A nano antibody targeting IL-23A and its application

Abstract

The present invention discloses a nano antibody targeting IL-23A and its application, and relates to the field of biomedical technology. The nano antibody VVH1 includes a framework region and a complementary determining region; the framework region includes VVH1FR-H1, VVH1FR-H2, VVH1FR-H3 and VVH1FR-H4 whose amino acid sequences are respectively shown in SEQ ID NO.4-7; the complementary determining region includes VVH1CDR-H1, VVH1CDR-H2 and VVH1CDR-H3 whose amino acid sequences are respectively shown in SEQ ID NO.1-3. The nano antibody has high binding activity with IL-23A, and has the characteristics of small molecular weight, good stability, good tissue infiltration and weak immunogenicity compared with traditional antibodies. It can be used to prepare antibody drugs targeting IL-23A, and then treat related diseases by binding with IL-23A.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a nanobody targeting IL-23A and its applications. Background Art

[0002] IL-23 is a member of the IL-12 cytokine family, and is a heterodimeric pro-inflammatory cytokine composed of two subunits, IL-23A (p19) and IL-12 / 23B (sharing p40 with IL-12), which are covalently bound by disulfide bonds. In 2003, studies found that the two subunits of IL-23 have different functions. Among them, IL-23A is mainly produced by macrophages and dendritic cells, and has also been shown to coordinate the activities of immune cells and play a key role in the pathogenesis of immune inflammatory diseases.

[0003] Traditional antibodies are composed of two identical heavy chains and two identical light chain polypeptides. The heavy chain consists of 1 variable region and 3 constant regions, and the light chain consists of 1 variable region and 1 constant region. In 1993, Belgian scientist Hamers-Casterman and his team first reported a heavy chain antibody naturally lacking a light chain found in the blood of camelids. It only contains one variable region of the heavy chain (VHH) and two conventional constant regions. Cloning its variable region can obtain a single-domain antibody composed only of the variable region of the heavy chain, called a nanobody (Nanobodies, Nbs).

[0004] As a new type of antibody, nanobodies have biological characteristics such as small size and molecular weight, strong tissue permeability, and weak immunogenicity. They also have production characteristics such as low production cost, high yield, being able to be mass-produced and expressed in a variety of microbial systems, and not requiring post-translational modification. Moreover, they have application advantages such as being able to specifically recognize some hidden antigenic epitopes, laying a good foundation for their clinical development or industrial production.

[0005] Based on the above content, IL-23A can be a potential target for specific treatment of immune diseases. However, current clinical research on drugs targeting this target is still very limited. Therefore, developing new antibodies targeting this target is of great significance. Summary of the Invention

[0006] The object of the present invention is to provide a nanobody targeting IL-23A and its applications to solve the problems existing in the above-mentioned prior art. This nanobody has high binding activity with IL-23A, and has characteristics such as small molecular weight, good stability, good tissue infiltration, and weak immunogenicity compared with traditional antibodies, and can be used in the preparation of antibody drugs targeting IL-23A.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a nanobody VVH1 targeting IL-23A. The nanobody VVH1 includes a framework region and complementarity-determining regions. The framework region includes VVH1 FR-H1, VVH1 FR-H2, VVH1 FR-H3, and VVH1 FR-H4 with amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7 respectively. The complementarity-determining regions include VVH1 CDR-H1, VVH1 CDR-H2, and VVH1 CDR-H3 with amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 respectively.

[0009] The present invention also provides a coding gene for the above-mentioned nanobody VVH1.

[0010] The present invention also provides a recombinant vector comprising the above-mentioned coding gene.

[0011] The present invention also provides a recombinant host cell comprising the above-mentioned recombinant vector.

[0012] The present invention also provides the use of the above-mentioned coding gene, recombinant vector or recombinant host cell in the preparation of the above-mentioned nanobody VVH1.

[0013] The present invention also provides the use of the above-mentioned nanobody VVH1 in the preparation of an antibody drug targeting IL-23A.

[0014] The present invention also provides an antibody drug targeting IL-23A, the active ingredient of which comprises the above-mentioned nanobody VVH1.

[0015] Furthermore, the antibody drug also comprises pharmaceutically acceptable excipients.

[0016] The present invention discloses the following technical effects:

[0017] Through a large number of studies, the present invention has screened and obtained the nanobody VVH1 targeting IL-23A. This nanobody has high binding activity with IL-23A and has characteristics such as small molecular weight, good stability, good tissue infiltration, and weak immunogenicity compared with traditional antibodies. It can be used in the preparation of antibody drugs targeting IL-23A, and further treat related diseases (such as inflammatory diseases or autoimmune diseases) by binding to IL-23A. Description of the Drawings

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0019] Figure 1 It is the sedimentation equilibrium experimental result of the binding of nanobody VHH1 to IL-23A;

[0020] Figure 2 It is the predicted analysis diagram of the epitope where the nanobody binds to IL-23A; among them, A is the schematic diagram of the binding of nanobody VHH1 to the truncated protein EpitopeA; B is the schematic diagram of the binding of nanobody VHH1 to the truncated protein Epitope B;

[0021] Figure 3 It is the schematic diagram of the complex structure of the docking of nanobody VHH1 to IL-23A; among them, yellow is IL-23A, magenta is the antigen epitope region where the predicted antibody may bind, pure white is the nanobody, and the regions marked with red, green, and blue on the antibody correspond to the CDR-H1, CDR-H2, and CDR-H3 regions of the antibody respectively. Detailed implementation manners

[0022] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0024] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0025] Without departing from the scope or spirit of the present invention, various modifications and variations to the specific embodiments of the description of the present invention will be apparent to those skilled in the art. Other embodiments obtained from the description of the present invention will be apparent to those skilled in the art. The description and examples of the present invention are merely exemplary.

[0026] Regarding the use of "comprising", "including", "having", "containing", etc. in this text, they are all open-ended terms, meaning including but not limited to.

[0027] The description of the sequences involved in this application is provided in Table 1.

[0028] Table 1 Sequence Information

[0029]

[0030] Example 1

[0031] 1. Preparation of Nanobody

[0032] According to the general formula of the nanobody combined with mutagenesis treatment, a variety of nanobodies were designed, expressed and purified. These nanobodies are composed of 4 framework regions (FRs) and 3 complementarity-determining regions (CDRs), called FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3 and FR-H4.

[0033] Among them, the general formula of the nanobody is as follows:

[0034] CDR-H1, which has the following structure: LRLSCAASGX 1 X 2 X 3 X 4 X 5 X 6 X 7 ;

[0035] CDR-H2, which has the following structure: IX 8 X 9 X 10 GX 11 -T;

[0036] CDR-H3, which has the following structure: X 12 AX 13 X 14 X 15 X 16 X 17 X 18 X 19 --X 20 X 21 X 22 X23 DY;

[0037] wherein,

[0038] X 1 is selected from (i) the amino acid residues F, R, S and (ii) amino acid residues that are conservative substitutions relative to (i);

[0039] X 2 is selected from (i) the amino acid residues I, T and (ii) amino acid residues that are conservative substitutions relative to (i);

[0040] X 3 is selected from (i) the amino acid residues F, Y, L and (ii) amino acid residues that are conservative substitutions relative to (i);

[0041] X 4 is selected from (i) the amino acid residues T, R, S and (ii) amino acid residues that are conservative substitutions relative to (i);

[0042] X 5 is selected from (i) the amino acid residues L, T, G, F and (ii) amino acid residues that are conservative substitutions relative to (i);

[0043] X 6 is selected from (i) the amino acid residues N, Y and (ii) amino acid residues that are conservative substitutions relative to (i);

[0044] X 7 is selected from (i) the amino acid residues T, E, F and (ii) amino acid residues that are conservative substitutions relative to (i);

[0045] X 8 is selected from (i) the amino acid residues T, N, S and (ii) amino acid residues that are conservative substitutions relative to (i);

[0046] X 9 is selected from (i) the amino acid residues I, W, S and (ii) amino acid residues that are conservative substitutions relative to (i);

[0047] X 10 is selected from (i) the amino acid residues D, R, S and (ii) amino acid residues that are conservative substitutions relative to (i);

[0048] X 11 is selected from (i) the amino acid residues G, I, S and (ii) amino acid residues that are conservative substitutions relative to (i);

[0049] X 12 is selected from (i) the amino acid residues K, A, N and (ii) amino acid residues that are conservative substitutions relative to (i);

[0050] X 13 Selected from (i) amino acid residues D, R, E and (ii) amino acid residues that are conservative substitutions relative to (i);

[0051] X 14 Selected from (i) amino acid residues R, G and (ii) amino acid residues that are conservative substitutions relative to (i);

[0052] X 15 Selected from (i) amino acid residues F, R, W and (ii) amino acid residues that are conservative substitutions relative to (i);

[0053] X 16 Selected from (i) amino acid residues I, G, E and (ii) amino acid residues that are conservative substitutions relative to (i);

[0054] X 17 Selected from (i) amino acid residues M, I, S and (ii) amino acid residues that are conservative substitutions relative to (i);

[0055] X 18 Selected from (i) amino acid residues L, R and (ii) amino acid residues that are conservative substitutions relative to (i);

[0056] X 19 Selected from (i) amino acid residues T, I and (ii) amino acid residues that are conservative substitutions relative to (i);

[0057] X 20 Selected from (i) amino acid residues M, P, T and (ii) amino acid residues that are conservative substitutions relative to (i);

[0058] X 21 Selected from (i) amino acid residues R, A, N and (ii) amino acid residues that are conservative substitutions relative to (i);

[0059] X 22 Selected from (i) amino acid residues T, L, Q, R and (ii) amino acid residues that are conservative substitutions relative to (i);

[0060] X 23 Selected from (i) amino acid residues Y, P, Q and (ii) amino acid residues that are conservative substitutions relative to (i).

[0061] 2. Screening of Nanobodies with Specific Binding

[0062] The reactivity of the purified nanobody with the important functional protein of IL-23A was detected by ELISA. The nanobodies with positive ELISA results and higher OD values were selected. The positive clones were subjected to colony PCR, and the plasmids of the nanobodies with positive PCR results were extracted and sequenced. The sequencing results are shown in Table 1. After analyzing the sequencing results by DNAMAN, the amino acid sequence of a nanobody that can specifically react with IL-23A was obtained, which was named VVH1, and the amino acid sequence is shown in Table 1.

[0063] 3. Calculation of binding free energy

[0064] To determine the binding free energy between the nanobody VHH1 and IL-23A, the MM-PBSA method was used for calculation. The binding affinity between the two was calculated. Molecular docking can predict the main binding mode between the antibody and IL-23A at the atomic level. The interactions between the two usually include hydrogen bonds, salt bridges, and π-π stacking, etc. The binding energy of the nanobody VVH1 prepared in the present invention is -89.47866 kcal / mol, which can prove the stable binding of the antibody and the antigen.

[0065] 4. Sedimentation equilibrium experiment

[0066] The sedimentation equilibrium experiment showed that the molecular weights of the analogs were all close to the calculated values of the cross-linked double-stranded monomers ( Figure 1 ), confirming that there was no oligomerization.

[0067] 5. Confirmation of binding sites

[0068] To confirm the binding site of the nanobody to IL-23A, the truncated protein of IL-23A was expressed using the Escherichia coli E. coil prokaryotic expression system with the existing IL-23A plasmid in the laboratory as a template. First, IL-23A was truncated into two equal segments in the middle. After designing primers to construct recombinant plasmids to prokaryotically express the two truncated proteins (Epitope A and Epitope B), they were respectively subjected to Western blotting reaction with the nanobody VVH1 to determine the binding ability of the nanobody VVH1 to different epitopes of IL-23A. The results are shown in Figure 2 .

[0069] 6. Overall spatial structure of the nanobody and IL-23A and spatial distribution of key sites

[0070] To more intuitively display the identified epitopes in the spatial structure, AlphaFold2 was used to perform homology modeling on the nanobody and IL-23A, and the modeled data was processed and analyzed to display the overall spatial structure of the nanobody VVH1 and IL-23A and the spatial distribution of key sites. The results are shown in Figure 3 .

[0071] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the spirit of the present invention's design, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A nanobody VVH1 targeting IL-23A, characterized in that: The nanobody VVH1 includes a framework region and a complementary determining region; the framework region includes VVH1 FR-H1, VVH1 FR-H2, VVH1 FR-H3 and VVH1 FR-H4 whose amino acid sequences are shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively; the complementary determining region includes VVH1CDR-H1, VVH1CDR-H2 and VVH1 CDR-H3 whose amino acid sequences are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

2. A gene encoding the Nanobody VVH1 as claimed in claim 1.

3. A recombinant vector, characterized in that: Comprising the coding gene described in claim 2.

4. A recombinant host cell, characterized in that Comprising the recombinant vector according to claim 3.

5. Use of the encoding gene according to claim 2, the recombinant vector according to claim 3 or the recombinant host cell according to claim 4 in the preparation of the nanobody VVH1 according to claim 1.

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