Nanobody targeting il-23a and use thereof

Abstract

The present invention belongs to the technical field of biomedicine. Provided are a nanobody targeting IL-23A and the use thereof. The nanobody comprises a framework region and a complementarity determining region, wherein the framework region comprises FR-H1, FR-H2, FR-H3 and FR-H4 having amino acid sequences as shown in SEQ ID NOs. 4-7, respectively; and the complementary determining region comprises CDR-H1, CDR-H2 and CDR-H3 having amino acid sequences as shown in SEQ ID NOs. 1-3, respectively. The nanobody can be used in the preparation of an antibody drug targeting IL-23A, which is capable of treating related diseases by means of binding to IL-23A.

Description

A nanobody targeting IL-23A and its application Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a nanobody targeting IL-23A and its application. Background Technology

[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 (which shares p40 with IL-12), linked by covalent disulfide bonds. A 2003 study found that the two subunits of IL-23 have different functions. IL-23A is mainly produced by macrophages and dendritic cells and 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 consist of two identical heavy chains and two identical light chains. The heavy chain comprises one variable region and three constant regions, while the light chain comprises one variable region and one constant region. In 1993, Belgian scientist Hamers-Casterman and his team first reported the discovery of a naturally occurring heavy chain antibody lacking the light chain in the blood of camels. This antibody contains only one heavy chain variable region (VHH) and two conventional constant regions. Cloning its variable region yields single-domain antibodies consisting only of the heavy chain variable region, called nanobodies (Nbs).

[0004] Nanobodies, as a novel type of antibody, possess biological characteristics such as small size and molecular weight, strong tissue permeability, and weak immunogenicity. They also have advantages such as low production cost, high yield, large-scale production and expression in various microbial systems, and no need for post-translational modification. Furthermore, they can specifically recognize some hidden antigenic epitopes, laying a good foundation for their clinical development or industrial production.

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

[0006] The purpose of this invention is to provide a nanobody targeting IL-23A and its application, thereby addressing the problems existing in the prior art. This nanobody exhibits high binding activity to IL-23A, and possesses a small molecular weight and good stability. Compared to traditional antibodies, it has advantages such as good tissue infiltration and weak immunogenicity, and can be used to prepare antibody drugs targeting IL-23A.

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

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

[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-described encoding gene.

[0011] The present invention also provides a recombinant host cell comprising the above-described 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 the application of the above-mentioned nanobody VVH1 in the preparation of antibody drugs targeting IL-23A.

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

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

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

[0017] Through extensive research and screening, this invention has obtained the nanobody VVH1 that targets IL-23A. This nanobody has high binding activity to IL-23A and, compared with traditional antibodies, has the characteristics of small molecular weight, good stability, good tissue infiltration and weak immunogenicity. It can be used to prepare antibody drugs that target IL-23A, and then treat related diseases (such as inflammatory diseases or autoimmune diseases) by binding to IL-23A. Attached Figure Description

[0018] 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.

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

[0020] Figure 2 shows the predicted analysis of nanobody binding to the IL-23A epitope; where A is a schematic diagram of the binding of nanobody VHH1 to truncated protein EpitopeA; and B is a schematic diagram of the binding of nanobody VHH1 to truncated protein EpitopeB.

[0021] Figure 3 is a schematic diagram of the complex structure of the nanobody VHH1 docked with IL-23A; in the figure, yellow represents IL-23A, purple-red represents the antigenic epitope region that the antibody is predicted to bind to, pure white represents the nanobody, and red, green and blue regions are marked on the antibody, which correspond to the CDR-H1, CDR-H2 and CDR-H3 regions of the antibody, respectively. Detailed Implementation

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

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

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

[0028] Table 1 Sequence Information

[0029] Describing sequences: CDR-H1 general formula LRLSCAASGX1X2X3X4X5X6X7; CDR-H2 general formula IX8X9X. 10 GX 11 -TCDR-H3 General Formula X 12 AX 13 X 14 X 15 X 16 X 17 X 18 X 19 --X 20 X 21 X 22 X 23 DYVHH1 CDR-H1LRLSCATSAFIFRLNT(SEQ ID NO.1)VHH1 CDR-H2VTIDGST(SEQ ID NO.2)VHH1 CDR-H3KADRRIMMRTYDY(SEQ ID NO.3)VHH1 FR-H1EVQLVESGGGLVQAGGSLRLSCATS(SEQ ID NO.4)VHH1 FR-H2MGWYRQAPGKQRELVAM(SEQ ID NO.5)VHH1 FR-H3NYADSVKGRFTISRDSTKNTMYLQMNSLKPEDTAVYYC(SEQ ID NO.6)VHH1 FR-H4WGPGTQVTVSSEPKTPKPQP(SEQ ID NO.7) VHH1 amino acid sequence EVQLVESGGGLVQAGGSLRLSCATSAFIFRLNTMGWYRQAPGKQRELVAMVTIDGSTNYADSVKGRFTISRDSTKNTMYLQMNSLKPEDTAVYYCKADRRIMMRTYDYWGPGTQVTVSSEPKTPKPQP (SEQ ID NO.8)

[0030] Example 1

[0031] 1. Preparation of Nanobodies

[0032] Based on the general formula of nanobodies, various nanobodies were designed, expressed, and purified. These nanobodies consist of four framework regions (FRs) and three complementarity-determining regions (CDRs), and are named FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, and FR-H4.

[0033] The general formula for nanobodies is as follows:

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

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

[0036] CDR-H3 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 X 23 DY;

[0037] in,

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

[0039] X2 is selected from amino acid residue I (i), and amino acid residues T and (ii) are conserved substitutions relative to (i).

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

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

[0042] X5 is selected from (i) amino acid residues L, T, G, F and (ii) amino acid residues that are conserved substitutions relative to (i).

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

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

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

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

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

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

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

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

[0051] X 14 The amino acid residues selected from (i) are amino acid residues R and G and (ii) are amino acid residues that are conserved substitutions relative to (i);

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

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

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

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

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

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

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

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

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

[0061] 2. Screening of nanobodies that specifically bind to the target antibody

[0062] The reactivity of purified nanobodies with the important functional protein IL-23A was detected by ELISA. Nanobodies with positive ELISA results and high OD values ​​were selected. Positive clones were subjected to bacterial culture PCR, and the plasmids of 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 one nanobodies that can specifically react with IL-23A was obtained and named VVH1. The amino acid sequence is shown in Table 1.

[0063] 3. Combined with free energy calculation

[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 modes of the antibody and IL-23A at the atomic level. The interactions between the two typically include hydrogen bonding, salt bridging, and π-π stacking. The binding energy of the nanobody VVH1 prepared in this invention is -89.47866 kcal / mol, which demonstrates the stability of the antibody-antigen binding.

[0065] 4. Settlement equilibrium experiment

[0066] Sedimentation equilibrium experiments showed that the molecular weights of the analogues were close to the calculated values ​​of the cross-linked double-chain monomers (Figure 1), confirming that there was no oligomerization.

[0067] 5. Confirmation of binding sites

[0068] To confirm the binding sites of the nanobody and IL-23A, a truncated IL-23A protein was expressed using an existing IL-23A plasmid as a template and the E. coli prokaryotic expression system. First, IL-23A was truncated into two segments. Primers were designed to construct a recombinant plasmid for prokaryotic expression of the two truncated protein segments (Epitope A and Epitope B). These segments were then subjected to Western blotting 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 and spatial distribution of key sites of nanobodies and IL-23A

[0070] To more intuitively display the identified epitopes in spatial structure, AlphaFold2 was used to perform homology modeling on the nanobody and IL-23A. The modeled data was then processed and analyzed to show 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 merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined 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 complementation-determining region; the framework region includes amino acid sequences VVH1 FR-H1, VVH1 FR-H2, VVH1 FR-H3, and VVH1 FR-H4 as shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, respectively; the complementation-determining region includes amino acid sequences VVH1CDR-H1, VVH1CDR-H2, and VVH1 CDR-H3 as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

2. The encoding gene of the nanobody VVH1 as described in claim 1.

3. A recombinant vector, characterized in that, Includes the coding gene as described in claim 2.

4. A recombinant host cell, characterized in that, Includes the recombinant vector as described in claim 3.

5. The use of the encoding gene as described in claim 2, the recombinant vector as described in claim 3, or the recombinant host cell as described in claim 4 in the preparation of the nanobody VVH1 as described in claim 1.

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