Specific binding protein and application thereof

Abstract

The invention belongs to the technical field of antibodies, and discloses a specific binding protein and application thereof. An extracellular structural domain of RNF149 protein is purified through eukaryotic expression, a monoclonal antibody is obtained through an immune mouse and hybridoma technology, and then a high-affinity humanized recombinant antibody, namely the specific binding protein, is obtained through humanized transformation. The specific binding protein can accurately target an extracellular region of the RNF149 protein through epitopes, reduces cross reaction with non-target protein, has relatively high RNF149 protein binding specificity, has binding affinity less than or equal to 10 <-9 > M with the RNF149 protein, and has sub-nanomole-level binding affinity. The specific binding protein also has higher titer, still has better binding capacity when the dilution ratio is 1: 551, and is suitable for high-sensitivity detection. The invention provides a recombinant antibody which can be specifically combined with RNF149 protein and is relatively high in sensitivity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of antibodies, and relates to a specific binding protein and application thereof. BACKGROUND

[0002] RNF149 (RING finger protein 149) protein belongs to the PA-TM-RING protein family, has ubiquitin ligase activity, is a ubiquitin E3 ligase encoded by a human RNF149 gene, and plays an important role in protein ubiquitination modification, lysosomal degradation pathway and cell proliferation regulation. The protein includes an N-terminal PA domain (Protease-Associated domain), a transmembrane domain (Transmembrane domain, TM) and a C-terminal RING domain (Really Interesting New Gene Domain). It has been found in the prior art that RNF149 protein marks target proteins through K48-linked polyubiquitination, so that the target proteins are degraded by proteasomes or lysosomes to regulate the ubiquitin-proteasome system. It has also been found in the prior art that knockdown of RNF149 leads to accumulation of CD9 and inhibits the growth of certain cancer cells. Since CD9 plays an important role in tumor metastasis, it is speculated that RNF149 protein may affect cancer progression by regulating CD9.

[0003] At present, the antibodies against RNF149 protein on the market are mainly provided by Abnova, Thermo Fisher, Sigma and other companies. The commercially available antibodies still have some defects, which are specifically as follows: the insufficient specificity of the antibodies leads to cross-reaction with non-target proteins, thereby producing false positive detection results; the detection sensitivity for endogenous proteins is insufficient; and the performance in special application scenarios such as multiplex detection and fixed tissue staining is poor. Therefore, it is of great significance to develop an antibody that can specifically bind to RNF149 protein and has high sensitivity. SUMMARY

[0004] To solve the above technical problems, in some embodiments, a specific binding protein and application thereof are provided.

[0005] In one aspect, in some embodiments, a specific binding protein is provided, which specifically binds to at least part of the extracellular domain of a target protein, the target protein including RNF149 protein.

[0006] In some embodiments, the extracellular domain of the RNF149 protein includes amino acids 33-201 of the RNF149 protein; the amino acids 33-201 of the RNF149 protein are shown in SEQ ID NO: 1.

[0007] Exemplarily, the specific binding protein comprises an antibody of the target protein or an antigen binding fragment of the target protein.

[0008] In some embodiments, the antibody comprises a heavy chain variable region comprising at least one of VH-CDR1, VH-CDR2, and VH-CDR3.

[0009] In some embodiments, the heavy chain variable region comprises VH-CDR1, VH-CDR2, and VH-CDR3.

[0010] In some embodiments, the VH-CDR1 comprises an amino acid sequence represented by Formula I:

[0011] S-Y-W-X1-N (Formula I);

[0012] X1in Formula I is selected from any one of I, M;

[0013] In some embodiments, the VH-CDR2 comprises an amino acid sequence represented by Formula II:

[0014] X2-I-X3-P-S-D-S-X4-T-X5-X6-N-Q-K-F-X8-D (Formula II);

[0015] X2in Formula II is selected from any one of N, M;

[0016] X3in Formula II is selected from any one of Y, H;

[0017] X4in Formula II is selected from any one of Y, E;

[0018] X5in Formula II is selected from any one of N, R;

[0019] X6in Formula II is selected from any one of Y, L;

[0020] X7in Formula II is selected from any one of S, F;

[0021] X8in Formula II is selected from any one of K, M;

[0022] In some embodiments, the VH-CDR3 comprises an amino acid sequence represented by Formula III:

[0023] X9-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X16 -X 17 -X 18 -X 19 -X 20 (Formula III);

[0024] X9in Formula III is selected from any one of D, G;

[0025] X10in Formula III is selected from any one of G, L; 10

[0026] X11in Formula III is selected from any one of Y, S; 11

[0027] X12in Formula III is selected from any one of G, S; 12

[0028] X13in Formula III is selected from any one of D, N; 13 -X 18 selected from any one of natural amino acids;

[0029] X14in Formula III is selected from any one of V, S. 19

[0030] X15in Formula III is selected from any one of D, N. 20

[0031] In some embodiments, the VH-CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 16.

[0032] In some embodiments, the VH-CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 17 or SEQ ID NO: 18.

[0033] In some embodiments, the VH-CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 19.

[0034] In some embodiments, the antibody further comprises a light chain variable region;

[0035] In some embodiments, the light chain variable region comprises at least one of VL-CDR1, VL-CDR2, VL-CDR3.

[0036] In some embodiments, the light chain variable region comprises VL-CDR1, VL-CDR2, and VL-CDR3.

[0037] In some embodiments, the VL-CDR1 comprises an amino acid sequence as set forth in Formula IV:

[0038] R-X​​​​​21 -S-Q-D-I-G-X 22 -S-L-X 23 (Formula IV);

[0039] X in Formula IV 21 is selected from any one of S, A;

[0040] X in Formula IV 22 is selected from any one of T, I;

[0041] X in Formula IV 23 is selected from any one of H, N;

[0042] In some embodiments, the VL-CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 21.

[0043] In some embodiments, the VL-CDR3 comprises an amino acid sequence as set forth in Formula V:

[0044] L-Q-Y-A-X 24 -X 25 -P-Y-T (Formula V);

[0045] X in Formula V 24 is selected from any one of T, S;

[0046] X in Formula V 25 is selected from any one of F, S.

[0047] In the amino acid sequences herein, “-” is used only to connect amino acids and has no other meaning.

[0048] In some embodiments, the VL-CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 20.

[0049] In some embodiments, the VL-CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 22.

[0050] In some embodiments, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3.

[0051] In some embodiments, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 4, SEQ ID NO: 5.

[0052] In some embodiments, the heavy chain of the specific binding protein comprises an amino acid sequence as set forth in SEQ ID NO: 12, SEQ ID NO: 14.

[0053] In some embodiments, the light chain of the specific binding protein comprises an amino acid sequence as set forth in SEQ ID NO: 13, SEQ ID NO: 15.

[0054] In some embodiments, it is found by Western-Blot detection that the RNF149#1 antibody and the RNF149#2 antibody can not only specifically recognize the extracellular domain of the RNF149 protein, but also recognize the RNF149 protein in a native conformation.

[0055] In some embodiments, the specific binding protein is a mono-specific binding protein or a multi-specific binding protein.

[0056] For example, the multi-specific binding protein includes but is not limited to a bi-specific binding protein, a tri-specific binding protein, a tetra-specific binding protein, i.e., a single binding protein can simultaneously bind two, three or four different antigens / target proteins; in some embodiments, the target protein species targeted by the multi-specific binding protein is not specifically limited, and those skilled in the art can determine the target protein species targeted by the multi-specific binding protein according to the target protein species targeted by the antibodies fused in the multi-specific binding protein.

[0057] In some embodiments, an isolated nucleic acid or a nucleic acid group is provided, which encodes the specific binding protein described above.

[0058] In some embodiments, a host cell comprising the nucleic acid or the nucleic acid group described above is provided.

[0059] In some embodiments, a method for producing the specific binding protein described above is provided, which comprises culturing the host cell described above.

[0060] In some embodiments, the specific binding protein is further recovered from the host cell.

[0061] In some embodiments, the specific binding protein described above is provided for use in the preparation of an anti-tumor drug.

[0062] In some embodiments, a pharmaceutical composition comprising the specific binding protein described above and a pharmaceutically acceptable carrier is provided.

[0063] In some embodiments, the pharmaceutical composition is used for preventing and / or treating diseases;

[0064] In some embodiments, the diseases include cancer;

[0065] In some embodiments, the cancer includes but is not limited to primary liver cancer, renal papillary cell carcinoma or nasopharyngeal carcinoma.

[0066] In some embodiments, the use of the specific binding protein described above in the preparation of a reagent for detecting RNF149 protein is provided.

[0067] In some embodiments, the recombinant antibody (specific binding protein) provided is an antibody developed against the extracellular domain of RNF149 protein, and thus the recombinant antibody can directly bind to the target protein RNF149 without the need for cell membrane breaking or fixation / permeabilization steps. At the same time, because the recombinant antibody can precisely target the extracellular region through epitope, it reduces cross-reaction with non-target proteins, and is suitable for drug development (such as mAb, diabody) and as a diagnostic reagent.

[0068] In some embodiments, because the recombinant antibody provided can directly target the extracellular region without the need for a delivery system, the convenience of clinical translation is improved, and it also becomes a potential choice for therapeutic antibody development.

[0069] In some embodiments, a kit for detecting RNF149 protein is provided, comprising the specific binding protein, or the specific binding protein encoded by the nucleic acid or nucleic acid group described above, or the specific binding protein produced by the host cell described above.

[0070] In some embodiments, the kit provided further comprises one or more reagents for expressing or purifying the specific binding protein and / or one or more reagents for incubating the specific binding protein with the sample to be tested in vitro to allow specific binding to the target protein.

[0071] In some embodiments, the sample to be tested includes but is not limited to a cell or tissue sample.

[0072] In some embodiments, the recombinant antibody provided has a high binding affinity to RNF149 protein and can specifically recognize RNF149 protein.

[0073] In some embodiments, it is found through ELISA detection that when the dilution volume ratio of RNF149#1 antibody and RNF149#2 antibody is 1:51200, the OD 450 values of both are greater than 0.1, indicating that the RNF149 antibody has a sub-nanomolar binding affinity to the antigen, and the RNF149 antibody has a very high titer, which is suitable for high-sensitivity detection.

[0074] In some embodiments, it is found through surface plasmon resonance technology to detect the affinity of RNF149 antibody to antigen that the KD(M) of RNF149#1 antibody is 2.13e -10 and the KD(M) of RNF149#2 antibody is 7.79e -12, the binding affinity of the RNF149 antibody is less than or equal to 10 -9 M (nM level), which can reduce the dosage and reduce side effects, while meeting the needs of low concentration and high efficiency binding of tumor targeted therapy, effectively reducing off-target effects.

[0075] In some embodiments, a method for detecting RNF149 protein in a sample is provided, which comprises incubating the sample with the specific binding protein or the kit described above.

[0076] Compared with the prior art, the technical scheme at least has the following beneficial effects or advantages:

[0077] In some embodiments, the provided recombinant antibody (specific binding protein) can also recognize the full-length protein of RNF149.

[0078] In some embodiments, the extracellular domain of RNF149 protein is purified by eukaryotic expression, and the monoclonal antibody is obtained by immunizing mice and hybridoma technology, and then the high-affinity humanized recombinant antibody is obtained by humanization. The recombinant antibody of the present application is an antibody developed against the extracellular domain of RNF149 protein, so it can directly bind to the target protein RNF149 without the need for cell membrane breaking or fixation / permeabilization steps. At the same time, because the recombinant antibody of the present application can precisely target the extracellular region through epitope, it reduces the cross-reaction with non-target proteins, and is suitable for drug development (such as monoclonal antibody, double antibody) and as a diagnostic reagent. Because the recombinant antibody of the present application can directly target the extracellular region without the need for a delivery system, it improves the convenience of clinical transformation, and also makes it a potential choice for therapeutic antibody development.

[0079] In some embodiments, the provided recombinant antibody has a high binding affinity to RNF149 protein, can specifically recognize RNF149 protein, and can be applied to the diagnosis or targeted therapy of diseases related to abnormal expression of RNF149 (such as tumors), and has important clinical transformation prospects.

[0080] In some embodiments, it is found by ELISA detection that when the dilution volume ratio of RNF149#1 antibody and RNF149#2 antibody is 1:51200, the OD 450 nm values are all greater than 0.1, indicating that the RNF149 antibody has sub-nanomolar binding affinity to the antigen, and the RNF149 antibody has very high titer, which is suitable for high-sensitivity detection.

[0081] In some embodiments, it is found by surface plasmon resonance technology that the KD (M) of RNF149#1 antibody is 2.13e -10, KD(M) of RNF149#2 antibody = 7.79e -12 , the binding affinity of RNF149 antibody is all ≤10 -9 M (nM level), which can reduce the dose of administration and reduce side effects, while meeting the needs of low concentration and high efficient binding of tumor targeted therapy, effectively reducing off-target effects.

[0082] In some embodiments, it is found by Western-Blot detection that RNF149#1 antibody and RNF149#2 antibody can not only specifically recognize the extracellular domain of RNF149 protein, but also recognize RNF149 protein in natural conformation. BRIEF DESCRIPTION OF DRAWINGS

[0083] Figure 1 The sequence prediction results of RNF149 antibody heavy chain variable region and light chain variable region.

[0084] Figure 2 The structure prediction of RNF149 antibody.

[0085] Figure 3 The SDS-PAGE detection results of RNF149#1 antibody. NR is the band of RNF149#1 antibody molecule, R is the band of heavy chain and light chain obtained after reducing RNF149#1 antibody molecule, and M is protein marker.

[0086] Figure 4 The SEC-HPLC detection results of RNF149#1 antibody.

[0087] Figure 5 The SDS-PAGE detection results of RNF149#2 antibody. NR is the band of RNF149#2 antibody molecule, R is the band of heavy chain and light chain obtained after reducing RNF149#2 antibody molecule, and M is protein marker.

[0088] Figure 6 The SEC-HPLC detection results of RNF149#2 antibody.

[0089] Figure 7 The ELISA detection results of RNF149 antibody and RNF149 protein binding.

[0090] Figure 8 The standard curve of RNF149#1 antibody content logarithmic value and OD 450 nm value.

[0091] Figure 9 The standard curve of RNF149#2 antibody content logarithmic value and OD 450 nm value.

[0092] Figure 10 Figure 2 is a graph showing the result of surface plasmon resonance assay for detecting the binding of RNF149#1 antibody to RNF149 protein.

[0093] Figure 11 Figure 3 is a graph showing the result of surface plasmon resonance assay for detecting the binding of RNF149#2 antibody to RNF149 protein.

[0094] Figure 12 Figure 4 is a graph showing the result of Western-Blot assay for detecting the extracellular domain of RNF149 protein and RNF149-GFP by using RNF149#1 antibody and RNF149#2 antibody. Figure 12 Figure 4A is a graph showing the result of Western-Blot assay for detecting the extracellular domain of RNF149 protein by using RNF149#1 antibody and RNF149#2 antibody. Figure 12 Figure 4B is a graph showing the result of Western-Blot assay for detecting RNF149-GFP by using RNF149#1 antibody and RNF149#2 antibody. RNF149-GFP is full-length RNF149 protein with GFP tag.

[0095] Figure 13 Figure 5 is a graph showing the result of immunofluorescence assay for RNF149#1 antibody and RNF149#2 antibody in HeLa cells and HepG2 cells. DETAILED DESCRIPTION

[0096] The technical solutions of the present application will be described in combination with examples. However, the present application is not limited to the following examples. The experimental methods and detection methods described in the following examples are all conventional methods unless otherwise specified. The reagents and materials described in the following examples can be purchased on the market unless otherwise specified.

[0097] In this document, the term "antibody" refers to an immunoglobulin molecule capable of binding to a specific antigen.

[0098] An antibody is a symmetrical structure of four polypeptide chains, two identical heavy chains (H chains) of larger relative molecular mass and two identical light chains (L chains) of smaller relative molecular mass. The chains are linked by disulfide bonds and noncovalent bonds to form a monomeric molecule consisting of four polypeptide chains. There are two types of light chains, kappa and lambda, and five types of heavy chains, mu, delta, gamma, epsilon and alpha, which define the isotypes of antibodies as IgM, IgD, IgG, IgA and IgE, respectively. The amino-terminal (N-terminal) amino acid sequences of the polypeptide chains vary greatly and are called variable regions (V regions), while the carboxy-terminal (C-terminal) sequences are relatively stable and are called constant regions (C regions). The V regions of L and H chains are called variable light (VL) and variable heavy (VH), respectively. VL / VH is a part of immunoglobulin (antibody) light / heavy chain responsible for recognizing and binding to specific antigens. VH and VL together form the antigen-binding site of an antibody, which is responsible for specifically recognizing and binding to an epitope of an antigen.

[0099] In the variable region, some regions have a higher degree of variation in amino acid composition and sequence, which are called hypervariable regions (HVRs). The HVRs are the positions where antigens and antibodies bind, and are therefore also called complementarity-determining regions (CDRs). There are three CDR regions on the variable region of the heavy chain and the variable region of the light chain. HVR1, HVR2 and HVR3 of VL and VH are also called CDR1, CDR2 and CDR3, respectively. CDR3 has a higher degree of hypervariability, and H chain plays an important role in binding to antigens.

[0100] In this document, the term "antigen-binding fragment" of an antibody refers to a polypeptide that comprises a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as "antigen-binding portion". In this document, unless the context clearly indicates otherwise, when the term "antibody" is referred to, it not only includes intact antibodies, but also antigen-binding fragments of antibodies.

[0101] The term "antigen" refers to a molecule or a portion of a molecule that is capable of being bound by a selective recognition or binding agent of an antigen-binding molecule protein, such as an antibody. An antigen can have one or more epitopes capable of interacting with different antigen-binding protein molecules, such as antibodies.

[0102] In this document, the term "affinity" refers to the overall strength of the noncovalent interactions between a single binding site of a molecule, such as an antibody, and its binding partner, such as an antigen. Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects the 1:1 interaction between members of a binding pair (e.g., antibody and antigen).

[0103] Herein, the term "KD" refers to the equilibrium dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka) and is expressed as a molar concentration (M).

[0104] Herein, the term "room temperature" refers to 23 °C ± 2 °C.

[0105] Example 1

[0106] This example provides the construction of RNF149 recombinant antibodies.

[0107] 1. Gene cloning

[0108] A gene fragment encoding the extracellular domain of RNF149 protein (33-201 AA of RNF149 protein) was designed, the amino acid sequence of the extracellular domain of RNF149 is shown in SEQ ID NO: 1, and it was constructed into the eukaryotic cell expression vector pcDNA3.4-Fc (donated by Dr. Chen Zhicheng of Shenzhen Bay Laboratory). The day before transfection, the HEK 293F cells (donated by Dr. Chen Zhicheng of Shenzhen Bay Laboratory) were adjusted to a density of 1 x 10 6 cells / mL; on the day of transfection, the cells were counted, and the HEK 293F suspension cells were adjusted to a density of 2 x 10 6 cells / mL using the medium. For a 100 mL system, the plasmid mixture was 5 mL Opti-MEM (purchased from Gibco, catalog number: 31985070) + 100 μg DNA. For a 100 mL system, the transfection reagent mixture was 5 mL Opti-MEM + 400 μL of PEI with a concentration of 1 μg / mL (purchased from Yeasen, catalog number: 40816ES08), which was added to the plasmid mixture, inverted up and down, mixed well, and incubated at room temperature for 15 min to obtain the transfection mixture; the transfection mixture was added to the HEK 293F cells with a density of 2 x 10 6 cells / mL, gently mixed, and incubated in a 37 °C suspension incubator containing 8% CO2 at 125 rpm for 7 days to obtain the suspension culture medium, and 3.5 vol% of 293 suspension cell culture supplement (purchased from Yiqiao God, catalog number: M293-SUPI) was added to the cell culture medium at 20-24 h after transfection, and 3.5 vol% of 293 suspension cell culture supplement was added every other day thereafter.

[0109] 2. Antigen purification

[0110] The supernatant was collected by centrifugation of the suspension culture at 4000 rpm for 15 min; the supernatant was filtered using a 0.22 μm filter; the AKTA protein purification instrument (brand: CYTIVA, model: AKTA PURE) was opened, and the Protein A column was equilibrated using PBS at a flow rate of 3 mL / min; then the filtered supernatant was loaded onto the Protein A column at a flow rate of 3 mL / min; non-specifically bound proteins on the Protein A column were washed away using PBS at a flow rate of 3 mL / min; the bound antigen on the Protein A column was eluted using a glycine buffer at pH = 3.0 at a flow rate of 1 mL / min; the eluate was collected to obtain the purified RNF149 protein.

[0111] 3. Antibody immunization

[0112] The purified RNF149 protein was adjusted to a concentration of 1 mg / mL using PBS (purchased from Wuhan Pons Life Science Co., Ltd., Catalog No.: PB180327), mixed with Freund's complete adjuvant (CFA) (purchased from Sigma, Catalog No.: F5881-10 mL) at a volume ratio of 1:1 (the first immunization used contains inactivated tubercle bacillus), and repeatedly aspirated to form a stable "water-in-oil" emulsion. Then the antigen was slowly released by subcutaneous injection and multi-site injection (neck back, groin) of the mouse. The basic immunization was on day 0, and then every two weeks, the immunization was enhanced by injection mixed with Freund's incomplete adjuvant (IFA) (purchased from Sigma, Catalog No.: F5506-10 mL). On day 7-10 after the first booster, 50-100 μL of blood was taken from the tail vein or orbit, and then the antibody titer was verified by ELISA until the titer met the standard, i.e. the OD 450 >0.2.

[0113] 4. Cell collection and hybridoma cell construction

[0114] On day 3 after the last booster, the mouse was sacrificed to prepare hybridoma cells. The spleen of the immunized mouse was taken to prepare a single cell suspension, which was fused with myeloma cells SP2 / 0, and the hybridoma cells were plated and cultured in DMEM medium (purchased from Gibco, Catalog No.: C11995500BT) containing HAT (purchased from Gibco, Catalog No.: 21060017). The positive cell wells were screened by indirect Elisa method. The positive cell strain was selected and subcloned, and after 2-3 rounds of subcloning, the monoclonal cell strain was obtained when all the subcloned wells were antibody positive and the signal was uniform.

[0115] 5. Antibody sequence analysis

[0116] The cDNA of the hybridoma cell expressing the RNF149 antibody was extracted using the RNA extraction kit (RC112-01) of Nanjing Nuo Weizan Biotechnology Co., Ltd. and the reverse transcription kit (11141ES60) of Yixing Bio. The VH and VL genes were amplified using 5'-GAGGTGCAGCTGCAGGAGTC TGG-3' (leader sequence primer) and 5'-GGGCCAGTGGATAGACCGATGGG-3' (constant region reverse primer). After amplification, the amplified fragments were connected to the intermediate vector pMD TM 19-T (purchased from Takara, item number: 6013). Then sequencing was performed to obtain the corresponding light chain gene sequence of the targeting antibody and the heavy chain gene sequence of the targeting antibody and the amino acid sequence, the amino acid sequence of the heavy chain variable region (1-VH, 2-VH) is shown as SEQ ID NO: 2 and SEQ ID NO: 3, and the amino acid sequence of the light chain variable region (1-VL, 2-VL) is shown as SEQ ID NO: 4 and SEQ ID NO: 5.

[0117] 6. Antibody preparation

[0118] The obtained original heavy chain gene sequence of the targeting antibody was subjected to humanization modification, and the modified nucleotide sequence is shown as SEQ ID NO: 6 and SEQ ID NO: 7, 5'AscI (GGCGCGCC) was added, and the gene was cloned into the heavy chain empty vector pAb20-hCHIgG1 (Ampicillin resistance) by 5'AscI using a recombination method, and the nucleotide sequence is shown as SEQ ID NO: 8, and the plasmid was extracted using the Endo-Free Plasmid Miniprep Kit to prepare the recombinant plasmid DNA of medium scale. The obtained light chain gene sequence of the targeting antibody was subjected to humanization modification, and the modified nucleotide sequence is shown as SEQ ID NO: 9 and SEQ ID NO: 10, 5'AscI was added, and the gene was cloned into the light chain empty vector pAb20-hCK (Ampicillin resistance) by 5'AscI using a recombination method, and the nucleotide sequence is shown as SEQ ID NO: 11, and the plasmid was extracted using the Endo-Free Plasmid Miniprep Kit to prepare the recombinant plasmid DNA of medium scale.

[0119] The supernatant was collected after 5 days of expression in a 37°C suspension incubator with 8% CO2 at 125 rpm. The antibody was purified using a Protein A affinity column. Finally, the antibody bound to the Protein A column was eluted with a citric acid buffer at pH = 3.4 at a flow rate of 1 mL / min, and the eluate was collected in tubes, about 500 μL per tube. The purified antibody (RNF149#1 antibody and RNF149#2 antibody) was obtained. The amino acid sequence of the heavy chain (1-RNF149-LH) of the RNF149#1 antibody is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain (1-RNF149-LC) is shown in SEQ ID NO: 13. The humanized framework region in SEQ ID NO: 12 is from position 122 to position 451, and the humanized framework region in SEQ ID NO: 13 is from position 108 to position 214. The amino acid sequence of the heavy chain (2-RNF149-LH) of the RNF149#2 antibody is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain (2-RNF149-LC) is shown in SEQ ID NO: 15. The humanized framework region in SEQ ID NO: 14 is from position 122 to position 451, and the humanized framework region in SEQ ID NO: 15 is from position 108 to position 214. After obtaining the heavy chain and light chain sequences corresponding to the RNF149#1 antibody and the RNF149#2 antibody, the CDR region sequences were predicted using ABodyBuilder-ML, and the results are shown in FIG. (1). Figure 1 The variable amino acid positions and variable amino acid types in the amino acid sequences of the RNF149 antibody heavy chain variable region VH-CDR1, VH-CDR2, VH-CDR3 and the RNF149 antibody light chain variable region VL-CDR1, VL-CDR2, VL-CDR3 are shown in FIG. (2).

[0120] The antibody structure was predicted using Alphafold2, and the corresponding antibody epitope was analyzed by Pymol 3.1. Among them, the antigen antibody binding epitope is completely exposed on the surface of the antibody. Figure 2 The purified antibody was absorbed into a dialysis bag and dialyzed in a beaker containing 1xPBS. The absorbance value at 280 nm was read using a Nano Drop instrument. The purity of the purified antibody was detected by SDS-PAGE and SEC-HPLC (less than 1 mg of protein amount was not detected by SEC-HPLC). The molecular weight of the protein was determined by SDS-PAGE using Marker (purchased from Biorad, product number: 1610394), and the results are shown in FIG. (3). Figures 3 to 6 Figure 3 and Figure 5 ​The middle NR is Non-reduced condition, indicating that the protein sample is not added to the reducing agent during the loading process, and the band is the size of the antibody molecule; R is Reduced, indicating that the protein sample is added to the reducing agent during the loading process, breaking the disulfide bond in the protein, and the bands running out are heavy chain (~ 50 kD) and light chain (~ 25 kD) size. The results show that the purity of RNF149#1 antibody is SDS-PAGE > 90%, SEC-HPLC 280nm = 99.25%; the purity of RNF149#2 antibody is SDS-PAGE > 90%, SEC-HPLC 280nm = 100%.

[0121] Example 2

[0122] This example detects the binding ability of RNF149 antibody (RNF149#1 antibody, RNF149#2 antibody) to RNF149 protein by ELISA. Dilute RNF149 protein to 1 μg / mL with coating solution (Solarbio #C1050), coat RNF149 protein (1 μg / mL, 100 μL / well) on a 96-well ELISA plate (NEST #504201) at 4°C overnight. The next day, remove the liquid in the plate, dry the plate on absorbent paper, add 150 μL blocking solution (coating buffer containing 2% BSA) to each well, incubate at 37°C for 1 hour, discard the blocking solution, and wash three times with washing solution (PBS + 0.05% Tween 20). Dilute RNF149 antibody from the initial dilution (e.g. 1:2500 by volume) by a factor of 1:4. Add 100 μL of diluted RNF149 antibody to each well, add primary antibody to negative control wells without coated antigen, add antibody diluent to negative control wells with coated antigen, and add known titer antibody samples developed by the laboratory to positive control wells. Each dilution has duplicate wells. Incubate at 37°C for 1 hour, and wash three times with washing solution. Dilute HRP-labeled goat anti-human IgG (purchased from Shengong Bioengineering (Shanghai) Co., Ltd., Catalog No.: D110150-0100) or AffiniPure Fc-specific Goat Anti-Human IgG (H+L) (Jackson ImmunoResearch, Catalog No.: 109-035-098) with blocking solution, and add 100 μL to each well. Incubate at 37°C for 1 hour, and wash three times with washing solution. Add 100 μL of TMB substrate (Solarbio #C0010) to each well, and incubate at room temperature for 10-20 minutes. Add 100 μL of stop solution (Solarbio #C0011) to each well, and measure the absorbance at 450 nm within 30 minutes. TMF(ab)2Fragment Goat Anti-Human IgG, F(ab)2fragment specific (purchased from JACKSON, item number: 109-036-097), dilution volume ratio 1:5000, 100 μL of diluted secondary antibody was added to each well, and incubated at 37°C for 1 hour, and washed 3 times with washing solution. Color development and termination: 100 μL of TMB substrate (Solarbio #PR1200) was added to each well, and reacted at room temperature for 10 minutes in the dark (positive wells were observed to be blue). Immediately, 50 μL of ELISA termination solution (Solarbio #C1058) was added to each well, and the color changed to yellow. Immediately, the OD value at 450 nm was measured with an enzyme-labeled instrument (Bio Tek, model: Synergy H1), and the results are shown in Figure 7 , a standard curve graph of the logarithmic value of the antibody content and the OD 450 nm value is shown in Figure 8 and Figure 9 .

[0123] As can be seen from Figure 7 , when the dilution volume ratio of RNF149#1 antibody and RNF149#2 antibody is 1:51200, the OD 450 nm value is greater than 0.1, indicating that the RNF149 antibody has a sub-nanomolar binding affinity to the antigen, and the RNF149 antibody has a very high titer, which is suitable for high-sensitivity detection.

[0124] Example 3

[0125] In this example, the affinity of RNF149 antibody to antigen was measured by surface plasmon resonance technology.

[0126] The affinity KD and kinetic data ka, kd of RNF149 antibody (RNF149#1 antibody, RNF149#2 antibody) to RNF149 protein binding were detected by surface plasmon resonance (SPR) molecular interaction analyzer Biacore 8K.

[0127] Protein A (purified RNF149 protein) and protein B (RNF149#1 antibody, 0.29 mg / mL and RNF149#2 antibody, 0.32 mg / mL) were used as analytes to detect affinity and kinetics by directly coupling S series CM5 chips (item number: BR-1005-30, Cytiva), and the affinity between antigen and antibody was detected by single cycle kinetics mode, and the results are shown in Figure 10 and Figure 11 .

[0128] As can be seen from Figure 10 and Figure 11The SPR experiment results show that the RNF149 antibody has a strong affinity for the RNF149 protein, with the KD(M) of the RNF149#1 antibody being 2.13 e. -10 The KD(M) of the RNF149#2 antibody is 7.79e. -12 The binding affinity of RNF149 antibodies is ≤10. -9 M (nM level) can reduce the dosage and side effects, while meeting the needs of tumor targeted therapy for low-concentration and high-efficiency binding, effectively reducing off-target effects.

[0129] Example 4

[0130] This example provides Western-Blot identification of the RNF149 antibody.

[0131] Take an appropriate amount of purified RNF149 protein (33-201AA) and cell samples (cell samples fused to express RNF149-GFP, where RNF149-GFP is the full-length RNF149 protein with a GFP tag), add 5×SDS and boil at 95℃ for 10 min, perform SDS-PAGE analysis, transfer to PVDF membrane, and block with blocking buffer (1×TBST + 5% skim milk) at room temperature for at least 1 h. After washing the membrane three times with 1×TBST, it was immersed in a diluted primary antibody solution using RNF149#1 antibody / RNF149#2 antibody diluted 1:500 (v / v) as the primary antibody (1×TBST + 2% BSA antibody dilution), and incubated overnight at 4°C. After washing the membrane three times with 1×TBST, secondary antibody HRP-labeled goat anti-human IgG (purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number: D110150-0100) was added, and incubated at room temperature for 45–60 min. After washing the membrane four times with 1×TBST, chemiluminescence imaging was performed, and the results are as follows. Figure 12 As shown, the red arrow indicates the target protein band.

[0132] Depend on Figure 12 It is known that the RNF149#1 antibody and the RNF149#2 antibody can not only specifically recognize the extracellular domain of the RNF149 protein, but also recognize the RNF149 protein in its native conformation.

[0133] Example 5

[0134] This embodiment provides immunofluorescence (IF) identification of RNF149 antibody.

[0135] First, the tweezers were sterilized at high temperature, then the tweezers were used to clamp the climbing sheet horizontally into the 24-well plate, and 500 μL of complete medium was added; by taking an appropriate amount of HeLa cells and HepG2 cells (density about 20%) and inoculating them in the 24-well plate with the climbing sheet, the next day the cells were fused to express RNF149-GFP (green fluorescence) (pLD3334 in Deng Lab at SZBL), and when the target confluence was 60%, the subsequent experiment was carried out; on the third day, the culture medium was discarded, and 1xPBS (purchased from Wuhan Ponsay Life Technology Co., Ltd., Catalog No. PB180327) was used to wash 3 times, then 500 μL of 4% paraformaldehyde (Biosharp, Catalog No: P0099-500mL) was added, and it was placed at room temperature for 15 min; then the fixing solution was discarded, and 1xPBS was used to wash 3 times, then 500 μL of PBS containing 0.5% Triton X-100 (purchased from Shenguo Bioengineering (Shanghai) Co., Ltd., Catalog No: A600198-500) was added, and it was placed for 10 min; the membrane breaking solution was discarded, and 1xPBS was used to wash 3 times, then 500 μL of 5% bovine serum albumin (Bovine Serum Albumin, BSA) (purchased from Yeasen, Catalog No: 36101ES76) was added for blocking, and it was placed at room temperature for 1 h; incubation of primary antibody (RNF149#1 and RNF149#2): the primary antibody was diluted with 3% BSA (the dilution ratio of the antibody was 1:100), the cell surface of the cell climbing sheet was covered with the primary antibody, and it was placed in a wet box, incubated at 4°C overnight, after the incubation was completed, the primary antibody was recovered, 500 μL of PBS containing 0.05% Triton-X100 was added, and it was washed 3 times for 5 min each time; incubation of secondary antibody YSFluor TM 647 Goat Anti-Human lgG(H+L) (purchased from Yeasen, Catalog No: 33223ES60): the secondary antibody was diluted with 3% BSA (the dilution ratio of the antibody was 1:200), the cell surface of the cell climbing sheet was covered with the secondary antibody, and it was placed in a wet box, incubated at room temperature for 1 h, after the incubation was completed, the secondary antibody was recovered, 500 μL of PBS containing 0.05% Triton X-100 was added, and it was washed 3 times for 5 min each time; mounting: 10 μL of mounting agent (purchased from MCE, Catalog No: HY-K1042) was added to the slide, the climbing sheet was gently inverted on the slide, and nail polish was used for fixation, after the cell climbing sheet was dried, subsequent microscopic imaging (Nikon laser confocal scanning microscope A1) was carried out, and the results are shown in Figure 13 .

[0136] As can be seen from Figure 13 , the RNF149#1 antibody and the RNF149#2 antibody can recognize RNF149 and have the same localization as the fused RNF149-GFP.

[0137] The present application utilizes eukaryotic expression to purify the extracellular domain of RNF149 protein, obtains a monoclonal antibody through immunization of mice and hybridoma technology, and then obtains a high-affinity humanized recombinant antibody through humanization modification. The recombinant antibody can accurately target the extracellular region of RNF149 protein through epitope, reduces cross-reaction with non-target proteins, has high RNF149 protein binding specificity, the binding affinity of the recombinant antibody of the present application with RNF149 protein is ≤10 -9 M, with sub-nanomolar binding affinity. The recombinant antibody of the present application also has high titer, and still has good binding ability when the dilution ratio is 1:51200, and is suitable for high-sensitivity detection. The present application provides a recombinant antibody capable of specific binding with RNF149 protein and having high sensitivity, which can reduce the dosage and side effects of drug administration, and effectively reduce off-target effects.

[0138] The numbered sequence information in this paper is as follows:

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145] As described above, the basic principles, main features and advantages of the present application are better described. The above examples and specification only describe the preferred embodiments of the present application, and the present application is not limited by the above examples. Without departing from the spirit and scope of the present application, various changes and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the scope of protection of the present application.

Claims

1. A specifically binding protein, characterized in that, The specific binding protein specifically binds to at least a portion of the extracellular domain of the target protein, which includes the RNF149 protein.

2. The specific binding protein according to claim 1, characterized in that, The extracellular domain of the RNF149 protein includes amino acids 33 to 201 of the RNF149 protein; And / or, amino acids 33 to 201 of the RNF149 protein are as shown in SEQ ID NO:

1.

3. The specific binding protein according to claim 1, characterized in that, The specific binding protein includes an antibody against the target protein or an antigen-binding fragment of the target protein.

4. The specific binding protein according to claim 3, characterized in that, The antibody includes a heavy chain variable region, which includes at least one of VH-CDR1, VH-CDR2, and VH-CDR3. The VH-CDR1 comprises the amino acid sequence shown in Formula I: SYW-X1-N (Formula I); In Equation I, X1 is selected from either I or M; The VH-CDR2 comprises the amino acid sequence shown in Formula II: X2-I-X3-PSDS-X4-T-X5-X6-NQK-X7-X8-D(Formula II); In Formula II, X2 is selected from either N or M; In Formula II, X3 is selected from either Y or H; In Equation II, X4 is selected from either Y or E; In Formula II, X5 is selected from either N or R; In Formula II, X6 is selected from either Y or L; In Formula II, X7 is selected from either S or F; In Formula II, X8 is selected from either K or M; The VH-CDR3 comprises the amino acid sequence shown in Formula III: X9-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 (Formula III); In Formula III, X9 is selected from either D or G; X in Formula III 10 Choose from either G or L; X in Formula III 11 Choose either Y or S; X in Formula III 12 Choose from either G or S; X in Formula III 13 -X 18 Selected from any natural amino acid; X in Formula III 19 Choose from either D or N; X in Formula III 20 Choose from either V or S; And / or, the VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO:16; And / or, the VH-CDR2 comprises an amino acid sequence as shown in SEQ ID NO:17 or SEQ ID NO:18; And / or, the VH-CDR3 comprises an amino acid sequence as shown in SEQ ID NO:

19.

5. The specific binding protein according to claim 3, characterized in that, The antibody further includes a light chain variable region, which includes at least one of VL-CDR1, VL-CDR2, and VL-CDR3. The VL-CDR1 comprises the amino acid sequence shown in Formula IV: R-X 21 -S-Q-D-I-G-X 22 -S-L-X 23 (Formula IV); X in Formula IV 21 Choose from either S or A; X in Formula IV 22 Choose from either T or I; X in Formula IV 23 Choose from either H or N; The VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO:21; The VL-CDR3 comprises the amino acid sequence shown in Formula V: L-Q-Y-A-X 24 -X 25 -P-Y-T (Formula V); X in Equation V 24 Choose from either T or S; X in Equation V 25 Choose from either F or S; And / or, the VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO:20; And / or, the VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO:

22.

6. The specific binding protein according to any one of claims 1 to 5, characterized in that, The specific binding protein is either a single-specific binding protein or a multi-specific binding protein.

7. An isolated nucleic acid or nucleic acid genome, characterized in that, The isolated nucleic acid or nucleic acid genome encodes the specific binding protein as described in any one of claims 1 to 5.

8. A host cell, characterized in that, Includes the nucleic acid or nucleic acid group as described in claim 7.

9. A method for producing the specific binding protein according to any one of claims 1 to 5, characterized in that, Includes culturing the host cells as described in claim 8; And / or, may also include the recovery of specific binding proteins from the host cells.

10. The use of the specific binding protein according to any one of claims 1 to 5 in the preparation of an antitumor drug.

11. A pharmaceutical composition, characterized in that, It includes the specific binding protein as described in any one of claims 1 to 5 and a pharmaceutical carrier.

12. The use of the specific binding protein according to any one of claims 1 to 5 in the preparation of reagents for detecting RNF149 protein.

13. A reagent kit, characterized in that, Includes the specific binding protein as described in any one of claims 1 to 5, or the specific binding protein encoded by the nucleic acid or nucleic acid group as described in claim 7, or the specific binding protein produced by the host cell as described in claim 8; And / or, the kit may further include one or more reagents for expressing or purifying the specific binding protein and / or one or more reagents for incubating the specific binding protein with the test sample in vitro to achieve specific binding to the target protein.

14. A method for detecting RNF149 protein in a sample, characterized in that, The method includes incubating the sample with the specific binding protein according to any one of claims 1 to 5 or the kit according to claim 13.

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