CN117229393A - Antibodies that specifically bind adeno-associated virus 5 and uses thereof - Google Patents
Antibodies that specifically bind adeno-associated virus 5 and uses thereof Download PDFInfo
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Abstract
The invention relates to the technical field of antibodies, in particular to an antibody specifically binding adeno-associated virus 5 and application thereof. The antibody capable of specifically binding the adeno-associated virus 5 has high affinity to the adeno-associated virus 5, has no cross reaction to adeno-associated viruses of other serotypes, can realize high-specificity and high-sensitivity detection of the adeno-associated virus 5, provides a more accurate quantitative method for titer detection of the adeno-associated virus 5, and has important significance for development of adeno-associated virus 5 gene therapy related preparations.
Description
Technical Field
The invention relates to the technical field of antibodies, in particular to an antibody specifically binding adeno-associated virus 5 and application thereof.
Background
Adeno-Associated Virus (AAV) is a small non-enveloped Virus belonging to the parvoviridae family, externally presenting a 20-sided structure, approximately 26nm in diameter, whose capsid proteins consist of three proteins VP1, VP2 and VP 3. The genome of AAV is a single stranded linear DNA of about 4700bp, comprising two Open Reading Frames (ORFs) upstream and downstream: rep and Cap are located between 2T-shaped Inverted Terminal Repeats (ITRs) each consisting of 145 nucleotides. The ITR functions as a viral replication origin and packaging signal, the Rep gene is involved in viral replication and integration, encodes viral replication proteins, and the Cap gene is responsible for encoding the three viral capsid proteins. Rep and Cap genes exist on the genome of natural wild adeno-associated virus existing in nature, and adeno-associated virus vectors are gene delivery tools modified by taking AAV genome as a framework. Compared with other common virus vectors such as lentiviral vectors, adenovirus vectors, retrovirus vectors and the like, the AAV vector has the advantages of high safety, low immunogenicity, wide host cell range (infected dividing and non-dividing cells), easy production, high penetrability, long-term expression, site-directed integration and the like, and is considered as the most promising gene therapy vector.
Different mutants of capsid proteins produce different AAV subtypes, which can be separated into different serotypes according to the results of the serum test. At present, 13 AAV serotypes (AAV 1-AAV 13) have been isolated from human and monkey bodies, and different AAV serotypes have different capsid protein space structures, sequences and tissue specificities, so that the recognition and the combination of cell surface receptors are also greatly different, and the transfected tissue types, cell types and infection efficiency of different serotypes are different, and AAV viruses of corresponding serotypes need to be selected according to different tissue organs in the process of applying AAV viruses.
Among the usual serotypes of vectors used for gene therapy, AAV5 has a good infection efficiency in retina, nervous system, joint synovium, lung, etc., and has been widely used in the treatment of various rare diseases. Accurate AAV titer determination is an important component of the substance control of AAV gene therapeutic drugs, and is also a precondition for developing preclinical studies and clinical studies. However, there is no accurate kit capable of detecting the titer of AAV5 with high sensitivity, and development of an antibody that specifically binds to AAV5 is of great importance for development of a detection reagent thereof.
Disclosure of Invention
The present invention provides antibodies that specifically bind adeno-associated virus 5 (AVV 5) and uses thereof.
Specifically, the invention provides the following technical scheme:
the present invention provides an antibody or antigen-binding fragment thereof against adeno-associated virus 5, which is any one of the following (1) to (2):
(1) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1. 2 and 3; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 4. 5 and 6;
(2) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 7. 8 and 9; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 10. 11, 12.
Preferably, the antibody or antigen binding fragment thereof is any one of the following (1) - (2):
(1) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14;
(2) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: shown at 16.
Preferably, the antibody or antigen binding fragment thereof is selected from the group consisting of monoclonal antibodies, fab ', F (ab') 2, fd, fv, dAb, complementarity determining region fragments, single chain antibodies, animal-derived antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies, or multispecific antibodies.
In some embodiments of the invention, antibodies are provided with clone number 9A2H3, the amino acid sequences of complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region of which are set forth in SEQ ID NOs: 1. 2 and 3; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 4. 5 and 6; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14.
In some embodiments of the invention, an antibody clone number 10B4E6 is provided having the amino acid sequences of complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region of SEQ ID NO: 7. 8 and 9; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 10. 11, 12; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: shown at 16.
The antibody or antigen binding fragment thereof of the adeno-associated virus 5 provided by the invention can specifically bind adeno-associated virus 5, has no cross reaction with adeno-associated viruses of other serotypes, and has higher affinity to AAV 5.
The present invention provides nucleic acid molecules encoding antibodies or antigen binding fragments thereof to adeno-associated virus 5 as described above.
Based on the amino acid sequences of the above antibodies or antigen binding fragments thereof, the skilled artisan can obtain nucleotide sequences of nucleic acid molecules encoding the above antibodies or antigen binding fragments thereof. Because of the degeneracy of the codons, the nucleotide sequences of the nucleic acid molecules encoding the antibodies or antigen binding fragments thereof are not unique, and all nucleic acid molecules capable of encoding the production of the antibodies or antigen binding fragments thereof are within the scope of the invention.
The invention also provides biological materials containing the nucleic acid molecules; the biological material is an expression cassette, a vector or a host cell.
The above-mentioned expression cassette can be obtained by ligating a transcription or translation regulatory element such as a promoter upstream of the nucleic acid molecule and/or ligating a transcription or translation regulatory element such as a terminator downstream thereof.
Such vectors include, but are not limited to, plasmid vectors, phage vectors, viral vectors, artificial chromosome vectors, and the like.
The host cells include microbial cells, insect cells, or other animal cells.
The invention provides an antibody conjugate, which is obtained by coupling an antibody or an antigen binding fragment thereof of adeno-associated virus 5 with a marker, wherein the marker is one or more selected from enzyme markers, biotin markers, fluorescent dye markers, chemiluminescent dye markers and radioactive markers.
The present invention provides an antibody composition of adeno-associated virus 5 comprising the antibodies in (1) - (2) below:
(1) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1. 2 and 3; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 4. 5 and 6;
(2) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 7. 8 and 9; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 10. 11, 12.
The two antibodies in (1) and (2) above bind to different epitopes in the antigen space.
The antibody composition can be used as a pairing antibody for detecting adeno-associated virus 5 by a double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) method, and two antibodies in the antibody composition are respectively used as a capture antibody and a detection antibody in the double-antibody sandwich ELISA method. Wherein the detection antibody may also carry a detectable label.
In some embodiments of the invention there is provided an antibody composition consisting of the antibody of (1) above or an antibody conjugate thereof and the antibody of (2) above or an antibody conjugate thereof.
The invention provides a detection kit comprising an antibody or antigen-binding fragment thereof to adeno-associated virus 5 as described above, or comprising said antibody conjugate, or comprising said antibody composition.
The kit can be used as a detection kit for adeno-associated virus 5.
In some embodiments of the invention, the detection kit is a double antibody sandwich ELISA detection kit.
Based on the antibody or antigen binding fragment thereof or antibody conjugate or antibody composition, the content of adeno-associated virus 5 in samples such as culture medium, cell lysate and the like can be specifically detected by a double-antibody sandwich ELISA method, and the antibody has higher sensitivity.
In the above kit, the antibody or antigen-binding fragment thereof may further comprise a detectable label; the kit may further comprise a second antibody carrying a detectable label to detect the antibody or antigen binding fragment thereof.
For ease of detection, the kit may also contain other reagents for ELISA detection including, but not limited to, adeno-associated virus 5 standards, PBST washes, blocking solutions, chromogenic solutions, stop solutions, and the like.
Based on the function of the antibody or antigen binding fragment thereof of the invention, the invention provides the use of any of the following of the antibody or antigen binding fragment thereof of adeno-associated virus 5 or the nucleic acid molecule or the biological material or the antibody conjugate or the antibody composition or the detection kit:
(1) Use in detecting the presence or level of adeno-associated virus 5 in a sample;
(2) The application in the quality control of gene therapeutic agent with adeno-associated virus 5 as carrier;
(3) The application in the preparation and/or detection of gene therapeutic drugs with adeno-associated virus 5 as a vector;
(4) Use in the production of adeno-associated virus 5;
(5) The application in the quality control of vaccine using adeno-associated virus 5 as carrier.
In the above (1), the use comprises detecting the presence or level of adeno-associated virus 5 in the sample using the antibody or antigen-binding fragment thereof or the antibody conjugate or the antibody composition or the detection kit.
The method for detecting adeno-associated virus 5 using the antibody or antigen-binding fragment thereof or the antibody conjugate or the antibody composition or the detection kit may use detection methods such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, and the like.
The sample includes a culture medium, a cell lysate, and the like.
In some embodiments of the invention, the antibody or antigen binding fragment thereof is used to detect adeno-associated virus 5 in a double antibody sandwich ELISA.
In the above (2), the application includes: the quality of the gene therapy drug is controlled by detecting the titer of the adeno-associated virus 5 during the preparation of the gene therapy drug.
In the above (4) and (5), the application includes: detecting the titer of adeno-associated virus 5 using said antibody or antigen-binding fragment thereof or said antibody conjugate or said antibody composition or said detection kit.
The present invention provides a method for detecting adeno-associated virus 5, the method comprising: detecting the adeno-associated virus 5 in the sample to be detected by using the antibody or the antigen binding fragment thereof of the adeno-associated virus 5 or the antibody conjugate or the antibody composition of the adeno-associated virus 5 or the detection kit.
The detection can be performed by enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography and the like.
In the present invention, the adeno-associated virus 5 includes naturally occurring adeno-associated virus 5 as well as artificially constructed recombinant adeno-associated virus 5. The recombinant adeno-associated virus 5 expresses a capsid protein of adeno-associated virus 5.
The beneficial effects of the invention at least comprise: the invention provides an antibody capable of specifically binding to adeno-associated virus 5, which has high affinity to adeno-associated virus 5, has no cross reaction to adeno-associated viruses of other serotypes, can realize high-specificity and high-sensitivity detection of adeno-associated virus 5, and is an ideal detection antibody for adeno-associated virus 5 content.
The invention develops the detection reagent of the adeno-associated virus 5 based on the antibody, can specifically detect the content of the adeno-associated virus 5 in a sample, has higher sensitivity, provides a more accurate quantitative method for the titer detection of the adeno-associated virus 5, and has important significance for the development of the adeno-associated virus 5 gene therapy related preparation.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a SDS-PAGE result of example 3 of the present invention, wherein the left panel shows the antibody of clone No. 9A2H3, and the right panel shows the antibody of clone No. 10B4E 6; m is the molecular weight of the protein and R is the antibody sample.
FIG. 2 shows the detection of AAV5 content by the double antibody sandwich ELISA method in example 3 of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention uses recombinant adeno-associated virus 5 as immunogen to perform mouse immunity, and obtains hybridoma cell strain for stably expressing mouse antibody through cell fusion and screening and cell subcloning. Experiments prove that the antibody can specifically identify the adeno-associated virus 5, does not cross other adeno-associated viruses, and can realize accurate detection of the titer of the adeno-associated virus 5. Further, the invention obtains the amino acid sequence of the antibody and the nucleotide sequence of the encoding gene thereof through hybridoma sequencing.
EXAMPLE 1 preparation of AAV5-specific antibodies
1. Immunization of mice: mice were immunized with recombinant adeno-associated virus 5 (AAV 5, available from Acrobiosystems) as an immunogen. After the immunization, the immune response level was determined by detecting serum of the immunized animal by ELISA. After the conventional immunization is finished, if the immunized animal can reach the immune response level aiming at the immunogen, cell fusion is carried out.
2. Screening: the supernatant of the fused cells is screened by ELISA method, and clones which are positive to the specific binding of AAV5 and have no binding with AAV of serotypes such as AAV1, AAV2, AAV3, AAV8, AAV9, AAVdj and the like are selected.
3. Cloning and expanding culture: positive master clone cells were transferred to 24 well plates for expansion culture. Supernatants were collected from each of the expanded clones and tested by ELISA.
4. Subcloning: subcloning positive parent clone by limiting dilution method and subcloning screening by ELISA method.
5. Hybridoma cell antibody gene sequencing: total RNA of the hybridoma cells is extracted, and the RNA is reversely transcribed into cDNA through an RT-PCR reaction. Cloning antibody light chain and heavy chain sequences, constructing the antibody light chain and heavy chain sequences on a T vector, and then carrying out DNA sequencing analysis to obtain antibody gene sequences.
6. Antibody production and purification: the antibody gene sequence obtained in the step 5 is transfected into CHO cells, and is subjected to expansion culture, the antibody is purified by adopting a protein A/G affinity chromatography method, and the purified antibody is stored in Phosphate Buffer (PBS) by adopting a dialysis method.
EXAMPLE 2 analysis of specificity of AAV5 antibodies
8 supernatants of different hybridoma cells were obtained by cell fusion and screening according to the method of example 1, and the AAV5 antibody obtained above was specifically analyzed by ELISA, as follows:
1. dilution of AAV5, AAV1, AAV2, AAV3, AAV8, AAV9, AAVdj to 5X 10 with CBS 9 vg/mL, add to the wells of the microplate, 100 μl per well. The plates were sealed with a plate and left to stand overnight at 4 ℃.
2. The wells were discarded, the ELISA plates were dried, washed with PBST wash solution, 300. Mu.L/Kong Jinpao, and the ELISA plates were dried and washed 3 times.
3. mu.L of blocking agent (PBST wash containing 5% BSA) was added to each well, membrane-sealed with a plate, incubated at 37℃and then washed.
4. The AAV5 antibody cell supernatant described above was added to the ELISA plate at 100. Mu.L per well. And (5) sealing the plates by using sealing plates, placing the plates at 37 ℃ for incubation, and then cleaning.
5. HRP-Anti-Mouse IgG was diluted to 0.05. Mu.g/mL with sample dilution, 100. Mu.L was added to each well, membrane sealed with sealing plate, incubated at 37℃and then washed.
6. 100 mu L of color development liquid is added into each hole, the plates are sealed by sealing plates, and the plates are placed at 37 ℃ for light-proof incubation.
7. 50 mu L of stop solution is added into each hole, and the ELISA plate is gently shaken until color development is uniform.
8. The absorbance values of 450 nm and 630nm were read by an ELISA reader, and the absorbance values were read by OD 450 Knot subtracts OD 630 The absorbance (OD) was obtained, and the measurement results are shown in table 1.
9. Clones that bound strongly to AAV5, weakly or not to AAV1, AAV2, AAV3, AAV8, AAV9, AAVdj were selected for subcloning. Selected clones were: 9A2H3, 10B4E6,2G2A5,6G4B2,3H5G2,9G1F4,9D9H5,8E2B6, where 9A2H3 and 10B4E6 showed high specificity, strong binding to AAV5, and no cross-reactivity with adeno-associated viruses of other serotypes.
TABLE 1 ELISA detection of different antibodies OD values
EXAMPLE 3 identification and functional analysis of AAV5-specific antibodies
In this example, AAV 5-specific antibodies (clone numbers 9A2H3, 10B4E 6) screened in example 2 were identified and functionally analyzed by methods known in the art, as follows:
1. SDS-PAGE identification result (figure 1) shows that the molecular weight of two bands of the antibody of clone No. 9A2H3 subjected to reduction electrophoresis is about 55kDa and 25kDa respectively, and the purity is more than 99%; the two bands of clone No. 10B4E6 were subjected to reduction electrophoresis with molecular weights of about 50kDa and 25kDa, respectively, and had purities of greater than 99%.
2. AAV5 is detected by a double antibody sandwich ELISA method by using antibodies of clone numbers 9A2H3 and 10B4E6, and quantitative detection experimental results (shown in fig. 2 and table 2) of AAV5 show that the antibodies of clone numbers 9A2H3 and 10B4E6 can quantitatively detect AAV5 by using the double antibody sandwich ELISA method, so that the content of AAV5 in a matrix is obtained, the linear correlation is good, and the sensitivity of detecting AAV5 can reach 2.97E+06 vg/mL.
TABLE 2 double antibody sandwich ELISA method for detecting AAV5
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. An antibody or antigen-binding fragment thereof to adeno-associated virus 5, wherein the antibody or antigen-binding fragment thereof is any one of the following (1) - (2):
(1) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1. 2 and 3; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 4. 5 and 6;
(2) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 7. 8 and 9; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 10. 11, 12.
2. The antibody or antigen-binding fragment thereof of adeno-associated virus 5 of claim 1, wherein the antibody or antigen-binding fragment thereof is any one of the following (1) - (2):
(1) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14;
(2) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: shown at 16.
3. The antibody or antigen-binding fragment thereof of adeno-associated virus 5 according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of monoclonal antibodies, fab ', F (ab') 2 Fd, fv, dAb, complementarity determining region fragments, single chain antibodies, animal-derived antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies or multispecific antibodies.
4. A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof against adeno-associated virus 5 according to any one of claims 1 to 3.
5. A biological material comprising the nucleic acid molecule of claim 4, wherein the biological material is an expression cassette, a vector or a host cell.
6. An antibody conjugate, which is characterized in that the antibody or antigen binding fragment thereof of the adeno-associated virus 5 according to any one of claims 1 to 3 is conjugated with a label, wherein the label is one or more selected from the group consisting of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label and a radioactive label.
7. An antibody composition of adeno-associated virus 5, comprising an antibody of (1) - (2) as follows:
(1) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1. 2 and 3; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 4. 5 and 6;
(2) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 7. 8 and 9; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 10. 11, 12.
8. A test kit comprising an antibody or antigen-binding fragment thereof against adeno-associated virus 5 according to any one of claims 1 to 3, or comprising an antibody conjugate according to claim 6, or comprising an antibody composition against adeno-associated virus 5 according to claim 7.
9. Use of an antibody or antigen binding fragment thereof of adeno-associated virus 5 according to any one of claims 1 to 3 or a nucleic acid molecule according to claim 4 or a biological material according to claim 5 or an antibody conjugate according to claim 6 or an antibody composition of adeno-associated virus 5 according to claim 7 or a detection kit according to claim 8, as follows:
(1) Use in detecting the presence or level of adeno-associated virus 5 in a sample;
(2) The application in the quality control of gene therapeutic agent with adeno-associated virus 5 as carrier;
(3) The application in the preparation and/or detection of gene therapeutic drugs with adeno-associated virus 5 as a vector;
(4) Use in the production of adeno-associated virus 5;
(5) The application in the quality control of vaccine using adeno-associated virus 5 as carrier.
10. A method of detecting adeno-associated virus 5, the method comprising: detection of adeno-associated virus 5 in a sample to be tested using an antibody or antigen-binding fragment thereof of adeno-associated virus 5 according to any one of claims 1 to 3 or an antibody conjugate according to claim 6 or an antibody composition of adeno-associated virus 5 according to claim 7 or a detection kit according to claim 8.
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