Nanometer antibody targeting IL-23A and application thereof

Abstract

The invention discloses a nano antibody targeting IL-23A and application of the nano antibody, and relates to the technical field of biological medicines. The nano antibody VVH1 comprises a framework region and a complementarity determining region, the framework region comprises VVH1FR-H1, VVH1FR-H2, VVH1FR-H3 and VVH1FR-H4, and the amino acid sequences of the VVH1FR-H1, the VVH1FR-H2, the VVH1FR-H3 and the VVH1FR-H4 are respectively shown as SEQ ID NO.4-7; the complementarity determining region comprises VVH1CDR-H1, VVH1CDR-H2 and VVH1CDR-H3, wherein the amino acid sequences of the VVH1CDR-H1, the VVH1CDR-H2 and the VVH1CDR-H3 are respectively shown as SEQ ID NO.1-3. The nano antibody has high binding activity with IL-23A, has the characteristics of small molecular weight, good stability, good tissue wettability, weak immunogenicity and the like compared with a traditional antibody, and can be applied to preparation of an IL-23A-targeted antibody drug so as to treat related diseases through combination with IL-23A.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a nano antibody targeting IL-23A and applications thereof. Background Art

[0002] IL-23 is a member of the IL-12 cytokine family. It is a heterodimeric pro-inflammatory cytokine composed of two subunits, IL-23A (p19) and IL-12 / 23B (shares p40 with IL-12), which are bound by covalent disulfide bonds. In 2003, a study found that the two subunits of IL-23 have different functions. IL-23A is mainly produced by macrophages and dendritic cells. It has also been shown to coordinate the activity 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 one variable region and three constant regions, and the light chain consists of one variable region and one constant region. In 1993, Belgian scientist Hamers-Casterman and his team first reported a heavy chain antibody with a natural lack of light chain found in the blood of camelids. It contains only one heavy chain variable region (VHH) and two conventional constant regions. Cloning its variable region can obtain a single-domain antibody composed only of the heavy chain variable region, called Nanobodies (Nbs).

[0004] As a new type of antibody, nanoantibodies have biological characteristics such as small size and molecular weight, strong tissue permeability, and weak immunogenicity. They also have the characteristics of low production cost, high yield, and can be mass-produced and expressed in a variety of microbial systems without the need for post-translational modification. They can also specifically recognize some hidden antigen epitopes and other application advantages, which lay a good foundation for their clinical development or industrial production.

[0005] Based on the above, IL-23A can be used as a potential target for specific treatment of immune diseases. However, clinical drug research targeting this target is still very limited. Therefore, it is of great significance to develop new antibodies targeting this target. Summary of the invention

[0006] The purpose of the present invention is to provide a nano antibody targeting IL-23A and its application to solve the problems existing in the above-mentioned prior art. The nano antibody has high binding activity with IL-23A, small molecular weight, good stability, and has the characteristics of good tissue infiltration and weak immunogenicity compared with traditional antibodies, and can be used to prepare 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, wherein the nanobody VVH1 comprises a framework region and a complementary determining region; the framework region comprises VVH1 FR-H1, VVH1 FR-H2, VVH1 FR-H3 and VVH1 FR-H4, whose amino acid sequences are respectively shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7; the complementary determining region comprises VVH1 CDR-H1, VVH1 CDR-H2 and VVH1 CDR-H3, whose amino acid sequences are respectively shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.

[0009] The present invention also provides a gene encoding 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 encoding gene, recombinant vector or recombinant host cell in the preparation of the above-mentioned nanobody VVH1.

[0013] The present invention also provides a use of the nano antibody 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 includes the above-mentioned nano antibody VVH1.

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

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

[0017] After extensive research, the present invention screened and obtained the nano antibody VVH1 targeting IL-23A. The nano antibody has high binding activity with IL-23A. Compared with traditional antibodies, it has the characteristics of small molecular weight, good stability, good tissue infiltration and weak immunogenicity. It can be used to prepare antibody drugs targeting IL-23A, and then treat related diseases (such as inflammatory diseases or autoimmune diseases) by binding to IL-23A. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The results of sedimentation equilibrium experiments of nanobody VHH1 binding to IL-23A;

[0020] Figure 2 It is a prediction analysis diagram of the nanobody binding to the IL-23A epitope; wherein A is a schematic diagram of the binding of the nanobody VHH1 to the truncated protein EpitopeA; B is a schematic diagram of the binding of the nanobody VHH1 to the truncated protein Epitope B;

[0021] Figure 3 Schematic diagram of the complex structure of the nanoantibody VHH1 docked with IL-23A; yellow represents IL-23A, purple represents the antigen epitope region that the antibody is predicted to bind to, pure white represents the nanoantibody, and the red, green and blue regions on the antibody correspond to the CDR-H1, CDR-H2 and CDR-H3 regions of the antibody, respectively. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] A 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 Nanobodies

[0032] According to the general formula of nanoantibodies combined with mutation treatment, a variety of nanoantibodies were designed, expressed and purified. These nanoantibodies 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 nanobody is as follows:

[0034] CDR-H1, which has the following structure: LRLSCAASGX1X2X3X4X5X6X7;

[0035] CDR-H2 has the following structure: IX8X9X 10 GX 11 -T;

[0036] CDR-H3 has the following structure: 12 AX 13 X 14 X 15 X 16 X 17 X 18 X 19 --X 20 X 21 X 22 X 23 DY;

[0037] in,

[0038] X1 is selected from the group consisting of (i) amino acid residues F, R, S and (ii) amino acid residues that are conservative substitutions relative to (i);

[0039] X2 is selected from (i) amino acid residues I, T and (ii) an amino acid residue that is a conservative substitution relative to (i);

[0040] X3 is selected from the group consisting of (i) amino acid residues F, Y, L and (ii) amino acid residues that are conservative substitutions relative to (i);

[0041] X4 is selected from the group consisting of (i) amino acid residues T, R, S and (ii) amino acid residues that are conservative substitutions relative to (i);

[0042] X5 is selected from the group consisting of (i) amino acid residues L, T, G, F and (ii) amino acid residues that are conservative substitutions relative to (i);

[0043] X6 is selected from (i) amino acid residues N, Y and (ii) amino acid residues that are conservative substitutions relative to (i);

[0044] X7 is selected from the group consisting of (i) amino acid residues T, E, F and (ii) amino acid residues that are conservative substitutions relative to (i);

[0045] X8 is selected from the group consisting of (i) amino acid residues T, N, S and (ii) amino acid residues that are conservative substitutions relative to (i);

[0046] X9 is selected from (i) amino acid residues I, W, S and (ii) amino acid residues that are conservative substitutions relative to (i);

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

[0048] X 11 An amino acid residue selected from (i) G, I, S and (ii) a conservative substitution relative to (i);

[0049] X 12 An amino acid residue selected from (i) amino acid residues K, A, N and (ii) an amino acid residue that is a conservative substitution relative to (i);

[0050] X 13 An amino acid residue selected from (i) amino acid residues D, R, E and (ii) an amino acid residue that is a conservative substitution relative to (i);

[0051] X 14 is 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 An amino acid residue selected from (i) amino acid residues M, I, S and (ii) an amino acid residue that is a conservative substitution 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 is selected from (i) amino acid residues T, I and (ii) amino acid residues that are conservative substitutions relative to (i);

[0057] X 20 An amino acid residue selected from (i) amino acid residues M, P, T and (ii) an amino acid residue that is a conservative substitution relative to (i);

[0058] X 21 An amino acid residue selected from (i) R, A, N and (ii) a conservative substitution relative to (i);

[0059] X 22 An amino acid residue selected from (i) T, L, Q, R and (ii) a conservative substitution relative to (i);

[0060] X 23 The amino acid residue is 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 specific binding nanobodies

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

[0063] 3. Binding free energy calculation

[0064] In order to determine the binding free energy between nano antibody VHH1 and IL-23A, the MM-PBSA method was used for calculation. The binding affinity of the two was calculated. Molecular docking can predict the main binding mode of antibodies and IL-23A at the atomic level. The interaction between the two usually includes hydrogen bonds, salt bridges and π-π stacking. The binding energy of the nano antibody VVH1 prepared by the present invention is -89.47866 kcal / mol, which can prove that the antibody and antigen bind stably.

[0065] 4. Sedimentation equilibrium experiment

[0066] Sedimentation equilibrium experiments showed that the molecular weights of the analogs were close to the calculated values ​​of the cross-linked double-chain monomers ( Figure 1 ), confirming the absence of oligomerization.

[0067] 5. Confirmation of Binding Sites

[0068] In order to confirm the binding site of the nanobody with IL-23A, the existing IL-23A plasmid in the laboratory was used as a template to express the IL-23A truncated protein using the E. coli prokaryotic expression system. First, IL-23A was truncated into two segments from the middle, and primers were designed to construct recombinant plasmids for prokaryotic expression of the two truncated proteins (Epitope A and Epitope B). Western blotting reactions were performed with the nanobody VVH1 to determine the binding affinity of the nanobody VVH1 to different epitopes of IL-23A. The results are shown in Figure 2 .

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

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

[0071] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all 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 VVH1 CDR-H1, VVH1 CDR-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.

6. Use of the nanobody VVH1 as claimed in claim 1 in the preparation of an antibody drug targeting IL-23A.

7. An antibody drug targeting IL-23A, characterized in that: The active ingredient comprises the nanobody VVH1 described in claim 1.

8. The antibody drug according to claim 7, characterized in that The antibody drug also includes pharmaceutically acceptable excipients.

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