Antibody targeting FOXA3 and application thereof

Abstract

The invention discloses an antibody targeting FOXA3 and an application of the antibody. The antibody or the antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of which the amino acid sequences are respectively shown as SEQ ID NO: 1, SEQ ID NO: 2 and MDY; the light chain variable region comprises an LCDR1, an LCDR2 and an LCDR3, wherein the amino acid sequences of the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7. The anti-FOXA3 monoclonal antibody can be used for detecting the expression level of FOXA3 protein in cells in vitro, is good in specificity and high in antibody concentration and purity, can be used for analyzing the expression level of FOXA3 protein in protein or tissue in vitro, and has good stability.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an antibody targeting FOXA3 and an application thereof. Background Art

[0002] FOXA3, the full name of forkhead box A3, is a member of the forkhead class of DNA-binding proteins. FOXA3 is a member of the hepatocyte nuclear transcription factor family and plays a key role in metabolic homeostasis. It is a transcriptional activator of liver-specific transcripts (such as albumin and transthyretin) and interacts with chromatin. [1] In the process of transdifferentiation of fibroblasts into hepatocytes, FOXA3 participates as an important transcription factor, which is essential for the acquisition of drug metabolism and protein expression capabilities of hepatocytes. Currently, there are few types of FOXA3 antibodies on the market, which cannot meet market demand. Summary of the invention

[0003] The technical problem to be solved by the present invention is to overcome the technical problem that the prior art lacks a stable FOXA3 antibody with high sensitivity and good specificity, and to provide an antibody targeting FOXA3 and its application. The anti-FOXA3 monoclonal antibody of the present invention can be used to detect the expression level of FOXA3 protein in cells in vitro, has good specificity, high antibody concentration and purity, can be used to analyze the expression level of FOXA3 protein in proteins or tissues in vitro, and has good stability.

[0004] The present invention solves the above technical problems through the following technical solutions.

[0005] The first aspect of the present invention provides an antibody or an antigen-binding fragment thereof targeting FOXA3, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and MDY, respectively; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively.

[0006] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:3 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:3.

[0007] In some embodiments of the present invention, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:8 or is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:8.

[0008] In some embodiments of the present invention, the antibody is a full-length antibody, Fab, Fab', F(ab')2, scFv, Fv or a multispecific antibody.

[0009] In the present invention, when the antibody is a full-length antibody, the heavy chain constant region of the full-length antibody is derived from the heavy chain of a human antibody or a variant thereof, and the light chain constant region of the full-length antibody is derived from the κ chain or λ chain of a human antibody or a variant thereof.

[0010] In some specific embodiments of the present invention, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:23 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:23.

[0011] In some specific embodiments of the present invention, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:25 or is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:25.

[0012] The second aspect of the present invention provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof as described in the first aspect.

[0013] In some specific embodiments of the present invention, the nucleotide sequence encoding the HCDR1 is shown as SEQ ID NO: 10, the nucleotide sequence encoding the HCDR2 is shown as SEQ ID NO: 11, and the nucleotide sequence encoding the HCDR3 is ATGGACTAC.

[0014] In some specific embodiments of the present invention, the nucleotide sequence encoding the LCDR1 is shown as SEQ ID NO:14, the nucleotide sequence encoding the LCDR2 is shown as SEQ ID NO:15, and the nucleotide sequence encoding the LCDR3 is shown as SEQ ID NO:16.

[0015] In some examples of the present invention, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:12.

[0016] In some examples of the present invention, the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:17.

[0017] The third aspect of the present invention provides a recombinant expression vector, which comprises the nucleic acid as described in the second aspect.

[0018] In some embodiments of the present invention, the recombinant expression vector is a plasmid, cosmid, phage or viral vector.

[0019] In some specific embodiments of the present invention, the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

[0020] The fourth aspect of the present invention provides a cell, wherein the cell comprises the recombinant expression vector as described in the third aspect or the nucleic acid as described in the second aspect; or expresses the antibody or antigen-binding fragment thereof as described in the first aspect.

[0021] In some embodiments of the present invention, the cell is a prokaryotic cell or a eukaryotic cell.

[0022] In some preferred embodiments of the present invention, the cell is selected from yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof; the mammalian cell is, for example, HEK293 cell.

[0023] The fifth aspect of the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof targeting FOXA3, the method comprising culturing the cell as described in the fourth aspect, and obtaining the antibody or the antigen-binding fragment thereof targeting FOXA3 from the culture.

[0024] A sixth aspect of the present invention provides a kit, comprising the antibody or antigen-binding fragment thereof as described in the first aspect.

[0025] In some embodiments of the present invention, the kit further comprises a reagent for detecting the binding of the antibody or antigen-binding fragment thereof to an antigen.

[0026] The seventh aspect of the present invention provides a method for detecting FOXA3, comprising the step of contacting the antibody or antigen-binding fragment thereof as described in the first aspect with a sample to be tested.

[0027] In some embodiments of the present invention, the method is for non-diagnostic purposes, such as antibody detection and development of immunological detection methods in the laboratory.

[0028] The eighth aspect of the present invention provides a use of the antibody or antigen-binding fragment thereof according to the first aspect, the nucleic acid according to the second aspect, the recombinant expression vector according to the third aspect, or the cell according to the fourth aspect in preparing a FOXA3 detection reagent.

[0029] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0030] The reagents and raw materials used in the present invention are commercially available.

[0031] The positive and progressive effects of the present invention are:

[0032] The anti-FOXA3 monoclonal antibody of the present invention has high sensitivity, good specificity, high antibody concentration and purity, can be used for in vitro analysis of the expression level of FOXA3 protein in proteins or tissues, has good stability, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the immunofluorescence staining effect of commercially available FOXA3 antibody (1:200) (positive sample).

[0034] Figure 2 Schematic diagram of the immunofluorescence staining effect of FOXA3 antibody (1:10000) (positive sample) in Example.

[0035] Figure 3 Schematic diagram of the immunofluorescence staining effect of FOXA3 antibody (1:10000) (negative sample) in Example. DETAILED DESCRIPTION

[0036] definition

[0037] The term "complementarity determining region" or "CDR region" or "CDR" as used herein is a region of an antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen epitopes and are numbered sequentially from the N-terminus and include CDR1, CDR2, and CDR3. In a given heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems. It is well known to those skilled in the art that the CDR of an antibody can be defined in the art by a variety of methods, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (World Wide Web imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. It will be appreciated by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions as defined by any of the above-mentioned known schemes described in the present invention.

[0038] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDRs are different between antibodies, only a limited number of amino acid positions in CDRs are directly involved in antigen binding. Using at least two of the Kabat, Chothia, IMGT, AbM and Contact methods, the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of a CDR. As those skilled in the art will appreciate, the residues of the rest of the CDR sequence can be determined by the structure and protein folding of the antibody. Therefore, the present invention also contemplates variants of any CDR given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, and the remaining CDR residues defined according to Kabat or Chothia or AbM can be replaced by conservative amino acid residues.

[0039] As used herein, "percent (%) sequence identity" and "sequence identity" of amino acid sequences have a definition recognized in the art, which refers to the percentage of identity between two polypeptide sequences determined by sequence alignment (e.g., by manual inspection or a publicly known algorithm). It can be determined using methods known to those skilled in the art, such as using publicly available computer software such as BLAST, BLAST-2, Clustal Omega and FASTA software.

[0040] In the present invention, unless the context clearly indicates otherwise, when the term "antibody" is mentioned, it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0041] The term "isolated" as used herein refers to something obtained artificially from a natural state. If a certain "isolated" substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated from this natural state is called "isolated". The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0042] As used herein, "vector" refers to a construct that is capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0043] The term "host cell" as used herein refers to cells that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

[0044] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0045] Example 1

[0046] The amino acid sequences of the FOXA3 antibodies screened in this example are shown in Table 1, and the CDRs are defined using Kabat.

[0047] Table 1 Antibody sequences

[0048]

[0049]

[0050] In the table, VH is the heavy chain variable region, VL is the light chain variable region; HC is the heavy chain, and LC is the light chain.

[0051] In the antibody obtained in this example, the signal peptide sequence of the heavy chain is:

[0052] MYLGLNCVFIVFLLKGVQS(SEQ ID NO:19)

[0053] The nucleotide sequence encoding it is shown in SEQ ID NO:20.

[0054] The signal peptide sequence of the light chain is:

[0055] MRFQVQVLGLLLLWISGAQC(SEQ ID NO:21)

[0056] The nucleotide sequence encoding it is shown in SEQ ID NO:22.

[0057] Example 2

[0058] This example verifies the effect of the antibody by immunofluorescence staining. The immunofluorescence operation steps are as follows:

[0059] 1. Take out the fixed 24-well plate hiHep cells [2], after discarding the supernatant, 300 μl / well 0.25% Triton X-100 was added for permeabilization for 15 min;

[0060] 2. After discarding the supernatant, add 300 μl / well 3% BSA to block for 45 minutes;

[0061] 3. After discarding the supernatant, add 300 μl / well primary antibody (FOXA3 antibody prepared in Example 1) (1:10000) and incubate at room temperature for 2 h;

[0062] 4. Discard the supernatant and add 500 μl / well 1× PBS to wash 3 times;

[0063] 5. Add 300 μl / well secondary antibody (Alexa Fluor 488AffiniPure Goat Anti-Mouse IgG, 115-545-205, Jackson ImmunoResearch) (1:500) incubate at room temperature in the dark for 1 h;

[0064] 6. Discard the supernatant and add 500 μl / well 1X PBS to wash 3 times;

[0065] 7. Add 300 μl / well DAPI (1:3000) and incubate at room temperature in the dark for 3 minutes;

[0066] 8. After discarding the supernatant, add 500 μl / well 1×PBS and wash 3 times, then add 1 ml 1×PBS to take fluorescent photos.

[0067] like Figure 1 and Figure 2 As shown, when the FOXA3 antibody of this example is used at a ratio of 1:10000, it can achieve the staining level of 1:200 of the commercially available antibody (Abcam, ab222245), indicating that the antibody has higher purity and better sensitivity.

[0068] And if Figure 3 As shown, the FOXA3 antibody of this example has no signal in the negative control cells, indicating that the antibody has good specificity.

[0069] Example 3: Antibody stability test

[0070] The samples used in this example are hiHep cells made by the company. [2] The positive rate was calculated by counting with imageJ software: positive rate = (number of FOXA3 positive cells / total number of DAPI cells) * 100%

[0071] After the antibodies were stored at -20°C, 2-8°C and 37°C for different periods of time with 0.02% ProClin 300, the positive rates of FOXA3 in the samples were detected. The test results are shown in Table 2.

[0072] The FOXA3 antibody of the present invention can maintain good detection sensitivity and stability under storage conditions of -20°C; the stability is also good under storage conditions of 2-8°C for 2 weeks; under 37°C, the stability can only be maintained for 3 days. Compared with commercially available antibodies stored at 4°C for 1-2 weeks, the stability is comparable.

[0073] Table 2 Antibody storage stability experiment

[0074]

[0075] The same sample was tested three times independently. As shown in Table 3, the CVs of the three test results were 0.5% and 0.2%, respectively. The coefficient of difference between the three tests was small, indicating that the antibody detection stability was high.

[0076] Table 3 Antibody detection stability experiment

[0077] Sample 1 Sample 2 First test 20.6％ 19.8％ Second test 19.7％ 19.4％ The third test 20.2％ 19.6％ CV 0.5％ 0.2％

[0078] References

[0079] 1.Liu C, Zhou B, Meng M, et al. FOXA3 induction under endoplasmicreticulum stress contributes to non-alcoholic fatty liver disease[J]. Journal of Hepatology, 2021.

[0080] 2. Huang P, Zhang L, Gao Y, He Z, Yao D, Wu Z, et al. Direct reprogramming of human fibroblasts to functional and expandable hepatocytes. Cell stem cell2014;14:370-84.

Claims

1. An antibody or antigen-binding fragment thereof targeting FOXA3, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and MDY, respectively; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in amino acid sequences of SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:3, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:8, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:

8.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein: The antibody is a full-length antibody, Fab, Fab', F(ab')2, scFv, Fv or a multispecific antibody; Preferably, when the antibody is a full-length antibody, the heavy chain constant region of the full-length antibody is derived from the heavy chain of a human antibody or a variant thereof, and the light chain constant region of the full-length antibody is derived from the κ chain or λ chain of a human antibody or a variant thereof; More preferably, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:23, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:23; and / or the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:25, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:

25.

4. An isolated nucleic acid, characterized in that The nucleic acid encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; Preferably, the nucleotide sequence encoding the HCDR1 is shown in SEQ ID NO: 10, the nucleotide sequence encoding the HCDR2 is shown in SEQ ID NO: 11, and the nucleotide sequence encoding the HCDR3 is ATGGACTAC; and / or, the nucleotide sequence encoding the LCDR1 is shown in SEQ ID NO: 14, the nucleotide sequence encoding the LCDR2 is shown in SEQ ID NO: 15, and the nucleotide sequence encoding the LCDR3 is shown in SEQ ID NO: 16; More preferably, the nucleotide sequence encoding the heavy chain variable region is shown as SEQ ID NO:12; and / or the nucleotide sequence encoding the light chain variable region is shown as SEQ ID NO:

17.

5. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid according to claim 4; Preferably, the recombinant expression vector is a plasmid, cosmid, phage or viral vector; More preferably, the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

6. A cell, characterized in that The cell contains the recombinant expression vector according to claim 5 or the nucleic acid according to claim 4; or expresses the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; Preferably, the cell is a prokaryotic cell or a eukaryotic cell; More preferably, the cell is selected from yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof; the mammalian cell is, for example, HEK293 cell.

7. A method for preparing an antibody or an antigen-binding fragment thereof targeting FOXA3, characterized in that: The method comprises culturing the cell according to claim 6, and obtaining an antibody or an antigen-binding fragment thereof targeting FOXA3 from the culture.

8. A kit, characterized in that: The kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; Preferably, the kit further comprises a reagent for detecting the binding of the antibody or antigen-binding fragment thereof to an antigen.

9. A method for detecting FOXA3, characterized in that: The method comprises the steps of contacting the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 with a sample to be tested; Preferably, the method is for non-diagnostic purposes.

10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid according to claim 4, the recombinant expression vector according to claim 5 or the cell according to claim 6 in preparing a FOXA3 detection reagent.

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