added is relatively small, gently tap the 96-well plate after adding the reagent to mix thoroughly.

[0115] 6) Incubate for 1–4 hours. Observe the color change during the incubation process. The reaction is complete when the color changes to orange-yellow.

[0116] 7) Measure the absorbance at 450 nm using a microplate reader.

[0117] 8) The curve was fitted using a four-parameter fitting method and the half-inhibitory concentration (IC 50 ).

[0118] The experimental results showed that NbBCMA-PE38-1D7 had a significant killing effect on BCMA high-expressing cells, and its IC 50The concentration of NbCD36-PE38-1E5 was 1135 ng / mL. However, NbCD36-PE38-1E5 had no obvious killing effect.

[0119] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A VHH chain of a nanobody targeting BCMA, characterized in that The variable region CDRs of the VHH chain include: CDR1 with an amino acid sequence as shown in SEQ ID NO.1, CDR2 with an amino acid sequence as shown in SEQ ID NO.2, and CDR3 with an amino acid sequence as shown in SEQ ID NO.

3.

2. The VHH chain of the BCMA-targeting Nanobody according to claim 1, characterized in that The VHH chain also includes a framework region FR.

3. The VHH chain of the BCMA-targeting Nanobody according to claim 2, characterized in that: The framework region FR includes: FR1 with an amino acid sequence as shown in SEQ ID NO.4, FR2 with an amino acid sequence as shown in SEQ ID NO.5, FR3 with an amino acid sequence as shown in SEQ ID NO.6, and FR4 with an amino acid sequence as shown in SEQ ID NO.

7.

4. The VHH chain of the BCMA-targeting Nanobody according to claim 1, characterized in that The amino acid sequence of the VHH chain is as shown in SEQ ID NO.8; or, the VHH chain is an amino acid sequence as shown in SEQ ID NO.8 with one or more amino acids added, deleted or substituted, and the sequence similarity reaches 80% and still exerts similar physiological activity.

5. A nanobody targeting BCMA, characterized in that: It is a single-domain antibody targeting the BCMA epitope and has the VHH chain according to any one of claims 1 to 4.

6. A polynucleotide characterized by: The polynucleotide encodes a protein selected from the group consisting of the VHH chain of the BCMA-targeting Nanobody according to any one of claims 1 to 4 or the BCMA-targeting Nanobody according to claim 5.

7. The polynucleotide according to claim 6, wherein The nucleotide sequence of the polynucleotide is shown in SEQ ID NO.

9.

8. An expression vector, characterized in that: The expression vector contains the polynucleotide according to claim 6 or 7.

9. A host cell, characterized in that The host cell contains the expression vector according to claim 8, or the polynucleotide according to claim 6 or 7 is integrated into its genome.

10. A method for producing a nanobody targeting BCMA, characterized in that: The steps include: (a) culturing the host cell according to claim 9 under conditions suitable for the production of Nanobodies, thereby obtaining a culture containing the Nanobodies targeting BCMA; and (b) isolating or recovering the BCMA-targeting Nanobody from the culture.

11. The method according to claim 10, wherein: The method comprises the following steps: (c) purifying the BCMA-targeting nanoantibody obtained in step (b).

12. The method according to claim 10, wherein: The BCMA-targeting nanoantibody contains the amino acid sequence shown in SEQ ID NO.

8.

13. An immunoconjugate, characterized in that: contain: (a) the VHH chain of the BCMA-targeting Nanobody according to any one of claims 1 to 4 or the BCMA-targeting Nanobody according to claim 5; and (b) a conjugated moiety selected from the group consisting of a detectable label, a toxin, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.

14. An immunoconjugate, characterized in that: contain: (a) the VHH chain of the BCMA-targeting Nanobody according to any one of claims 1 to 4 or the BCMA-targeting Nanobody according to claim 5; and (b) a conjugation moiety selected from the group consisting of a radionuclide.

15. A use of the VHH chain of the BCMA-targeting Nanobody according to any one of claims 1 to 4 or the BCMA-targeting Nanobody according to claim 5, characterized in that: Used to prepare drugs for treating multiple myeloma; or used to prepare reagents for detecting BCMA.

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