CN111808198A - Antibody specifically binding RANKL targeted therapeutic drug and application thereof - Google Patents
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Abstract
The invention provides an antibody specifically binding to a RANKL targeted therapeutic drug, which consists of 2 identical heavy chains and 2 identical light chains, wherein the variable region of the heavy chain is provided with three epitope regions, and the amino acid sequence of the epitope region of the variable region of the heavy chain is selected from the amino acid sequences shown in SEQ ID NO.7-SEQ ID NO. 15; the variable region of the light chain has three antigenic determinant regions, and the amino acid sequence of the antigenic determinant regions of the variable region of the light chain is selected from the amino acid sequences shown as SEQ ID NO.16-SEQ ID NO. 24. The antibody of the invention can be specifically combined with the disitumumab, and no competition relationship exists among the antibodies, so that the concentration of the disitumumab in serum can be effectively and accurately detected.
Description
Technical Field
The invention belongs to the field of biochemistry, and particularly relates to an antibody specifically binding to a RANKL targeted therapeutic drug (such as denosumab and/or an analogue thereof), an antibody combination and application thereof.
Background
Denosumab (Denosumab) is a monoclonal antibody produced by american ann company for resisting osteoporosis and bone cancer metastasis, and the antibody can specifically bind to a nuclear factor-kappa B Receptor activator ligand (Receptor activator of nuclear factor kappa-B ligand, RANKL), thereby blocking the binding of RANKL to its membrane Receptor RANK and blocking the signal transduction activation signal of RANKL-RANK in osteoclasts.
The double antibody sandwich method refers to the sequential binding of a pair of noncompetitive antibodies of known antigens, which may be proteins, polypeptides or antibodies, to the antigen, so that a "sandwich" state of "antibody-antigen-antibody to be detected" is formed. When an enzyme-coupled substrate is added, a display reaction/signal change occurs, and the concentration of the protein to be detected is reflected by reading the absorbance/signal change, so that it is called a double antibody sandwich method. Double antibody sandwich method if performed by ELISA method, it is called ELISA double antibody sandwich method. In clinical tests, the double antibody sandwich method is generally used for testing various proteins, antibodies and other macromolecular antigens, such as HBsAg, HBeAg, AFP, hCG and the like. The double-site sandwich method has high specificity and sensitivity, and can reach the ng level generally; and the detected sample and the enzyme-labeled antibody can be subjected to heat preservation reaction together for one-step detection.
Disclosure of Invention
The invention aims to solve the technical problems and provides an antibody capable of being specifically combined with a RANKL targeted therapeutic drug so as to effectively and accurately detect the concentration of the RANKL targeted therapeutic drug in serum.
In order to achieve the above object, the present invention provides an antibody specifically binding to RANKL targeted therapeutic drugs, the antibody consisting of 2 identical heavy chains and 2 identical light chains, wherein the variable region of the heavy chain has three epitope regions, and the amino acid sequence of the epitope region of the variable region of the heavy chain is selected from the amino acid sequences shown in SEQ ID No.7 to SEQ ID No. 15; the variable region of the light chain has three antigenic determinant regions, and the amino acid sequence of the antigenic determinant regions of the variable region of the light chain is selected from the amino acid sequences shown as SEQ ID NO.16-SEQ ID NO. 24.
Preferably, the RANKL targeted therapeutic includes, but is not limited to, denosumab.
The antibody is preferably a murine monoclonal antibody obtained by a mouse immunization method.
Preferably, the amino acid sequence of the variable region of the heavy chain is selected from the amino acid sequences shown as SEQ ID No.1-SEQ ID No.3, and the amino acid sequence of the variable region of the light chain is selected from the amino acid sequences shown as SEQ ID No.4-SEQ ID No. 6.
In a preferred embodiment, the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.1, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO. 4.
In a preferred embodiment, the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.2, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO. 5.
In a preferred embodiment, the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.3, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO. 6.
In another aspect, the invention also provides a kit comprising one or more of the antibodies. Preferably, the kit further comprises a washing solution, a color development solution, a RANKL targeted therapeutic drug standard and a stop solution.
In another aspect, the present invention also provides an antibody combination that specifically binds to a RANKL-targeted therapeutic drug comprising a first antibody and a second antibody, wherein said first and said second antibody are said antibodies and the amino acid sequence of the variable regions of the heavy and light chains of the first antibody is different from the amino acid sequence of the variable regions of the heavy and light chains of the second antibody.
Preferably, the first antibody consists of 2 identical heavy chains and 2 identical light chains, the amino acid sequence of the variable region of the heavy chain of the first antibody is shown as SEQ ID NO.1, and the amino acid sequence of the variable region of the light chain of the first antibody is shown as SEQ ID NO. 4; the second antibody consists of 2 identical heavy chains and 2 identical light chains, the amino acid sequence of the variable region of the heavy chain of the second antibody is shown as SEQ ID No.3, and the amino acid sequence of the variable region of the light chain of the second antibody is shown as SEQ ID No. 6. Or, preferably, the first antibody consists of 2 identical heavy chains and 2 identical light chains, the amino acid sequence of the variable region of the heavy chain of the first antibody is shown as SEQ ID NO.2, and the amino acid sequence of the variable region of the light chain of the first antibody is shown as SEQ ID NO. 5; the second antibody consists of 2 identical heavy chains and 2 identical light chains, the amino acid sequence of the variable region of the heavy chain of the second antibody is shown as SEQ ID No.3, and the amino acid sequence of the variable region of the light chain of the second antibody is shown as SEQ ID No. 6.
In another aspect, the invention also provides a kit comprising the antibody combination. Preferably, the first antibody is used as a detection antibody and the second antibody is used as a coating antibody.
The invention also provides nucleotide sequences encoding the antibodies.
The invention also provides a vector comprising the nucleotide sequence.
The invention also provides a host cell comprising the vector and used for expressing the antibody.
On the other hand, the invention also provides application of the antibody or the antibody combination in preparing a molecular diagnosis or treatment reagent for specifically binding the RANKL targeted treatment drug.
On the other hand, the invention also provides a method for detecting the RANKL targeted therapeutic drug by using a double-antibody sandwich method, in particular a method for detecting the concentration of the RANKL targeted therapeutic drug in serum, which adopts the antibody as a coating antibody and a detection antibody.
Preferably, according to said method, said coated antibody is a label-free antibody and is coated on a solid support.
Preferably, according to said method, said detection antibody is labeled with biotin.
Preferably, according to the method, the sensitivity of the detected concentration is 50ng/ml, with an upper limit of detection of 1280 ng/ml.
The invention provides an antibody capable of being specifically combined with a RANKL targeted therapeutic drug (such as denosumab), and a non-competitive anti-antibody is obtained by combining the antibody, so that a double-antibody sandwich method is constructed to detect the concentration of the RANKL targeted therapeutic drug in serum, and no competitive activity exists between the antibodies. When the concentration of the RANKL targeted therapeutic drug in serum is detected, 2 antibody pairing modes are adopted, one antibody becomes a capture antibody (coating antibody), the other antibody becomes a detection antibody, the sensitivity of the detection concentration reaches 50ng/ml or lower, and the upper limit of detection is 1280ng/ml or higher, which shows that the antibody and antibody combination can effectively and accurately detect the concentration of the RANKL targeted therapeutic drug in the serum.
Drawings
Fig. 1 is a binding curve of 4D11 with diskinumab, showing the binding force EC50 constant of both.
Fig. 2 is a binding curve of 12a6 with diskinumab, showing the binding force EC50 constant of both.
Fig. 3 is a binding curve of 14a10 with diskinumab, showing the binding force EC50 constant of both.
FIG. 4 is a standard curve for a 12A10 coated plate.
Fig. 5 is a standard curve for a 4D11 coated panel.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples, which will become apparent from the following description. It is noted that some conventional technical procedures, reagents and apparatuses are not described in detail in the following examples for the sake of brevity and clarity, but it is understood that the conventional technical procedures, reagents and apparatuses are obvious to those skilled in the art if not specifically stated.
The RANKL targeted therapeutic drug provided by the invention refers to a drug having a targeted blocking effect on RANKL, and includes but is not limited to monoclonal antibodies, pharmaceutical preparations containing monoclonal antibodies as active ingredients, fusion proteins, antisense nucleotides, kinase inhibitors, compounds, traditional Chinese medicine extracts, traditional Chinese medicine compound extracts, or a combination thereof. More preferably, the RANKL targeted therapeutic includes, but is not limited to, denosumab. The following examples illustrate Denosumab (Denosumab), and an antibody targeted to bind Denosumab may be referred to as "anti-Denosumab".
Preparation method of anti-disitumumab (antibody of targeting combined disitumumab)
Dessuzumab was mixed with Freund's Complete Adjuvant (CFA) at a ratio of 1:1 to prepare immunogen, and Balb/c mice were immunized by a first subcutaneous injection according to the monoclonal antibody preparation method, at 50. mu.L per injection point, 300. mu.L per mouse. After 14 days, diskinumab was mixed with incomplete adjuvant (IFA) at a 1:1 volume ratio to prepare an immunogen, and Balb/c mice were immunized by a second subcutaneous injection at 50. mu.L per injection point, 300. mu.L/mouse. After 14 days, diskinumab was mixed with incomplete adjuvant IFA to prepare an immunogen, and Balb/c mice were immunized by a third subcutaneous injection at 50. mu.L per injection point, 300. mu.L per mouse. After a total of 3 subcutaneous immunizations, the tail blood titers of the mice were measured.
Coating an enzyme label plate (1 mu g/mL) with the disitumumab, adding 100 mu L of the enzyme label plate into each hole, and reacting at 4 ℃ overnight; the plate was washed 3 times with PBS solution and blocked with 5% (w/v) mil-PBS for 1h at room temperature. Then, the plate was washed 1 time with PBS solution, and then mouse tail blood was added in a gradient dilution of 1:1000, 1:3000, 1:9000, 1:27000, 1:81000, and 1243000, and reacted at room temperature for 1 hour. The plate was then washed 3 times with PBS solution, patted dry, and then a goat anti-mouse Fc secondary antibody labeled with horseradish peroxidase (HRP) diluted 1:2000 (by volume) was added and reacted at room temperature for 1 h. Washing the plate with PBS solution for 5 times, drying, adding TMB (tetramethylbenzidine) substrate, and reacting for 20min in dark at room temperature; then 50 μ L of stop solution was added, mixed well and then OD450 value was read on a microplate reader. Spleen cells and SP2/0 cell fusions were performed on mice with tail blood titers all exceeding 1:50000 to obtain hybridoma antibodies.
After the mice meeting the conditions are sacrificed by cervical dislocation, splenocytes and SP2/0 cells are taken to be fused and planted in 15 96-well plates, the cells are cultured in a selective culture medium to be monoclonal cell strains, then the selected clones are picked and cloned, and the binding activity of the picked clones is detected. The enzyme-linked plate (10. mu.g/mL) was coated with diskinumab, and 100. mu.L of the coated plate was added to each well, and the reaction was performed overnight at 4 ℃. The plate was washed 3 times with PBS solution and blocked with 5% mik-PBS for 1h at room temperature; the plate was then washed 1 time with PBS solution. The monoclonal cell supernatant was mixed with 5% mil-K-PBS containing 10. mu.g/ml human IgG at a volume ratio of 1:1, reacted at room temperature for 1 hour, and then added to the wells and reacted at room temperature for 1 hour. The plate was then washed 3 times with PBS solution, patted dry, and then a 1:2000 dilution of HRP-labeled goat anti-mouse Fc secondary antibody was added and reacted at room temperature for 1 h. Washing the plate with PBS solution for 5 times, drying, adding TMB reaction substrate, keeping out of the sun, and reacting for 20min at room temperature; then 50 μ L of stop solution was added, mixed well and then OD450 value was read on a microplate reader. A total of 48 positive clones with OD ≥ 2.0 were initially screened from 1410 clones in 15 96-well plates. The results of the positive clone tests are shown in Table 1.
Table 1: preliminary screening positive clone detection result
| Clone# | 14G6 | 1E10 | 13F2 | 15A3 | 13F8 | 6G3 | 14E8 | 14A9 | 15A10 | 15A2 | 14A4 | 13E4 |
| OD(450nm) | 2.937 | 2.935 | 2.868 | 2.866 | 2.837 | 2.831 | 2.828 | 2.804 | 2.804 | 2.758 | 2.758 | 2.751 |
| Clone# | 15E1 | 12A5 | 12A6 | 13F7 | 7B2 | 7E2 | 4C2 | 4D11 | 6H9 | 7B1 | 14A5 | 14A10 |
| OD(450nm) | 2.742 | 2.732 | 2.732 | 2.688 | 2.687 | 2.673 | 2.667 | 2.642 | 2.638 | 2.622 | 2.618 | 2.618 |
| Clone# | 15E3 | 3G6 | 12A9 | 12D5 | 12A8 | 4C3 | 7B10 | 15B5 | 4B7 | 4C1 | 7B4 | 4C4 |
| OD(450nm) | 2.590 | 2.557 | 2.554 | 2.547 | 2.538 | 2.537 | 2.519 | 2.501 | 2.499 | 2.482 | 2.478 | 2.477 |
| Clone# | 15D8 | 7B11 | 15C6 | 7B3 | 1D5 | 15C12 | 1D8 | 12F2 | 15F12 | 12D6 | 13F6 | 4F10 |
| OD(450nm) | 2.462 | 2.441 | 2.425 | 2.402 | 2.288 | 2.227 | 2.224 | 2.218 | 2.140 | 2.133 | 2.026 | 2.000 |
| Clone# | NC | PC | ||||||||||
| OD(450nm) | 0.053 | 2.536 |
Note: NC is negative control, PC is positive control 4# mouse heart blood.
These antibodies were all detected as IgG class antibodies.
The anti-dessumab of the present invention is a murine whole antibody (IgG) comprising two identical heavy chains and two identical light chains. Wherein the variable region of the heavy chain and the variable region of the light chain are each composed of 3 antigen Complementarity Determining Regions (CDRs) and 4 framework regions (Framwork). The antigen complementarity determining regions of the variable regions of the heavy chain and the variable regions of the light chain determine the binding specificity of the antibody for antigen. The amino acid sequences of the constant regions of the heavy and light chains generally do not affect the binding specificity of the antibody for the antigen. Preferably, the amino acid sequence of the constant region of the heavy chain is shown in SEQ ID NO.25, and the amino acid sequence of the constant region of the light chain is shown in SEQ ID NO. 26.
The amino acid sequence of the heavy chain variable region of the anti-dessuzumab disclosed by the invention can be shown as SEQ ID No.1, wherein the amino acid sequences of 3 antigen complementarity determining regions of the heavy chain variable region are shown as H _ CDR1(SEQ ID No.7), H _ CDR2(SEQ ID No.8) and H _ CDR2(SEQ ID No.9) (also can have various different tandem sequences). Alternatively, the amino acid sequence of the heavy chain variable region may be as shown in SEQ ID NO.2, wherein the amino acid sequences of the 3 antigen complementarity determining regions of the heavy chain variable region are as shown in H _ CDR4(SEQ ID NO.10), H _ CDR5(SEQ ID NO.11), H _ CDR6(SEQ ID NO.12) (there may also be a plurality of different tandem sequences); alternatively, the amino acid sequence of the heavy chain variable region may be as shown in SEQ ID NO.3, wherein the amino acid sequences of the 3 antigen complementarity determining regions of the heavy chain variable region are as shown in H _ CDR7(SEQ ID NO.13), H _ CDR8(SEQ ID NO.14), H _ CDR9(SEQ ID NO.15) (or there may be a plurality of different tandem sequences).
The amino acid sequence of the light chain variable region of the anti-disitumumab of the present invention can be shown as SEQ ID No.4, wherein the amino acid sequences of 3 antigen complementarity determining regions of the light chain variable region are shown as L _ CDR1(SEQ ID No.16), L _ CDR2(SEQ ID No.17), L _ CDR3(SEQ ID No.18) (also can have a plurality of different tandem sequences); alternatively, the amino acid sequence of the light chain variable region may be as shown in SEQ ID No.5, wherein the amino acid sequences of the 3 antigen complementarity determining regions of the light chain variable region are as shown in L _ CDR4(SEQ ID No.19), L _ CDR5(SEQ ID No.20), L _ CDR6(SEQ ID No.21) (there may also be a plurality of different tandem sequences); alternatively, the amino acid sequence of the light chain variable region may be as shown in SEQ ID NO.6, wherein the amino acid sequences of the 3 antigen complementarity determining regions of the light chain variable region are as shown in L _ CDR7(SEQ ID NO.22), L _ CDR8(SEQ ID NO.23), L _ CDR9(SEQ ID NO.24) (or there may be a plurality of different tandem sequences).
The amino acid sequence of any of the above heavy chain variable regions may be combined with the amino acid sequence of any of the light chain variable regions to construct an antibody comprising 2 heavy chains and 2 light chains. For example, the amino acid sequence of each heavy chain variable region of the antibody numbered 4D11 is SEQ ID No.1, and the amino acid sequence of each light chain variable region is SEQ ID No. 4; the amino acid sequence of each heavy chain variable region of the antibody with the number of 12A6 is SEQ ID NO.2, and the amino acid sequence of each light chain variable region is SEQ ID NO. 5; the amino acid sequence of each heavy chain variable region of the antibody numbered 14A10 is SEQ ID No.3, and the amino acid sequence of each light chain variable region is SEQ ID No. 6. The following three antibodies are used as examples to illustrate the application, but it should be understood that the antibodies of the present invention are not limited to these three specific examples.
The amino acid sequences of the antibodies in Table 1 above can be detected by commercial institutions or known methods, and the desired antibodies can be synthesized based on the corresponding amino acid sequences.
Example 1: detection of binding force of disitumumab to disitumumab
And (3) detecting the binding force of each anti-disitumumab antibody to the disitumumab by using an ELISA method.
Firstly, the denosumab is diluted to 5 mu g/ml by using a sodium carbonate buffer solution (CBS, pH9.0) with pH9.0, then a 5 mu g/ml denosumab solution is coated on an enzyme label plate, 50 mu L/hole and placed at 2-8 ℃ for overnight incubation. After incubation was complete, the plates were washed 4 times with PBST, 300. mu.L/well. 1% BSA was added to each well at 100. mu.L/well and incubated at 25 ℃ for 30 min. After incubation was complete, the plates were washed 4 times with PBST, 300. mu.L/well. BSA (bovine serum albumin) was set as a negative control, and each anti-dessumab (4D11, 12A6, 14A10) was set as a test group and incubated with the enzyme-labeled plate-coated dessumab at concentrations of 1000.00, 333.33, 111.11, 37.04, 12.35, 4.12, 1.37ng/ml, 25 degrees, 1 h. After incubation was complete, the plates were washed 4 times with PBST, 300. mu.L/well. Commercial anti-mouse antibody (Jackson, # 115-. After incubation was complete, the plates were washed 4 times with PBST, 300. mu.L/well. Adding 50 μ L of TMB color developing solution into each well for color development reaction, and incubating for 3-5 min. After the color development reached a predetermined end point, 50. mu.L of a reaction terminator was added. The absorbance at 450nm (OD450) was measured in a 96-well plate using a microplate reader.
Binding curves of each antibody to denosumab are shown in fig. 1-3, which show binding force EC50 constants of 4D11, 12a6, 14a10 to denosumab, respectively. As shown in the figure, the binding force EC50 of the anti-disitumumab 4D11 and the disitumumab is 6.07ng/ml, the binding force EC50 of the anti-disitumumab 12A6 and the disitumumab is 4.23ng/ml, and the binding force EC50 of the anti-disitumumab 14A10 and the disitumumab is 7.87 ng/ml.
Example 2: destuzumab dolizumab 14A10 and 12A6 detection on serum concentration of Destuzumab
In this example, the concentration of the quality control sample was measured by a standard curve method, and the standard deviation and recovery rate of the standard substance and the quality control substance were calculated.
The standard substance is prepared by dessuzumab according to 1280, 960, 640, 480, 320, 180, 90 and 50ng/ml, the quality control substance is prepared by dessuzumab according to 960, 720, 360, 200 and 90ng/ml, and the solvent is 10% v/v calf serum. The standard substance and the quality control substance are added into an enzyme label plate coated by a 12A6 antibody according to the design of 3 wells, and the mixture is incubated for 1h at 25 ℃. After incubation was complete, the plates were washed 4 times with PBST, 300. mu.L/well. Biotin-labeled 14A10 antibody was then added at 100. mu.L/well and incubated for 1h at 25 ℃. After incubation was complete, the plates were washed 4 times with PBST, 300. mu.L/well. Streptavidin-horseradish peroxidase (Streptavidin-HRP) substrate was then added at 100. mu.L/well and incubated for 1h at 25 ℃. After incubation was complete, the plates were washed 4 times with PBST, 300. mu.L/well. Adding 50 mu LTMB of developing solution into each well for developing reaction, and incubating for 3-5 min. After the color development reached a predetermined end point, 50. mu.L of a reaction terminator was added. The absorbance at 450nm (OD450) was measured in a 96-well plate using a microplate reader. Figure 4 shows a standard curve for a 12a6 coated plate.
After the standard curve is calibrated by using the standard substance, the theoretical value of each standard substance is subjected to back calculation analysis by using the obtained standard curve and each parameter, and the back calculation analysis result and the recovery rate are shown in table 2. In Table 2, SD denotes a standard deviation and CV denotes a coefficient of variation (coefficient of variation).
Table 2: calculating the concentration and recovery rate of each standard sample according to the fitted standard curve
The concentration of each quality control product was calculated and analyzed by back calculation using the obtained standard curve and each parameter, and the analysis results and recovery rate were shown in table 3.
Table 3: calculating the concentration and recovery rate of each quality control sample according to the fitted standard curve
Example 3: destuzumab dolizumab 14A10 and 4D11 detection of serum concentration
In this example, the concentration of the quality control sample was measured by a standard curve method, and the standard deviation and recovery rate of the standard substance and the quality control substance were calculated.
The standard substance is prepared by dessuzumab according to 1280, 960, 640, 480, 320, 180, 90 and 50ng/ml, the quality control sample is prepared by dessuzumab according to 960, 720, 360, 200 and 90ng/ml, and the solvent is 10% (v/v) calf serum. Adding the standard substance and the quality control substance into a 4D11 antibody-coated enzyme label plate according to the design of 3-time wells, and incubating for 1h at 25 ℃. After incubation was complete, the plates were washed 4 times with PBST (phosphate buffered saline (PBS) + Tween-20) at 300. mu.L/well. Biotin-labeled 14A10 antibody was then added at 100. mu.L/well and incubated for 1h at 25 ℃. After incubation was complete, the plates were washed 4 times with PBST, 300. mu.L/well. SA-HRP substrate was then added, 100. mu.L/well, and incubated for 1h at 25 ℃. After incubation was complete, the plates were washed 4 times with PBST, 300. mu.L/well. Adding 50 mu LTMB of developing solution into each well for developing reaction, and incubating for 3-5 min. After the color development reached a predetermined end point, 50. mu.L of a reaction terminator was added. The absorbance at 450nm (OD450) was measured in a 96-well plate using a microplate reader. Fig. 5 shows a standard curve for a 4D11 coated panel.
After the standard curve is calibrated by using the standard substance, the theoretical value of each standard substance is subjected to back calculation analysis by using the obtained standard curve and each parameter, and the back calculation analysis result and the recovery rate are shown in table 4.
Table 4: calculating the concentration and recovery rate of each standard sample according to the fitted standard curve
The concentrations of the quality control substances were calculated and analyzed by back calculation using the obtained standard curves and parameters, and the analysis results and recovery rate were shown in Table 5.
Table 5: calculating the concentration and recovery rate of each quality control sample according to the fitted standard curve
As can be seen from the above experimental data, the combination of anti-disitumumab 14a10 and 12a6 and the combination of anti-disitumumab 14a10 and 4D11 can effectively and accurately detect the concentration of the disitumumab in the serum.
The above embodiments are merely exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Sequence listing
<110> mountain Applance biomedical products Co., Ltd
<120> antibody specifically binding to RANKL targeted therapeutic drug and application thereof
<160>26
<170>SIPOSequenceListing 1.0
<210>1
<211>120
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>1
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asp Tyr
20 25 30
Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Val Ile Asn Pro Gly Ser Gly Val Thr Ser Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Ile Tyr Tyr Asp Tyr Asp Glu Tyr Tyr Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Leu Thr Val Ser Ser
115 120
<210>2
<211>120
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>2
Glu Val Met Leu Val Glu Ser Gly Gly Gly Ser Ser Glu Leu Glu Gly
1 5 10 15
Pro Glu Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala
20 25 30
Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala
35 40 45
Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 7075 80
Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Arg Leu Gly Gly Gly Tyr Asp Asp Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
<210>3
<211>119
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>3
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Met Lys Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Asp Ile Asn Pro Asn Asn Gly Tyr Thr Phe Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Leu Tyr Tyr Asp Ser Asp Gly Glu Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser
115
<210>4
<211>111
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>4
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser
20 25 30
Ser Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Leu Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln His Ser Trp
85 90 95
Glu Ile Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210>5
<211>112
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>5
Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Gln Leu Cys Arg Trp
1 5 10 15
Arg Glu Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Ser Asn Gln Lys Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Thr Tyr Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105 110
<210>6
<211>107
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>6
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210>7
<211>8
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>7
Gly Tyr Ala Phe Thr Asp Tyr Leu
1 5
<210>8
<211>8
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>8
Ile Asn Pro Gly Ser Gly Val Thr
1 5
<210>9
<211>13
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>9
Ala Ile Tyr Tyr Asp Tyr Asp Glu Tyr Tyr Phe Asp Tyr
1 5 10
<210>10
<211>8
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>10
Gly Phe Thr Phe Ser Ser Tyr Ala
1 5
<210>11
<211>8
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>11
Ile Ser Ser Gly Gly Ser Tyr Thr
1 5
<210>12
<211>14
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>12
Ala Arg Leu Gly Gly Gly Tyr Asp Asp Tyr Ala Met Asp Tyr
1 5 10
<210>13
<211>8
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>13
Gly Tyr Thr Phe Thr Asp Tyr Tyr
1 5
<210>14
<211>8
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>14
Ile Asn Pro Asn Asn Gly Tyr Thr
1 5
<210>15
<211>12
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>15
Ala Leu Tyr Tyr Asp Ser Asp Gly Glu Phe Asp Tyr
1 5 10
<210>16
<211>10
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>16
Gln Ser Val Ser Thr Ser Ser Tyr Ser Tyr
1 510
<210>17
<211>3
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>17
Tyr Ala Ser
1
<210>18
<211>9
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>18
Gln His Ser Trp Glu Ile Pro Arg Thr
1 5
<210>19
<211>12
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>19
Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Phe
1 5 10
<210>20
<211>3
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>20
Trp Ala Ser
1
<210>21
<211>8
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>21
Gln Gln Tyr Tyr Thr Tyr Leu Thr
1 5
<210>22
<211>6
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>22
Gln Asp Ile Ser Asn Tyr
1 5
<210>23
<211>3
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>23
Tyr Thr Ser
1
<210>24
<211>9
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>24
Gln Gln Gly Asn Thr Leu Pro Trp Thr
1 5
<210>25
<211>246
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>25
Ser Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Cys Pro Pro Cys Lys
1 5 10 15
Glu Cys His Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser Val
20 25 30
Phe Ile Phe Pro Pro Asn Ile Lys Asp Val Leu Met Ile Ser Leu Thr
35 40 45
Pro Lys Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp
50 55 60
Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln
65 70 75 80
Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Ile Arg Val Val Ser
85 90 95
Thr Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys
100 105 110
Cys Lys Val Asn Asn Lys Asp Leu Pro Ser Pro Ile Glu Arg Thr Ile
115 120 125
Ser Lys Ile Lys Gly Leu Val Arg Ala Pro Gln Val Tyr Ile Leu Pro
130 135 140
Pro Pro Ala Glu Gln Leu Ser Arg Lys Asp Val Ser Leu Thr Cys Leu
145 150 155 160
Val Val Gly Phe Asn Pro Gly Asp Ile Ser Val Glu Trp Thr Ser Asn
165 170 175
Gly His Thr Glu Glu Asn Tyr Lys Asp Thr Ala Pro Val Leu Asp Ser
180 185 190
Asp Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asn Met Lys Thr Ser Lys
195 200 205
Trp Glu Lys Thr Asp Ser Phe Ser Cys Asn Val Arg His Glu Gly Leu
210 215 220
Lys Asn Tyr Tyr Leu Lys Lys Thr Ile Ser Arg Ser Pro Gly Leu Asp
225 230 235 240
Leu Asp Asp Ile Cys Ala
245
<210>26
<211>103
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>26
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
1 5 10 15
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
20 25 30
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
35 40 45
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
50 55 60
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
65 70 75 80
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
85 90 95
Ser Phe Asn Arg Gly Glu Cys
100
Claims (10)
1. An antibody specifically binding to a RANKL targeted therapeutic drug, which consists of 2 identical heavy chains and 2 identical light chains, and is characterized in that the variable region of the heavy chain has three epitope regions, and the amino acid sequence of the epitope region of the variable region of the heavy chain is selected from the amino acid sequences shown as SEQ ID No.7-SEQ ID No. 15; the variable region of the light chain has three antigenic determinant regions, and the amino acid sequence of the antigenic determinant regions of the variable region of the light chain is selected from the amino acid sequences shown as SEQ ID NO.16-SEQ ID NO. 24.
2. The antibody of claim 1, wherein the amino acid sequence of the variable region of the heavy chain is selected from the group consisting of the amino acid sequences shown in SEQ ID No.1-SEQ ID No.3, and the amino acid sequence of the variable region of the light chain is selected from the group consisting of the amino acid sequences shown in SEQ ID No.4-SEQ ID No. 6.
3. A kit containing one or more of the antibodies of claim 1 or 2.
4. An antibody combination specifically binding to a RANKL-targeted therapeutic drug comprising a first antibody and a second antibody, characterized in that said first and second antibodies are antibodies according to claim 1 or 2 and the amino acid sequence of the variable regions of the heavy and light chains of said first antibody is different from the amino acid sequence of the variable regions of the heavy and light chains of said second antibody.
5. A kit comprising the antibody combination of claim 4.
6. A nucleotide sequence encoding the antibody of claim 1 or 2.
7. A vector comprising the nucleotide sequence of claim 6.
8. A host cell comprising the vector of claim 7.
9. Use of an antibody according to claim 1 or 2 for the preparation of a molecular diagnostic or therapeutic agent for the specific binding to RANKL targeted therapeutic drugs.
10. A method for detecting RANKL targeted therapeutic drugs using a double antibody sandwich method, characterized in that the antibody according to claim 1 or 2 is used as a coating antibody and a detection antibody.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112679614A (en) * | 2021-01-15 | 2021-04-20 | 广东安普泽生物医药股份有限公司 | Antibody for specifically binding RANKL targeted therapeutic drug and application thereof |
| CN114773478A (en) * | 2022-03-14 | 2022-07-22 | 深圳市国创纳米抗体技术有限公司 | anti-AFP antigen VHH domain and bispecific antibodies containing same |
| CN117264071A (en) * | 2023-11-22 | 2023-12-22 | 江苏迈威康新药研发有限公司 | Binding agent of anti-RANKL monoclonal antibody or derivative thereof and application thereof |
| CN117285637A (en) * | 2023-11-22 | 2023-12-26 | 江苏迈威康新药研发有限公司 | Anti-idiotype antibody and application thereof |
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| CN112679614B (en) * | 2021-01-15 | 2022-08-16 | 广东安普泽生物医药股份有限公司 | Antibody for specifically binding RANKL targeted therapeutic drug and application thereof |
| CN114773478A (en) * | 2022-03-14 | 2022-07-22 | 深圳市国创纳米抗体技术有限公司 | anti-AFP antigen VHH domain and bispecific antibodies containing same |
| CN114773478B (en) * | 2022-03-14 | 2023-05-23 | 深圳市国创纳米抗体技术有限公司 | anti-AFP antigen VHH domain and bispecific antibody containing same |
| CN117264071A (en) * | 2023-11-22 | 2023-12-22 | 江苏迈威康新药研发有限公司 | Binding agent of anti-RANKL monoclonal antibody or derivative thereof and application thereof |
| CN117285637A (en) * | 2023-11-22 | 2023-12-26 | 江苏迈威康新药研发有限公司 | Anti-idiotype antibody and application thereof |
| CN117285637B (en) * | 2023-11-22 | 2024-03-22 | 江苏迈威康新药研发有限公司 | Anti-idiotype antibody and application thereof |
| CN117264071B (en) * | 2023-11-22 | 2024-03-22 | 江苏迈威康新药研发有限公司 | Binding agent of anti-RANKL monoclonal antibody or derivative thereof and application thereof |
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|---|---|
| CN111808198B (en) | 2022-06-03 |
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Address after: Room 303, No.23 Jinzhong Road, Huangpu District, Guangzhou City, Guangdong Province Applicant after: Guangdong anpuze biomedical Co., Ltd Address before: 528231, Guangdong Province, Nanhai District, Foshan Province, 321 National Road, immortal Creek, Guangdong biological medicine industry base, B building, building six Applicant before: AMPO BIOTECHNOLOGY Inc. |
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