CN110642946A

CN110642946A - Humanized nano antibody and preparation method thereof

Info

Publication number: CN110642946A
Application number: CN201910939111.1A
Authority: CN
Inventors: 纪雪梅; 刘煜; 赵明军
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2019-09-30
Filing date: 2019-09-30
Publication date: 2020-01-03

Abstract

The invention relates to the field of biomedicine, and particularly discloses a humanized modification method of an anti-TNF alpha nano antibody, wherein the nucleotide sequence of the humanized modified anti-TNF alpha nano antibody is shown as SEQ ID No.11, and the amino acid sequence is shown as SEQ ID No. 12; the transformation method comprises the following steps: determining single mutation and combined mutation of three key amino acids in FR2 region, wherein the nucleotide sequence is shown as SEQ ID No.1, 3, 5, 7 and 9, and the amino acid sequence is shown as SEQ ID No.2, 4, 6, 8 and 10; further multiple sequence alignment to determine a humanization scheme; obtaining the humanized anti-TNF alpha nano antibody through the method; expressed by a yeast expression system. The humanized anti-TNF alpha nano antibody modified by the invention can retain higher antigen affinity, thermal stability and biological activity, and lays a foundation for the development of disease treatment drugs of humanized nano antibodies in the future.

Description

Humanized nano antibody and preparation method thereof

Technical Field

The invention belongs to a nano antibody, and particularly relates to a humanization method of an anti-TNF alpha nano antibody (NbTNF alpha) and a preparation method thereof.

Background

Tumor necrosis factor alpha (TNF α) is the most studied multi-effector cytokine, and is secreted mainly by activated macrophages, and also by lymphocytes, kupffer cells, smooth muscle cells, and various tumor cells such as melanoma and breast cancer. It is not only a key mediator of the inflammatory response, but also can cause apoptosis and necrosis of cells by modulating signal transduction. Recently, new researches show that the blocking agent targeting TNF alpha can cause various autoimmune diseases such as diabetes, psoriasis, multiple sclerosis and the like when treating related diseases. In addition, more researches show that the TNF alpha has close relation with the tumor, on one hand, the low-concentration TNF alpha can participate in the generation and development of the tumor through a plurality of signal pathways, and on the other hand, the high-concentration TNF alpha has stronger anti-tumor effect. TNF α is currently being studied more and more intensively, with TNF α being the most mature target. Various TNF α antibody drugs are currently on the market, including three imported (eke, enrie, sumile) and three homemade (yicepu, qiang, anbero).

The nanobody (nanobody) is a special small molecule antibody, which is a heavy chain antibody with a deletion of light chain and CH1 constant region separated from camel serum, and a heavy chain single domain antibody (VHH) can be obtained by cloning the variable region of the heavy chain single domain antibody. As a small molecule antibody, the antibody has the advantages that the traditional antibody does not have, 1) the molecular weight is small, is only 15Kd, is 1/10 of the traditional monoclonal antibody, has strong and fast penetrating power, is easy to penetrate through blood vessels or tissues to reach a target site, and provides a new method for brain administration because the antibody can penetrate through the blood brain barrier; 2) the stability is good, and researches show that the nano antibody still can keep more than 80% of antigen binding capacity when being placed at the temperature of 37 ℃ for more than one week, while the single-chain antibody is aggregated and inactivated; meanwhile, people put the nano antibody and the monoclonal antibody at 90 ℃ and under a strong denaturing agent for a long time, and find that the nano antibody still keeps high activity, but the monoclonal antibody is completely inactivated and undergoes irreversible polymerization, so that the nano antibody is easier to store and transport under normal temperature conditions and severe environment; 3) compared with a monoclonal antibody, the nano antibody has a longer CDR3 region, the CDR3 forms a convex ring shape and can specifically recognize hidden antigen epitopes which cannot be recognized by a conventional antibody; 4) the nano antibody has simple structure and can be expressed in bacteria and yeast in large quantity. These advantages are not possessed by conventional antibodies and other small molecule antibodies (Fab, Fv, scFv, etc.), so that the nanobody has great development opportunities in the treatment and diagnosis of diseases. At present, the nano antibody entering the clinical stage mainly appears in various forms such as bivalent, multivalent, PEG modification, Fc fusion, albumin fusion and the like, so that the treatment effect of the medicine is improved, and the half life of the medicine can be prolonged. However, with the increasing molecular weight, the immunogenicity of the nanobody has to be paid attention to, and the part of the nanobody currently in clinical trials is a humanized nanobody, so that people are getting more and more attentive to find a simple and convenient humanized modification method with little influence on activity. Like murine antibodies, the general principle of humanization of nanobodies is to ensure higher affinity, thermostability, activity, yield, etc., while effectively reducing immunogenicity. At present, the humanized modification of the nano antibody mainly comprises the following two methods:

surface amino acid humanization: according to the report of related documents, the heavy chain variable region structure of the nano antibody is very similar to that of a human antibody, and the homology is as high as 80-90%, and in addition, the camel VHH germ line gene sequence is analyzed, so that the camel VHH germ line gene sequence has very high homology with a human VH gene III family. Through sequence comparison, it is found that the framework regions have about ten or more amino acid differences, the most important of which is the FR2 framework region, the amino acids at positions 42, 49, 50 and 52 of VH of human antibody are mainly hydrophobic residues and relatively conserved, usually interact with VL and participate in forming VH-VL interface, and VHH is mainly hydrophilic residues, so that VHH has good water solubility in case of light chain deletion, and the research also finds that these several residues in the framework region of VHH FR2 are also closely related to CDR3 conformation. Therefore, for humanization, the influence of these several residues of FR2 on CDR conformation was first studied, and residues with larger influence were retained. And secondly, performing humanized mutation on the surface amino acids of FR1, FR3 and FR4, wherein the three FR regions have little influence on the conformation of the CDR, and the mutation sites can be selected by means of simulation analysis or sequence comparison.

VHH humanization general framework grafting: vincke et al obtained a maximally humanized NbBcII10FGLA nanobody (h-NbBcII10FGLA) by using an antibody germline gene DP-47 of a human VH gene III family as a template to perform humanized modification on a beta-lactamase nanobody (NbBcII 10). On the basis of reducing immunogenicity, the preparation method not only keeps the affinity and solubility of the antibody, but also improves the thermal stability to a certain extent. Meanwhile, related researches are carried out to graft CDR regions of nano antibodies from different sources to a framework region of a humanized universal framework h-NbBcII10FGLA to generate humanized nano antibodies, and comparison shows that the affinity and the thermal stability are not obviously changed before and after transplantation, and the influence of the biological activity is not obvious, so that the h-NbBcII10FGLA is an ideal humanized nano antibody universal template designed at present.

At present, the expression preparation and the research on anti-breast cancer tumor are carried out aiming at the TNF alpha nano antibody (patent number: CN 103333253A), but the humanization of the anti-TNF alpha nano antibody is not reported in the literature. The invention completes the humanization transformation of the TNF alpha and verifies the TNF alpha binding activity, obtains the TNF alpha humanization nano antibody with high affinity, and lays a foundation for further developing therapeutic TNF alpha nano antibody medicines aiming at autoimmune diseases and tumor diseases.

Disclosure of Invention

The invention aims to provide a method for humanizing an anti-TNF alpha nano antibody, which can lay a certain foundation for preparing a humanized nano antibody in the future.

The invention discloses a TNF alpha nano antibody FR2 region 42, 50, 52 key amino acid single mutation and combined mutation sequence, the nucleotide sequence is shown as SEQ ID NO.1, 3, 5, 7, 9; the amino acid sequence is shown in SEQ ID No.2, 4, 6, 8 and 10.

The invention discloses a humanized and reformed anti-TNF alpha nano antibody, the nucleotide sequence of which is shown in SEQ.ID.NO. 11; the amino acid sequence is shown as SEQ ID No. 12.

The invention also discloses a humanized transformation method of the anti-TNF alpha nano antibody, which comprises the following steps:

1) partitioning CDRs and FRs of the anti-TNF alpha nanobody by an IMGT antibody CDRs region Numbering scheme;

2) different combination mutations are carried out on the key amino acids at the 42 th, 50 th and 52 th positions of the FR2 framework region which has an influence on the CDR3 conformation, and the common mutation of the amino acids at the 50 th position and the 52 th position has been determined to have little influence on the conformation;

3) carrying out sequence similarity search on the sequence of the anti-TNF alpha nano antibody through IgBLAST, searching out a human antibody germ line gene sequence with higher sequence homology with the sequence, and carrying out multiple sequence comparison on the anti-TNF alpha nano antibody and the human antibody germ line gene sequence by using MEGA5.0 software;

4) preparation of a humanization protocol: according to the analysis results of the steps 2) and 3), reasonable humanized design is carried out on the anti-TNF alpha nano antibody, and the method comprises the following steps:

4.1) amino acid residues that cannot be substituted for the FR backbone region: phenylalanine at position 42 cannot be substituted; adjacent residues outside the CDR regions (typically two adjacent amino acid residues) tend to have a greater effect on CDR conformation;

4.2) residues that do not require substitution in the FR backbone region: residues within the range of sequence variation of the human antibody may be left unsubstituted;

4.3) residues that can be substituted for the FR backbone region: substitutions are contemplated which are unique to the sequence of the human antibody;

4.4) residues of the FR backbone region to consider: simultaneous substitution of several residues in series may have some effect on CDR conformation.

The invention also discloses a humanized modified TNF alpha nano antibody expression and activity verification method, which comprises the following steps:

1) the humanized and modified anti-TNF alpha nano antibody (h-NbTNF alpha) is connected to pPICZ alpha A plasmid in a way of PCR amplification and enzyme digestion and enzyme linkage;

2) linearizing the recombinant vector obtained in the step 1), electrically converting the linearized recombinant plasmid into a GS115 Pichia pastoris expression strain, performing methanol induced expression, and purifying by a nickel column to obtain a humanized anti-TNF alpha nano anti-protein;

3) detecting the affinity and the thermal stability of the humanized and reformed anti-TNF alpha nano antibody by using an indirect ELISA method, and comparing the affinity and the thermal stability with the parent NbTNF alpha nano antibody;

4) the MTT method is adopted to detect the L929 cytotoxicity inhibition effect induced by the hTNF alpha antigen of the humanized and modified anti-TNF alpha nano antibody, and the effect is compared with the parent NbTNF alpha nano antibody.

Drawings

FIG. 1 shows SDS-PAGE and WB analyses of the purified proteins (A: SDS-PAGE detection map, Lane 1: NbTNF. alpha. Nanobody target protein; Lane 2: NbTNF. alpha₄₂A nanobody target protein; lane 3: NbTNF alpha₅₀A nanobody target protein; lane 4: NbTNF alpha₅₂Nano antibody target protein: m: protein marker.b: WB identification Panel, Line1-4 corresponds to Line1-4 in Panel A C: SDS-PAGE detection Panel: lane1 is NbTNF alpha_50-52A nanobody target protein; lane2 is NbTNF alpha_42-50-52A nanobody target protein; m: protein marker.d: WB identification map: line1-2 corresponds to Line1-2 in FIG. C)

FIG. 2 indirect ELISA method for determining affinity of NbTNF alpha protein and mutant protein to TNF alpha antigen (A: NbTNF alpha, B: NbTNF alpha 42, C: NbTNF alpha 50, D: NbTNF alpha 52, E: NbTNF alpha 50-52, F: NbTNF alpha 42-50-52; subscript 1 represents TNF alpha coating concentration of 1. mu.g/ml, subscript 2 represents TNF alpha coating concentration of 2. mu.g/ml);

FIG. 3 is a diagram showing multiple sequence alignments of NbTNF α with human germline genes V and J segments;

FIG. 4A is a diagram showing the result of PCR amplification of h-NbTNF alpha gene sequence; b, H-NbTNF alpha nano antibody liquid PCR result;

identifying the linear plasmid electrically transformed yeast by a PCR method to obtain a positive strain;

FIG. 5 shows SDS-PAGE detection of protein expression of h-NbTNF α nanobody-positive yeast strains; b, electrophoresis picture of purified h-NbTNF alpha nano antibody protein;

FIG. 6 shows indirect ELISA method for determining affinity of antibody protein to TNF alpha antigen A: NbTNF alpha B: h-NbTNF alpha (subscript 1 represents TNF alpha coating concentration of 1. mu.g/ml, subscript 2 represents TNF alpha coating concentration of 2. mu.g/ml);

FIG. 7A shows that the h-NbTNF α nanobody treated at different temperatures for 1h has an effect on its activity; b, treating at 90 ℃ for different time to influence the activity of the h-NbTNF alpha nano antibody;

FIG. 8 is the neutralizing effect of h-NbTNF α nanobody on TNF α -mediated cytotoxicity.

Detailed description of the invention

The original sequence of the anti-TNF alpha nano antibody comes from an authorized patent (the name: a nano antibody fusion protein and a preparation method and application thereof, the patent number: CN 103333253A)

Example 1 NbTNF α Nanobody Key amino acid residue Studies

1. Determination of anti-TNF alpha Nanobody FR region

The FR regions of the anti-TNF alpha nano antibody are divided by adopting IMGT, after http:// www.imgt.org clicks IMGT/Collier-de-Perles, the amino acid sequence of the NbTNF alpha nano antibody is input into a search box, and CDRs and FRs regions are automatically divided on a website according to the number of the IMGT, as shown in Table 1.

TABLE 1 results of the partition of FR regions of anti-TNF α Nanobodies

Effect of the key amino acids at positions 42, 50 and 52 of the FR2 framework region on the structure of NbTNF alpha nanobody

1) Using a site-directed mutagenesis kit to obtain a single-point mutation plasmid and a combined optimized co-mutation plasmid (SEQ. ID. NO.1, 3, 5, 7, 9; seq 1Nb TNF alpha 42, Seq 3Nb TNF alpha 50, Seq 5Nb TNF alpha 52, Seq 7Nb TNF alpha 50-52, Seq 9NbTNF alpha 42-50-52), wherein said Seq 1Nb TNF alpha 42 refers to the 42-position amino acid mutation of Seq 1Nb TNF alpha, and the other nomenclature is the same as above.

2) The recombinant plasmid vectors are respectively linearized, 20 mu g of plasmid is adjusted to 44 mu L, then 1 mu L of Sac I enzyme digestion and 5 mu L of Buffer are added, enzyme digestion is carried out for 5h at 37 ℃, and 20 mu L of linearized plasmid is obtained by concentration.

3) Add 10. mu.l of linearized plasmid DNA (10. mu.g) to 80. mu.L of competent cells, mix gently, transfer to ice-precooled 0.2cm electroporation cuvette, place on ice for 10min, place the electroporation cuvette in the electroporation apparatus according to the electroporation parameters provided by Invitrogen (25. mu.F, 200. omega., 1.6kV,5ms), close the lid, shock. mu.L of the electrotransformation solution was applied to YPDS (100. mu.g/ml zeocin), and cultured in an incubator at 30 ℃ for 2 days or more until monoclonals appeared. Selecting monoclonal strain, boiling-freezing-boiling to crack the cell wall of the strain, and performing PCR identification on positive strain genome

4) Positive yeast strains were selected and inoculated in 20ml YPD medium and shaken overnight to serve as seed bottles. The next day, large 1% to 100ml flasks were inoculated to enrich the yeast for one day. Then, 1.5% methanol was added every day for 5 days. After induction, the supernatant was collected by centrifugation at 8000rpm for 15min in a high-speed refrigerated centrifuge.

5) Purifying protein by using a nickel ion affinity chromatography column: and (3) balancing the column by using a binding buffer solution, adding a supernatant which is filtered by a 0.45-micron microporous filter membrane when the buffer solution is drained, and washing away the unbound hybrid protein by using the binding buffer solution when the supernatant is about to drain. Finally, elution buffer solution containing 100mM imidazole is used for eluting the target protein, so that the nano antibody proteins (SEQ. ID. NO.2, 4, 6, 8, 10; Seq 2Nb TNF alpha 42, Seq 4Nb TNF alpha 50, Seq 6Nb TNF alpha 52, Seq8Nb TNF alpha 50-52, Seq 10NbTNF alpha 42-50-52) with different site amino acid mutations are obtained, the molecular weight of several mutant proteins is about 15kD (figure 1), eluent is collected and dialyzed by PBS, and the filtered eluent is stored at 80 ℃ below zero after being filtered by a 0.22 mu m sterile filter.

3. Indirect ELISA for determining different mutants and NbTNF alpha affinity constant

The enzyme-linked immunosorbent assay plate was coated with TNF-alpha at a concentration of 1. mu.g/ml and 2. mu.g/ml, incubated at 37 ℃ for 2 hours and washed 5 times with PBST. Incubate 2 hours with 3% BSA blocking solution at 37 ℃ and wash twice with PBST. NbTNF alpha nano antibody and different nano antibody mutants are diluted to 5ng/ml by 5 times, then added into each well of an enzyme label plate, incubated at 37 ℃ for 2 hours and washed 5 times by PBST. The primary antibody (anti-His-tagged rabbit antibody) was diluted 1000-fold, added to each well of the microplate, incubated at 37 ℃ for 2 hours, and washed 5 times with PBST. The secondary antibody (goat anti-rabbit) was added to the plate, incubated at 37 ℃ for 1 hour and washed 5 times with PBST. Adding TMB developing solution, incubating for 30min at 37 deg.C in dark, and immediately adding 2M sulfuric acid to terminate the reaction. Reading light absorption value by a microplate reader at the wavelength of 450nm, drawing a curve, and obtaining a Kaff ═ n-1)/2(n [ Ab']t-[Ab]t)。NbTNFα₄₂，NbTNFα₅₀，NbTNFα₅₂And affinity constant determination of the combinatorially optimized co-mutants are shown in figure 2 and table 2.

TABLE 2 Indirect ELISA method for NbTNF alpha and single mutant affinity

Example 2 NbTNF α Nanobody amino acid residue humanization Studies

1. Multiple sequence alignment

Human antibody germline gene sequences with higher homology to anti-TNF α nanobodies (antibody germline genes of the human VH gene family III) were automatically searched from the library using the IgBLAST program in the NCBI database and aligned for multiple sequences using MEGA5.0 software, as shown in fig. 3.

2. Generation of humanization protocol

The scheme for designing the humanized modification of the anti-TNF alpha nano antibody is as follows: 1) amino acid residues that cannot be substituted: f at position 42 cannot be replaced; residues adjacent to the outside of the CDR regions (typically two adjacent amino acid residues) tend to have a large effect on CDR conformation, including G at position 40 and AR at positions 54-55; 2) residues that do not require substitution: residues within the range of human antibody sequence variation, such as Q at position 1, L at position 5 and T at position 86; 3) residues that can be substituted: substitutions are contemplated which are unique to the human antibody sequence, including A15P, T24A, P45A, V87L, D92N, KP95-96RA, Q123L, and R50L and F52W (numbers represent amino acid positions, sequences from VHH to VH); 4) residues to be considered: simultaneous substitution of several consecutive residues may have some effect on CDR conformation, such as DEP at positions 68-70 and KAQ at positions 82-84, and because the effect of co-mutation of DEP at positions 68-70 and KAQ at positions 82-84 in FR3 region on CDR conformation is not determined, the humanization of anti-TNF α nanobodies does not take these several sites into account, as shown in table 3.

TABLE 3 summary of different residues in anti-TNF α Nanobodies and humanized antibody sequences

Construction, expression and purification of h-NbTNF alpha nano antibody yeast expression vector

1) H-NbTNF alpha gene sequence (SEQ. ID. NO.11) synthesized by Kingsley company is taken as a template, and a primer h-NbTNF alpha is used

FP 5' -CCGCTCGAGAAAAGAGAGGCTCAGGTGCAGCTGGTGGAG-3 and

PCR amplification was conducted under the guide of RP5 '-GCTCTAGATCAATGATGATGATGATGATGAGAGGAGACGGTGACCAGG-3', and the amplified band appeared around 400bp, which coincided with the expected target band 413bp (FIG. 4A). The humanized NbTNF alpha gene fragment and pPICZ alpha A plasmid are subjected to double digestion by endonucleases Xho I and Xba I at 37 ℃, the target product is recovered by gel cutting after agarose gel electrophoresis, the DNA fragments are connected at 16 ℃ for 16h to transform DH5 alpha, and positive clones are screened for bacterial liquid PCR (figure 4B).

2) The recombinant vector was linearized and then transformed into yeast GS115 competent cells. mu.L of the electrotransformation solution was applied to YPDS (100. mu.g/mL zeocin), and cultured in an incubator at 30 ℃ for 2 days or more until monoclonals appeared. The cell wall of a single clone strain is cracked by boiling-freezing-boiling, the PCR identification of the positive strain genome is carried out, a target band is determined to appear in 970bp, the result shows that the rest bands except No. 8 and No. 10 strains are all appeared in about 1000bp and are consistent with an expected band, the recombinant vector is integrated into the yeast genome and is a positive yeast strain (figure 4C), then the SDS-PAGE identification protein expression is carried out on the supernatant of 8 positive recombinant yeast strains induced for 5 days, the expression is found at 15kD, and the difference of the expression amount is not large (figure 5A).

3) And selecting a positive yeast strain, and performing induced expression and purification by the same method as the expression method in the first embodiment. Thus obtaining the humanized anti-NbTNF alpha nano antibody (SEQ. ID. NO.12), namely the protein molecular weight is about 15kD (figure 5B), collecting eluent, dialyzing the eluent by PBS, filtering the eluent by a 0.22 mu m sterile filter, and storing the filtered eluent at 80 ℃ below zero.

4) Indirect ELISA (enzyme linked immunosorbent assay) for measuring affinity constant of anti-TNF alpha nano antibody before and after humanization modification

The experiment was performed in the same manner as in example 1, and an antibody concentration-absorbance curve was plotted (FIG. 6), wherein the affinity constant of the anti-TNF α nanobody NbTNF α was measured to be 4.16nM, and the affinity constant of the humanized nanobody h-NbTNF α was measured to be 87.1nM, according to the affinity constant formula Kaff ═ n-1)/2(n [ Ab' ] t- [ Ab ] t ]. As in table 4.

TABLE 4 Indirect ELISA for NbTNF alpha and humanized NbTNF alpha affinities

Example 3 humanized Nanobody h-NbTNF α thermostability assay

Respectively diluting infliximab (positive control), NbTNF alpha and h-NbTNF alpha nano antibodies to the concentration of 500 mu g/ml by using PBS; incubating 3 antibodies at 25 deg.C, 50 deg.C, 60 deg.C, 65 deg.C, 70 deg.C, 80 deg.C and 90 deg.C for 1 hr, respectively, and cooling to room temperature of 25 deg.C, or incubating at 90 deg.C for 0, 15, 30, 45, 60, 75 and 90min, respectively, and cooling to room temperature; centrifuging at 5000g for 5min, discarding precipitate, measuring antibody concentration in supernatant with BCA kit, and diluting three antibodies to final concentration of 2 μ g/ml; the relative binding of infliximab, NbTNF α, humanized h-NbTNF α nanobody treated under different conditions was determined using a non-competitive ELISA method. With increasing temperature, infliximab activity decreased significantly. The original TNF alpha antibody activity is basically maintained, the humanized antibody activity starts to be reduced after 70 ℃, the activity is maintained above 60% (figure 7A), and the h-NbTNF alpha nano antibody can also maintain 70% of antigen binding capacity after being heated for 90min at 90 ℃ (figure 7B).

EXAMPLE 4 inhibitory Effect of humanized h-NbTNF α Nanobodies on hTNF α cytotoxicity

Taking L929 cells in logarithmic growth phase, preparing cell suspension, and adjusting cell density to 10 × 10⁴Inoculating each cell/ml into 96-well plate, culturing at 37 deg.C under 5% CO2 condition for 24 hr, wherein each well has 100 μ l; TNF alpha antigen was diluted to 500pg/ml with DMEM complete medium containing actinomycin D at 1. mu.g/ml, and 50. mu.l of TNF alpha at 500pg/ml was taken separately from 50. mu.l of TNF alpha at 500pg/mlThe antibody was mixed at concentration and incubated for 30 min. (NbTNF α was diluted from 5nM to 4.88pM, inflixine was diluted from 20nM to 9.77pM, h-NbTNF α was diluted from 50nM to 48.82pM, and IgG was diluted from 200nM to 97.625 pM). Setting a cell control group, a TNF alpha control group (TNF alpha + culture medium) and a blank control group at the same time, and incubating for 24 hours in an incubator at 37 ℃ and 100% relative humidity and 5% CO 2; adding 20 mul of 0.5 percent MTT into each hole, and continuing culturing for 4 h; stopping culturing, removing the culture medium by aspiration, adding 100 μ l DMSO into each well, shaking on a decolorization shaker for 10min to dissolve formazan in crystal state, and measuring absorbance at OD490nm of full-wavelength microplate reader; the experimental data are processed by Graphpad software (figure 8), the NbTNF alpha nano antibody and the positive drug infliximab can obviously inhibit L929 cytotoxicity induced by TNF alpha antigen, the IC50 NbTNF alpha of h-NbTNF alpha is increased by 20.5 times, but the h-NbTNF alpha has stronger capacity of combining with hTNF alpha antigen, dose dependence exists, and when the concentration of the h-NbTNF alpha is more than 6.25nM, the cytotoxicity neutralization rate can reach more than 90%, which shows that the h-NbTNF alpha is a relatively ideal humanized nano antibody.

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<400> 7

caggtgcagc tggtggagtc tggcggtggc ttggtgcaag ctggcggctc cctgagactg 60

tcctgtaccg cctctggaca aacaagcagc acggctgata tgggctggtt ccgccagcct 120

ccaggcaagg gcctggagtg ggtcgctaga attagcggca ttgacggtac cacctactac 180

gatgaaccgg tgaagggccg tttcaccatc tccagagaca aagcccaaaa caccgtgtat 240

ctgcaaatgg atagcctgaa gccggaggac acggctgtgt attactgtag aagccctcgt 300

tatgccgatc aatggagcgc ctatgattat tggggccaag gcacccaagt caccgtctcc 360

tct 363

<210> 8

<211> 121

<212> PRT

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 8

Gln 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 Thr Ala Ser Gly Gln Thr Ser Ser Thr Ala

20 25 30

Asp Met Gly Trp Phe Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ala Gln Asn Thr Val Tyr

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 9

<211> 363

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 9

caggtgcagc tggtggagtc tggcggtggc ttggtgcaag ctggcggctc cctgagactg 60

tcctgtaccg cctctggaca aacaagcagc acggctgata tgggctgggt gcgccagcct 120

ccaggcaagg gcctggagtg ggtcgctaga attagcggca ttgacggtac cacctactac 180

gatgaaccgg tgaagggccg tttcaccatc tccagagaca aagcccaaaa caccgtgtat 240

ctgcaaatgg atagcctgaa gccggaggac acggctgtgt attactgtag aagccctcgt 300

tatgccgatc aatggagcgc ctatgattat tggggccaag gcacccaagt caccgtctcc 360

tct 363

<210> 10

<211> 121

<212> PRT

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 10

Gln 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 Thr Ala Ser Gly Gln Thr Ser Ser Thr Ala

20 25 30

Asp Met Gly Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ala Gln Asn Thr Val Tyr

65 70 75 80

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

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 11

<211> 363

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 11

caggtgcagc tggtggagtc tggcggtggc ttggtgcaac ctggcggctc cctgagactg 60

tcctgtgctg cctctggaca aacaagcagc acggctgata tgggctggtt ccgccaggct 120

ccaggcaagg gcctggagtg ggtcgctaga attagcggca ttgacggtac cacctactac 180

gatgaaccgg tgaagggccg tttcaccatc tccagagaca aagcccaaaa caccctgtat 240

ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtag aagccctcgt 300

tatgccgatc aatggagcgc ctatgattat tggggccaag gcaccctggt caccgtctcc 360

tct 363

<210> 12

<211> 121

<212> PRT

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 12

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gln Thr Ser Ser Thr Ala

20 25 30

Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Ser Val

35 40 45

Ala Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Phe Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ala Gln Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

Claims

1. A humanized anti-TNF alpha nano antibody is characterized in that the nucleotide sequence of the humanized anti-TNF alpha nano antibody is shown in SEQ ID No. 11.

2. The humanized anti-TNF α nanobody of claim 1, wherein the amino acid sequence encoding the humanized anti-TNF α nanobody is shown in seq id No. 12.

3. The method of claim 1, comprising the steps of:

2) carrying out different combination mutation on key amino acids at positions 42, 50 and 52 of an FR2 framework region which has an effect on CDR3 conformation, wherein the nucleotide sequence of a mutant TNF alpha nano antibody is shown as SEQ ID No.1, 3, 5, 7 and 9, and the amino acid sequence is shown as SEQ ID No.2, 4, 6, 8 and 10, and determining that the co-mutation of the amino acids at positions 50 and 52 has little effect on the conformation;

3) carrying out sequence similarity search on the anti-TNF alpha nano antibody NbTNF alpha through IgBLAST, searching out a human antibody germ line gene sequence with higher sequence homology with the anti-TNF alpha nano antibody NbTNF alpha, and carrying out multiple sequence comparison on the anti-TNF alpha nano antibody and the human antibody germ line gene sequence by using MEGA5.0 software;

4) preparation of a humanization protocol: according to the analysis results of the steps 2) and 3), reasonable humanized design is carried out on the anti-TNF alpha nano antibody, and the design is carried out as follows:

4. the method for preparing a humanized anti-TNF alpha nanobody according to claim 3, wherein the humanized anti-TNF alpha nanobody is obtained by performing induction expression through a yeast expression vector and purifying.