阅读说明：本技术 一种人源化纳米抗体及其制备方法 (Humanized nano antibody and preparation method thereof ) 是由 纪雪梅 刘煜 赵明军 于 2019-09-30 设计创作，主要内容包括：本发明涉及生物医药领域,具体公开了一种抗TNFα纳米抗体的人源化改造方法,本发明公开的人源化改造的抗TNFα纳米抗体核苷酸序列如SEQ.ID.NO.11所示,氨基酸序列如SEQ.ID.NO.12；其改造方法包括以下步骤：确定FR2区三种关键氨基酸单突变与联合突变,核苷酸序列如SEQ.ID.NO.1,3,5,7,9所示,氨基酸序列如SEQ.ID.NO.2,4,6,8,10所示；进一步多重序列对比确定人源化方案；通过上述得到人源化抗TNFα纳米抗体；经酵母表达系统表达。本发明改造的人源化抗TNFα纳米抗体,能保留较高的抗原亲和力,热稳定性以及生物活性,为今后人源化纳米抗体的疾病治疗药物的开发奠定基础。(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.)

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:

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

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.

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)