Anti-il-6 humanized nanobodies and recombinant humanized nanobodies and uses

Abstract

The application belongs to the technical field of immunology, and particularly relates to an anti-IL-6 humanized nanobody, a recombinant humanized nanobody and application. The humanized nanobody 2H3 of the application comprises complementarity determining regions CDR1-CDR3, the amino acid sequence of CDR1 is shown as SEQ ID NO. 13, the amino acid sequence of CDR2 is shown as SEQ ID NO. 14, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 15. The humanized nanobody can block the combination of IL-6 and its receptor while keeping the affinity with IL-6, and has a wide application prospect in the preparation of drugs for preventing and / or treating tumors. The humanized nanobody has a low molecular weight, reduces the immunological risk caused by heterogeneity, is more conducive to penetrating the blood-brain barrier, and is easier to reach the inside of tumors to play a therapeutic effect.

Description

Technical Field

[0001] This invention belongs to the field of immunology technology, specifically relating to a humanized nanobody against IL-6, a recombinant humanized nanobody, and their applications. Background Technology

[0002] Nanobodies were first reported by Belgian scientists in Nature in 1993 as naturally occurring antibodies (VHHs) lacking a light chain, found in the peripheral blood of alpacas. They were subsequently discovered in cartilaginous fish such as nurse sharks, wrasse, and chimaeras in 1995. They are currently the smallest known units capable of binding to target antigens. With a molecular weight of only 15 kDa, VHHs are also known as nanobodies (Nb). Nanobodies possess unique advantages, including small molecular weight, high solubility, strong stability, high affinity, low immunogenicity, and good tissue permeability, easily crossing blood vessels or tissues to reach target sites. They have great potential for development and their applications are very broad, including clinical use in tumor treatment and as diagnostic tools.

[0003] IL-6 (interleukin 6) is released by various cell types, including macrophages, T cells, and fibroblasts. Its biological effects are mediated by a hexamer complex composed of IL-6, its receptor (IL-6R), and glycoprotein 130 (gp130), which activates intracellular signaling pathways on target cells. This signaling pathway is tightly regulated; dysregulation of IL-6 signaling can lead to chronic inflammation, autoimmune diseases, and other adverse events. In the classical pathway, IL-6 binds to its membrane-bound receptor mIL-6R and the membrane protein gp130 to form the IL-6-IL-6R-gp130 hexamer, triggering downstream signal transduction and gene expression. In the reverse pathway, the IL-6-IL-6R complex binds to gp130, initiating intracellular signal transduction. Subsequently, pathways such as JAK-STAT and RAS-RAF are activated, promoting cell proliferation, differentiation, oxidative stress, and immune regulation. However, when IL-6 levels are elevated, this signal is widely expressed due to the ubiquitous presence of gp130. IL-6 activates signal transducer and activator of transcription 3 (STAT3) through JAK kinases (JAKs). The STAT3 signaling pathway plays an important role in autoimmune diseases, tumor cell proliferation, angiogenesis, and tumor immune escape. Small molecule inhibitors targeting IL-6 / STAT3 may become potential drugs for the treatment of inflammatory diseases and tumors.

[0004] To date, over 100 murine monoclonal antibodies have been humanized through CDR transplantation. The basic principle is that the variable regions of the antibody heavy and light chains are mainly composed of CDRs and backbone regions (FRs). The six CDRs in the variable region are responsible for recognizing and binding antigens; they directly contact the antigen and determine the antibody's specificity. The backbone region, excluding the variable region, primarily supports the CDRs, and its amino acid composition and arrangement are relatively difficult to alter. Therefore, transplanting the CDRs of a murine monoclonal antibody into the backbone region of a human monoclonal antibody can potentially give the human monoclonal antibody the same antigen specificity as the murine antibody while minimizing the heterology of the murine antibody, thus obtaining a modified antibody. Compared to chimeric antibodies, modified antibodies further reduce the proportion of murine components in the antibody, lowering the level of human anti-mouse antibodies. However, some antibodies may still lead to the generation of anti-idiotypic antibodies and have limitations, such as relatively complex construction methods that are time-consuming and labor-intensive; significant problems exist in the antibody's crystal structure and computer-simulated antibody microstructure; and there are still many issues regarding reducing immunogenicity and maintaining antigen-binding activity.

[0005] Therefore, there is an urgent need to find a humanized antibody against IL-6 that can maintain high immunological activity. Summary of the Invention

[0006] The purpose of this invention is to provide a humanized nanobody against IL-6. The humanized nanobody provided by this invention can not only specifically bind to IL-6, but also block the binding of IL-6 to its receptor while maintaining its affinity for IL-6.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The present invention provides a humanized nanobody against IL-6, the nanobody comprising complementarity-determining regions CDR1-CDR3, the amino acid sequences of CDR1-CDR3 being shown in SEQ ID NO.13-15.

[0009] Preferably, the nanobody further includes four framework regions FR1-FR4 alternately linked with complementarity-determining regions CDR1-CDR3, the amino acid sequences of FR1-FR4 being as shown in SEQ ID NO.5-8.

[0010] Preferably, the amino acid sequence of the nanobody is shown in SEQ ID NO.1.

[0011] The nucleotide sequence encoding the above-mentioned humanized nanobody is shown in SEQ ID NO.2.

[0012] The present invention also provides a recombinant humanized nanobody against IL-6, wherein the recombinant humanized nanobody comprises the above-mentioned humanized nanobody.

[0013] Preferably, the amino acid sequence of the recombinant nanoparticles is as shown in SEQ ID NO.3.

[0014] Preferably, the nucleotide sequence of the recombinant nanobody is shown in SEQ ID NO.4.

[0015] The present invention also provides a recombinant vector comprising the above-mentioned recombinant humanized nanobody and an initial vector, wherein the initial vector is a pCZN1 plasmid vector.

[0016] The present invention also provides an IL-6 blocker, wherein the blocker comprises the above-described humanized nanobody or the above-described recombinant humanized nanobody.

[0017] The present invention also provides the application of the above-mentioned humanized nanobody or the above-mentioned recombinant humanized nanobody in the preparation of drugs for autoimmune diseases or anti-tumor diseases or tumor diagnostic reagents.

[0018] The beneficial effects of this invention are:

[0019] This invention provides a humanized anti-IL-6 nanobody 2H3. The sequence of the screened anti-IL-6 nanobody 2H3 was analyzed. Targeting the heavy chain of IL-6, the anti-IL-6 nanobody 2H3 includes a framework region (FR) and a complementary antigenic determinant (CDR). The CDR1, CDR2, and CDR3 regions of the screened anti-IL-6 nanobody 2H3 sequence were transplanted into a humanized hs2dAb backbone. This invention uses a recombinant nanobody fused with the gene encoding the humanized anti-IL-6 nanobody 2H3 and a His tag gene, capable of specifically recognizing the IL-6 antigen. This nanobody can be applied to the molecular diagnosis of autoimmune diseases and tumors, and to the preparation of drugs for autoimmune diseases and anti-tumor diseases. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1This is the first-round DNA electrophoresis image of the natural camel-derived nanobody gene library. From left to right, the DNA bands in the gel wells are: the first lane is a 2000bp marker (band sizes are 2000, 1000, 750, 500, 250, and 100bp respectively), and the second, third, and fourth lanes are PCR products with bands of approximately 700–900bp.

[0022] Figure 2 This is the second round of DNA electrophoresis images of the natural camel-derived nanobody gene library. From left to right, the DNA bands in the gel wells are: the first lane is a 2000bp marker (band size is the same as above), and the second, third, fourth, fifth, and sixth lanes are PCR products with bands of approximately 450bp.

[0023] Figure 3 The graph shows the results of colony PCR analysis to identify the insertion rate of the target fragment;

[0024] Figure 4 A schematic diagram for screening specific single positive clones using phage-ELISA: 1 is IL-6 antigen coated on an ELISA plate, 2 is phage supernatant, 3 is mouse anti-M13K07 antibody, 4 is goat anti-mouse IgG (AP) antibody, and 5 is TMB chromogenic solution.

[0025] Figure 5 The images show the purified humanized anti-IL-6-2H3 nanobody. The left image shows the SDS-PAGE electrophoresis staining of the fragmented sample, the effluent sample, and the eluted sample. The right image shows the SDS-PAGE electrophoresis staining of the purified humanized anti-IL-6-2H3 nanobody.

[0026] Figure 6 Western blot image of humanized nanobody against IL-6-2H3;

[0027] Figure 7 Affinity assay of anti-IL-6-2H3 humanized nanobody with IL-6 antigen (indirect ELISA);

[0028] Figure 8 This is a graph showing the affinity of the anti-IL-6-2H3 humanized nanobody for the IL-6 antigen (BLI). Detailed Implementation

[0029] This invention provides a humanized anti-IL-6 nanobody 2H3, the amino acid sequence of which is shown in SEQ ID NO.1; specifically as follows:

[0030] EVQLQASGGGFVQPGGSLRLSCAASGYTFSSVCMGWFRQAPGKEREFVSAISYTYTGATYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCATDSFGGCRANPLSDSEYNSYWGQGTQVTVSS

[0031] In this invention, the humanized anti-IL-6 nanobody comprises a humanized framework region (FR) and a complementary antigenic determinant region (CDR); in this invention, the humanized framework region (FR) comprises FR1, FR2, FR3, and FR4; the specific amino acid sequences are as follows:

[0032] FR1-AA:EVQLQASGGGFVQPGGSLRLSCAAS(SEQ ID NO.5)

[0033] FR2-AA:MGWFRQAPGKEREFVSA(SEQ ID NO.6)

[0034] FR3-AA:YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC

[0035] (SEQ ID NO.7)

[0036] FR4-AA:WGQGTQVTVSS(SEQ ID NO.8)

[0037] The corresponding nucleotide sequences are as follows:

[0038] FR1: GAGGTTCAGCTGCAGGCTAGCGGTGGTGGTTTTGTGCAGCC GGGTGGTAGCCTGCGTCTGAGCTGCGCAGCATCA (SEQ ID NO.9)

[0039] FR2: ATGGGTTGGTTTCGCCAGGCACCGGGCAAAGAACGCGAATT TGTGAGTGCC (SEQ IDNO.10)

[0040] FR3: TATTATGCAGATAGCGTTAAAGGTCGTTTCACCATTAGTCGTG ATAATAGCAAAAACACCGTGTATCTGCAGATGAATAGTCTGCGCGCCGA AGATACCGCCACCTATTATTGT (SEQ ID NO. 11)

[0041] FR4: TGGGGTCAGGGTACCCAGGTTACCGTGAGCAGCTAA (SEQ ID NO. 12).

[0042] In this invention, the antigenic determinant complementary region (CDR) includes CDR1, CDR2, and CDR3; the specific amino acid sequences are as follows:

[0043] CDR1-AA:GYTFSSVC(SEQ ID NO.13)

[0044] CDR2-AA:ISYTYTGAT(SEQ ID NO.14)

[0045] CDR3-AA: ATDSFGGCRANPLSDSEYNSY (SEQ ID NO. 15).

[0046] In this invention, the nucleotide sequences of CDR1, CDR2, and CDR3 are specifically as follows:

[0047] CDR1: GGTTATACCTTTAGTAGCGTTTGT (SEQ ID NO.16)

[0048] CDR2:ATTAGCTATACCTATACCGGTGCAACC(SEQ ID NO.17)

[0049] CDR3: GCAACCGATAGTTTTGGTGGCTGCCGCGCAAATCCGCTGA GTGATAGCGAATATAATAGCTAT (SEQ ID NO. 18).

[0050] In this invention, the humanized anti-IL-6 nanobody is obtained by screening using the following method: a natural camel-derived nanobody phage display library is constructed using phage surface display technology, and then screening is performed based on biotinylated IL-6 antigen to obtain IL-6-specific nanobody gene sequences. The CDR1, CDR2, and CDR3 regions of the screened anti-IL-6 nanobody 2H3 sequence are then transplanted into the humanized hs2dAb backbone.

[0051] In this invention, the preferred method for constructing the natural camel-derived heavy chain antibody phage display gene library includes the following steps: 1) extracting total RNA from camel peripheral blood mononuclear cells and reverse transcribing the total RNA to obtain cDNA; 2) using the cDNA as a template to perform nested PCR amplification to obtain the variable region fragment of the heavy chain antibody; 3) digesting the variable region fragment of the heavy chain antibody and the pCANTAB5E phage vector with enzymes respectively, and then ligating them to obtain the ligation product; 4) transforming the ligation product into competent TG1 cells to obtain the natural camel-derived nanobody phage display library.

[0052] In this invention, total RNA is extracted from camel peripheral blood mononuclear cells and reverse transcribed to obtain cDNA. The method for extracting total RNA from camel peripheral blood mononuclear cells is not particularly limited; conventional animal peripheral blood total RNA extraction methods in this art are acceptable. In this invention, the reverse transcription is preferably performed using the Thermo Scientific ReverAid First Strand cDNA Synthesis Kits.

[0053] After obtaining the cDNA, this invention uses the cDNA as a template to perform nested PCR amplification to obtain the variable region fragment of the heavy chain antibody. In this invention, the nested PCR preferably includes two rounds of PCR; the first round of PCR is used to amplify the fragment between the heavy chain antibody guide peptide and antibody CH2, and the primer sequences for the first round of PCR are preferably shown in SEQ ID NO. 19 and SEQ ID NO. 20; the second round of PCR is used to amplify the fragment between the heavy chain antibody FR1 region and the long and short hinge regions, and the primer sequences for the second round of PCR are preferably shown in SEQ ID NO. 21 and SEQ ID NO. 22.

[0054] In this invention, after obtaining the variable region fragment of the heavy chain antibody, the variable region fragment of the heavy chain antibody and the pCANTAB5e phage vector are digested with enzymes, and then ligated to obtain the ligation product. In this invention, the enzyme digestion is preferably a double digestion, and the enzyme digestion is preferably performed using restriction endonucleases SifI and NotI. In this invention, the enzyme digestion procedure is preferably as follows: digestion at 37°C for 1 hour; digestion at 50°C for 1 hour. In this invention, the ligation temperature is preferably 16°C, and the ligation time is preferably 4 hours.

[0055] After obtaining the ligation product, this invention transforms the ligation product into competent cells to obtain a natural camel-derived nanobody phage display library. In this invention, the competent cells are preferably *E. coli* competent cells TG1, which are preferably prepared by glycerol resuspension. The transformation method is preferably electroporation. This invention also includes a phage rescue process after transformation. This invention does not specifically limit the electroporation and phage rescue processes; detailed steps are described in the embodiments.

[0056] The present invention does not specifically limit the steps for screening anti-IL-6 nanobodies using the biotinylated IL-6 antigen. Conventional nanobodies screening methods in the art can be used. For detailed steps, please refer to the embodiments.

[0057] The present invention also provides a gene encoding the humanized nanobody, the nucleotide sequence of which is shown in SEQ ID NO.2, specifically as follows: GAGGTTCAGCTGCAGG CTAGCGGTGGTGGTTTTGTGCAGCCGGGTGGTAGCCTGCGTCTGAGCTGCGCAGCATCAGGTTATACCTTTAGTAGCGTTTGTATGGGTTGGTTTCGCCAGGCACCGGGCAAAGAACGCGAATTTGTGAGTGCCATTAGCTATACCTATACCGGTGCAACCTATTATGCAGATAGCGTTAAAGGTCGTTTCACCATTAGTCGTGATAATAGCAAAAACACCGTGTATCTGCAGATGAATAGTCTGCGCGCCGAAGATACCGCCACCTATTATTGTGTGCAACCGATAGTTTTGGTGGCTGCCGCGCAAATCCGCTGAGTGATAGCGAATATAATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTGAGCAGCTAATCTAGA.

[0058] This invention provides a recombinant humanized nanobody against IL-6, comprising the aforementioned anti-IL-6 humanized nanobody and a His tag fusion expression. In this invention, the amino acid sequence of the recombinant humanized nanobody (2H3-His-AA) is shown in SEQ ID NO.3, and is as follows:

[0059] HHHHHHMEVQLQASGGGFVQPGGSLRLSCAASGYTFSSVCMGWFR QAPGKEREFVSAISYTYTGATYYADSVKGRFTISRDNSKNTVYLQMNSLR AEDTATYYCATDSFGGCRANPLSDSEYNSYWGQGTQVTVSS.

[0060] The present invention also provides a gene encoding the recombinant humanized nanobody, comprising the gene for the humanized nanobody and a His tag fusion expression gene.

[0061] In this invention, the nucleotide sequence of the gene (2H3-His) of the recombinant humanized nanobody is shown in SEQ ID NO.4, specifically as follows:

[0062] CACCACCACCACCACCACATGGAGGTTCAGCTGCAGGCTAGCGGTGGTGGTTTTGTGCAGCCGGGTGGTAGCCTGCGTCTGAGCTGCGCAGCATCAGGTTATACCTTTAGTAGCGTTTGTATGGGTTGGTTTCGCCAGGCACCGGGCAAAGAACGCGAATTTGTGAGTGCCATTAGCTATACCTATACCGGTGCAACCTATTATGCAGA TAGCGTTAAAGGTCGTTTCACCATTAGTCGTGATAATAGCAAAAACACCGTGTATCTGCAGATGAATAGTCTGCGCGCCGAAGATACCGCCACCTATTATTGTGCAACCGATAGTTTTGGTGGCTGCCGCGCAAATCCGCTGAGTGATAGCGAATAATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTGAGCAGCTAATCTAGA.

[0063] The nucleotide sequence of the His tag gene is shown in SEQ ID NO.23, and is as follows: CACCACCACCACCACCACCAC

[0064] The present invention also provides a recombinant vector for expressing a humanized nanobody against IL-6, comprising the gene of the recombinant humanized nanobody and an initial vector.

[0065] In this invention, the initial vector is preferably a pCZN1 plasmid vector; the insertion site of the gene of the recombinant humanized nanobody is preferably located at the Nco I-Xho I site of the pCZN1 plasmid vector. This invention does not impose any particular limitation on the preparation method of the recombinant vector; conventional recombinant vector preparation methods in the art can be used.

[0066] This invention also provides a recombinant bacterial strain expressing a humanized anti-IL-6 nanobody, comprising the recombinant vector and *Escherichia coli* Arctic Express. Preferably, the recombinant bacterial strain is obtained by transforming the recombinant vector expressing the anti-IL-6 humanized nanobody into *Escherichia coli* Arctic Express. This invention does not specifically limit the preparation method of the recombinant bacterial strain; conventional methods for preparing recombinant bacterial strains in the art can be used.

[0067] This invention provides the application of the aforementioned anti-IL-6 humanized nanobody, the gene encoding the anti-IL-6 humanized nanobody, the recombinant humanized nanobody, the gene encoding the recombinant humanized nanobody, the recombinant vector, and the recombinant strain in the preparation of drugs for autoimmune diseases and antitumor diseases or tumor diagnostic reagents.

[0068] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0069] RNA purification kit was purchased from TIANGEN; restriction endonucleases were purchased from NEB; T4 DNA ligase was purchased from NEB.

[0070] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0071] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0072] Example 1: Construction of a natural camel-derived nanobody gene library

[0073] 1. Extract total RNA from camel peripheral blood mononuclear cells. The specific steps are as follows:

[0074] (1) Blood collection from Bactrian camels was performed by professionals. Percoll cell separation solution was used to separate peripheral blood mononuclear cells from non-immunized camels. The separated peripheral blood mononuclear cells were washed three times with PBS and 1 mL of Trizol solution was added.

[0075] (2) Add 0.2 mL of chloroform to each 1 mL of Trizol, shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes.

[0076] (3) Centrifuge the sample at 12000 rpm and 4℃ for 15 min, and transfer the upper layer to a new Ep tube.

[0077] (4) Add an equal volume of isopropanol and mix on ice for 20 minutes.

[0078] (5) Centrifuge the solution at 12000 rpm and 4℃ for 10 min and discard the supernatant.

[0079] (6) Add 1 mL of 75% ethanol prepared with DEPC water to wash the precipitate (add at least 1 mL of ethanol for every 1 mL of Trizol).

[0080] (7) Centrifuge the solution from the previous step at 10,000 rpm for 10 min at 4℃, discard the supernatant, and repeat the previous step once more.

[0081] (8) Centrifuge at 10000 rpm and 4℃ for 10 min, discard the supernatant, and dry for 10-15 minutes.

[0082] (9) Add an appropriate amount of ddH2O to dissolve the RNA precipitate, measure the RNA concentration, and store at -80℃.

[0083] The RNA was purified using an RNA purification kit, and cDNA was obtained by reverse transcription according to the Thermo Scientific ReverAid First Strand cDNA Synthesis Kits.

[0084] 2. Using cDNA as a template, nested PCR was used to amplify the variable region fragment of the heavy chain antibody;

[0085] First round of PCR:

[0086] Table 1. First round PCR reaction system

[0087]

[0088] The conditions for the first round of PCR amplification were: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 45 s, for 32 cycles; 72℃ for 10 min.

[0089] The fragment between the heavy chain antibody guide peptide and antibody CH2 was amplified. This primer pair was used to amplify the vh-CH1-CH2 fragment with a size of 900 bp and the vhh-CH2 fragment with a size of about 700 bp. The results showed that the size of the fragment was about 700-900 bp, that is, there were about two nanobody gene electrophoresis bands.

[0090] Second round PCR: using the products from the first round PCR as templates

[0091] F-2: 5'-TCGCGGCCCAGCCGGCCCAGGTCCAACTGCAGGAGTCTGG GG-3' (SEQ ID NO. 21)

[0092] R-2: 5'-ATAAGAATGCGGCCGCTGAGGAGACGGTGACCTGGGTCCC C-3' (SEQ ID NO. 22)

[0093] Table 2. Second round PCR reaction system

[0094]

[0095]

[0096] The second round of PCR amplification conditions were: 94℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 45 s, 25 cycles; 72℃ for 10 min. Fragments (long and short fragments) between the FR1 region and the long and short hinge regions of the heavy chain antibody were amplified to obtain a camel heavy chain antibody heavy chain variable region vhh fragment of approximately 400 bp from the 700 bp vhh-CH2 fragment. The results showed that the size of this fragment was approximately 450 bp, meaning the nanobody gene electrophoresis band was approximately 450 bp.

[0097] The pCANTAB5e phage vector and VHH fragment were digested with restriction endonucleases SifI and NotI, and the two fragments were ligated using T4 DNA ligase. The specific digestion and ligation systems are as follows:

[0098] Table 3 Enzyme digestion system

[0099]

[0100] Enzyme digestion conditions: digest at 37℃ for 1 hour, then digest at 50℃ for 1 hour.

[0101] Table 4 Connection System

[0102]

[0103] Connection conditions: Connect at 16℃ for 4 hours, then connect overnight at 4℃.

[0104] The ligation product was electroporated into electrocompetent TG1 cells to construct a natural camel-derived nanobody phage display library. After helper phage rescue, the library size reached 9.0 × 10⁻⁶. 13 .

[0105] The insertion rate of the library was detected by colony PCR. Second-round PCR primers were used, and the annealing temperature was 55℃. The results are as follows: Figure 3 The insertion rate is shown to be over 95% (target fragment insertion rate = number of colonies containing the target fragment / total number of colonies).

[0106] 3. The preparation method of competent TG1 cells is as follows:

[0107] Take Escherichia coli TG1 glycerol bacteria from the -80℃ freezer, streak them onto 2×YT solid plates and incubate at 37℃ for 10 h. Pick a single colony and inoculate it into 3 mL of 2×YT liquid medium and incubate overnight at 37℃ with shaking at 200 rpm.

[0108] The following day, the bacterial culture was scaled up at a 1:100 ratio in Erlenmeyer flasks containing 200 mL of 2×YT medium and cultured at 37°C until OD reached. 600 When the bacterial concentration reaches approximately 0.4, collect the bacterial culture in a 50 mL centrifuge tube, incubate on ice for 1 hour, centrifuge at 9000 rpm and 4°C for 10 minutes, discard the supernatant, resuspend the bacterial pellet in an equal volume of cold pure water, centrifuge, and repeat once. Then resuspend the bacterial pellet in pre-chilled 10% glycerol and centrifuge. Resuspend the bacterial pellet in 1 mL of 10% glycerol (pre-chilled pure water) and aliquot 100 μl into pre-chilled 1 mL Eppendorf tubes, then immediately transfer to a -80°C freezer for storage; this is the competent TG1 cell.

[0109] 4. The steps for assisting phage rescue are as follows:

[0110] (1) Take 100 μL of the library and inoculate it into 50 mL of 2×YT / Amp / Glu medium. Incubate at 37℃ with shaking at 200 rpm until the logarithmic OD phase. 600 It is approximately 0.4 to 0.5.

[0111] (2) Add helper phage M13KO7 with an infection multiplicity of 20:1 to the culture medium, mix well and let stand at 37°C for 30 min.

[0112] (3) Centrifuge the culture medium at 9000 rpm for 10 min at room temperature, discard the supernatant precipitate of bacteria, resuspend in 200 mL of 2×YT / Amp / Kana culture medium, and incubate overnight at 37℃ and 200 rpm.

[0113] (4) Centrifuge the culture medium at 4℃ and 9000rpm for 10min, take the supernatant, add 1 / 5 volume of PEG / NaCl, and let it stand at 4℃ for 6h.

[0114] (5) Centrifuge at 9000 rpm for 20 min, discard the supernatant, resuspend the precipitate with PBS (1 mL), and obtain the recombinant phage antibody library. Aliquot the phage into 1.5 mL Eppendorf tubes and store at 4 °C.

[0115] Example 2: Screening for anti-IL-6 nanobodies

[0116] phage library (1×10) 13 After incubating 50 μL of streptavidin magnetic beads with a phage on a rotating platform at room temperature for 1 h, the phage antibody was collected. In two 1 mL centrifuge tubes blocked with 2% PBSM, 500 μL of pre-thresholded phage antibody was added to each tube. 500 μL of 5 μg biotinylated IL-6 antigen diluted in PBS was added to one tube, and 500 μL of PBS buffer was added to the other tube as a negative control. Both tubes were incubated on a rotating platform at room temperature for 1 h. Then, 50 μL of pre-blocked streptavidin magnetic beads were added, and the tubes were incubated on a rotating platform at room temperature for 30 min. The magnetic beads were collected. The beads were washed 7 times with PBST, 2 times with PBSM, and 1 time with PBS. Eluting was performed with Glycine at pH 2.7, and neutralization was performed with 1 mol / L Tris-HCl at pH 9.1.

[0117] Add the above neutralization solution to 5 mL of TG1 (OD100) in the logarithmic growth phase. 600 In a concentration of 0.5, phages are generated and purified for the next round of screening. After three rounds of screening, positive clones will be continuously enriched, thereby achieving the goal of using phage display technology to screen IL-6 specific antibodies in the antibody library.

[0118] Phage-ELISA is used to screen for specific single positive clones.

[0119] The principle diagram of the filtering process is as follows: Figure 4 As shown, the specific method is as follows:

[0120] First, VHH phage monoclonal supernatant was prepared: 180 single colonies were randomly picked from solid plates after three rounds of screening and inoculated into 96-well plates of 2×YT medium containing 100 μg / mL ampicillin and 2% glucose, incubated at 220 rpm. Incubate overnight at 37°C. The next day, transfer 50 μL of bacterial culture to a new 96-well plate. Add 800 μL of 2×YT medium containing 100 μg / mL ampicillin and 2% glucose to each well. After growth to the logarithmic phase, add helper phage M13K07 with an infection multiplicity of 20:1. Infect at 37°C for 30 min, centrifuge at 10,000 rpm for 5 min, discard the supernatant, and resuspend the bacteria in 800 μL of fresh 2×YT medium containing 100 μg / mL ampicillin and 50 μg / mL kanamycin. Incubate at 37°C and 220 rpm for 12 h. The next day, centrifuge the bacterial culture at 10,000 rpm for 5 min. The supernatant is the supernatant of VHH phage monoclonal cells.

[0121] Dilute IL-6 antigen to 10 μg / mL with coating buffer, add 100 μL to each well, and coat overnight at 4°C. Negative and positive controls were also included. The next day, wash three times with PBST, block with 2% PBSM at 37°C for 2 h, wash three times with PBST, add 200 μL of pretreated VHH phage monoclonal supernatant, and incubate at 37°C for 1 h. Add 1:5000 of murine anti-M13KO7 / HRP secondary antibody diluted with 0.1% PBST, incubate at 37°C for 1 h, wash away unbound antibody, add TMB chromogenic buffer, and read the absorbance at 450 nm using a microplate reader. A sample well with an OD value more than twice that of a control well was considered a positive control well, and the positive bacterial culture was used for gene sequencing.

[0122] Sequence analysis and BLAST alignment were performed using Snapegene software, and strains with identical CDR1, CDR2, and CDR3 sequences were considered as the same clone. The CDR1, CDR2, and CDR3 regions of the selected anti-IL-6-2H3 nanobodies were transplanted into the humanized hs2dAb backbone, and a His tag was added to facilitate protein purification and identification. The nanobodies sequence shown in SEQ ID NO.1 was ultimately used for subsequent experiments.

[0123] Example 3: Expression and purification of humanized nanobody IL-6-2H3-Nb in host bacterium Escherichia coli

[0124] (1) The subclone pCZN1 plasmid vector of the humanized nanobody IL-6-2H3-Nb sequence obtained by sequencing analysis was transformed into Escherichia coli Arctic Express. Single clones on the transformation plate were picked and inoculated into test tubes containing 3 mL LB medium with 50 μg / mL Kan and shaken at 220 rpm overnight at 37℃.

[0125] (2) The next day, inoculate 1:100 into 30 mL of LB culture medium containing 50 μg / mL Kan, and shake at 37℃ and 220 rpm until the bacterial cells reach OD. 600 The concentration was 0.6–0.8, and IPTG was added to a final concentration of 0.5 mM. The mixture was then incubated overnight at 20°C and 220 rpm with shaking to induce fusion protein expression.

[0126] (3) Collect bacterial cells and sonicate them to obtain crude inclusion body protein fluid, which is then purified by Ni column affinity to obtain fusion protein. Figure 5 This is the purified anti-IL-6 humanized nanobody 2H3. The left image shows the SDS-PAGE electrophoresis staining of the purification process of the anti-IL-6 humanized nanobody 2H3: lane M is the protein molecular standard, lanes 1-2 are the post-fracture sample and the eluted sample, respectively, and lanes 3-4 are the eluted sample; the right image shows the SDS-PAGE electrophoresis staining of the purified anti-IL-6 humanized nanobody 2H3: lane M is the protein molecular standard, lane 1 is 0.5 mg / mL BSA as the concentration standard, and lane 2 is the purified anti-IL-6 humanized nanobody 2H3.

[0127] Example 4: Specificity verification of anti-IL-6 humanized nanobody 2H3

[0128] Western blot was performed using IL-6 antigen (with purified anti-IL-6 humanized nanobody 2H3 as the primary antibody and anti-mouse His / HRP as the secondary antibody). Figure 6 The image shows a Western blot diagram of the IL-6 antigen: lane M represents the protein molecule standard, and lane 1 represents the IL-6 antigen. This demonstrates that the anti-IL-6 humanized nanobody 2H3 provided by this invention can specifically bind to the IL-6 antigen.

[0129] Example 5: ELISA method for detecting the specific binding of IL-6 humanized nanobody 2H3 to IL-6 antigen.

[0130] IL-6 antigen was diluted to 2 μg / mL with ELISA coating buffer, and 100 μL was added to each well of a 96-well ELISA plate for coating at 4℃. PBS coating served as a negative control. The next day, the plate was washed three times with 0.05% PBST, blotted dry with absorbent paper, and 300 μL of 5% PBSM was added to each well for blocking at 37℃ for 2 h. After blocking, the plate was washed three times with PBST and blotted dry. 100 μL of experimental and control samples were added to each well, and the plate was incubated at 37℃ for 1 h. The plate was washed three times with PBST and blotted dry. Mouse His antibody was diluted 1:5000 with 0.05% PBST, and 100 μL was added to each well. The plate was incubated at 37℃ for 1 h, then washed and blotted dry. Goat anti-mouse HRP antibody was diluted 1:5000 with 0.05% PBST, and 100 μL was added to each well. The plate was incubated at 37℃ for 1 h, then washed and blotted dry. The detection results are as follows: Figure 7 As shown, the anti-IL-6-2H3-Nb humanized nanobody specifically binds to the IL-6 antigen.

[0131] Example 6: Specificity Detection of Anti-IL-6 Humanized Nanobody 2H3 and IL-6 Antigen

[0132] The affinity of nanobodies was determined using biomembrane interference (BLI) technology. Biotinylated humanized IL-6 antigen was bound to a Streptavidin (SA) probe. Six nanobodies concentration gradients of 2000, 1000, 500, 250, 125, and 62.5 nmol / L were established. The binding and dissociation of each concentration of nanobodies with the biotinylated humanized IL-6 antigen were measured using Octet Red96e. The detection data were fitted using a 1:1 binding model, and binding and dissociation curves of IL-6 nanobodies 2H3 and IL-6 antigen at each concentration were plotted. The KD value was calculated.

[0133] The results are as follows Figure 8 The display shows that its binding constant is 1.285 × 10⁻⁶. -4 The dissociation constant is 4.233 × 10⁻⁶. -3 The equilibrium dissociation constant (KD) was calculated to be 9.755 × 10⁻⁶. -7 The results suggest that the anti-IL-6 humanized nanobody 2H3 has a strong ability to specifically bind to the IL-6 antigen.

[0134] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A humanized nanobody against IL-6, characterized in that, The nanobody includes complementarity-determining regions CDR1-CDR3, and the amino acid sequences of CDR1-CDR3 are shown in SEQ ID NO.13-15, respectively.

2. The humanized nanobody according to claim 1, characterized in that, The nanobody also includes four framework regions FR1-FR4 alternately linked with complementarity-determining regions CDR1-CDR3, and the amino acid sequences of FR1-FR4 are shown in SEQ ID NO. 5-8, respectively.

3. The humanized nanobody according to claim 1, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO.

1.

4. A nucleic acid molecule encoding the humanized nanobody according to any one of claims 1-3, characterized in that, The sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

5. A recombinant humanized nanobody against IL-6, characterized in that, The recombinant humanized nanobody consists of the humanized nanobody described in any one of claims 1-3 and a His tag.

6. The recombinant humanized nanobody according to claim 5, characterized in that, The amino acid sequence of the recombinant humanized nanobody is shown in SEQ ID NO.

3.

7. The recombinant humanized nanobody according to claim 6, characterized in that, The nucleotide sequence encoding the recombinant humanized nanobody is shown in SEQ ID NO.

4.

8. A recombinant vector, characterized in that, The recombinant vector includes a gene encoding the recombinant humanized nanobody of claim 5 and an initial vector, wherein the initial vector is a pCZN1 plasmid vector.

9. An IL-6 blocker, characterized in that, The blocking agent includes the humanized nanobody according to any one of claims 1-3 or the recombinant humanized nanobody according to claim 5.

Images