Binding protein containing AAV-9 antigen binding domain

Abstract

The invention relates to a binding protein containing an AAV-9 antigen binding domain, and relates to the technical field of immunization. 1, the antigen binding domain comprises a complementary determining region CDR-VH1, a complementary determining region CDR-VH2 and a complementary determining region CDR-VH 3; the amino acid sequence of the complementary determining region CDR-VH1 is D-Y-T-M-X1, and X1 is any one of A, H, S, T or N; the amino acid sequence of the complementarity determining region CDR-VH2 is A-V-S-W-S-G-X2-T-X3-Y-A-D-S-V-X4-G, the X2 is any one of F, T, N, S or R, the X3 is any one of K, Y, F, H or D, and the X4 is any one of K, R, E, A or Q; the amino acid sequence of the complementarity determining region CDR-VH 3 is G-S-T-G-P-L-A-X5-R-T-S-P-G-D-Y-D-X6, the X5 is any one of A, Q, G, T or V, and the X6 is any one of R, Y, S, F or H. The invention also discloses a preparation method of the complementarity determining region CDR-VH 3. The binding protein provided by the invention is strong in activity, has very high affinity with AAV-9, and has better affinity compared with a common AVV-9 antibody in the market at present.

Description

Technical Field

[0001] The present invention relates to the field of immunotechnology, and in particular to a binding protein comprising an AAV-9 antigen binding domain. Background Art

[0002] Adeno-associated virus (AAV) was first discovered in the mid-1960s from laboratory adenovirus (AdV) preparations and was soon found in human tissues. Driven by pure scientific curiosity, a few research groups began to understand the process of basic AAV biology, without realizing its great potential as a human gene therapy platform. During the first 15-20 years of AAV research, several important aspects of AAV were characterized, including its genome configuration and composition, DNA replication and transcription, infection latency, and virion assembly.

[0003] AAV belongs to the genus Dependoparvovirus in the family Parvoviridae. Its life cycle depends on the presence of a helper virus, such as AdV. AAV is found in a variety of vertebrates, including humans and non-human primates (NHPs). The current consensus is that AAV does not cause any human disease. It consists of an icosahedral protein capsid with a diameter of about 26 nm and a single-stranded DNA genome of ~4.7 kb. The capsid contains three types of subunits VP1, VP2 and VP3, with a total of 60 copies in a ratio of 1:1:10 (VP1:VP2:VP3). At this stage, researchers have discovered 12 human AAV serotypes (AAV1 to AAV12) and more than 100 non-human primate AAV serotypes. Different AAV serotypes have different capsid protein spatial structures, sequences and tissue specificities, so the cell surface receptors they recognize and bind to are also very different, which also leads to different tissue types, cell types and infection efficiencies of different serotypes.

[0004] Today, recombinant AAV (rAAV) has become the main platform for in vivo gene therapy delivery. The first rAAV gene therapy product was uniQure's alipogene tiparvovec (Glybera), which was approved by the European Medicines Agency (EMA) in 2012 for the treatment of lipoprotein lipase deficiency; in 2018, the FDA approved voretigene neparvovec-rzyl (Luxturna), the first rAAV gene therapy licensed in the United States. For therapeutic AAV vectors, accurate titer determination is an important part of AAV gene therapy drug quality control, and stable and reliable titers can ensure accurate dosing.

[0005] The physical titer of AAV generally refers to the genome titer or capsid titer. The genome titer is often detected by qPCR and Digital droplet PCR (ddPCR). Due to differences in sample preparation, primer design, PCR efficiency, etc., different batches and laboratories of qPCR will produce experimental deviations. Although ddPCR can overcome some limitations of qPCR, different sample processing methods will still produce deviations. The capsid titer is mainly detected by ELISA, which has the advantages of reliability and good reproducibility. For AAV vectors of different serotypes used in gene therapy, especially AAV vectors that have been optimized and modified in sequence structure, there is a need to develop specific antibodies as capture and detection antibodies to determine the AAV titer by ELISA. In view of this, the present invention is specially proposed. Summary of the invention

[0006] The present invention relates to a novel binding protein comprising an AAV-9 antigen binding domain, and studies the preparation and application of the binding protein.

[0007] The antigen binding domain comprises at least one complementary determining region selected from the following amino acid sequences; or has at least 80% sequence identity with the complementary determining region of the following amino acid sequence and has an affinity with AVV-9 of KD ≤ 8.14 × 10 9:

[0008] The complementarity determining region CDR-VH1 is DYTM-X1, wherein X1 is any one of A, H, S, T or N.

[0009] The complementary determining region CDR-VH2 is AVSWSGS-X2-T-X3-YADSV-X4-G, wherein X2 is any one of F, T, N, S or R, X3 is any one of K, Y, F, H or D, and X4 is any one of K, R, E, A or Q;

[0010] The complementary determining region CDR-VH3 is GSTGPLA-X5-RTSPGDYD-X6, wherein X5 is any one of A, Q, G, T or V, and X6 is any one of R, Y, S, F or H.

[0011] An important advantage of the present invention is that the binding protein has strong activity, has a high affinity for AAV-9, and has better affinity than the common AAV-9 antibodies currently on the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the SDS-PAGE picture of the antibody obtained in Example 1. DETAILED DESCRIPTION

[0013] The present invention is described in further detail below.

[0014] Definition

[0015] "Binding protein comprising an antigen binding domain" generally refers to all proteins / protein fragments comprising a CDR region.

[0016] The term "antibody" includes polyclonal antibodies and monoclonal antibodies and antigen compound binding fragments of these antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies and antibody minimum recognition units, as well as single-chain derivatives of these antibodies and fragments. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD. In addition, the term "antibody" includes naturally occurring antibodies and non-naturally occurring antibodies, including, for example, chimeric, bifunctional and humanized antibodies, and related synthetic isoforms. The term "antibody" can be used interchangeably with "immunoglobulin".

[0017] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are generally the most variable parts of an antibody and contain the antigen binding site. The light or heavy chain variable region (VL or VH) consists of a framework region interrupted by three hypervariable regions called "complementarity determining regions" or "CDRs". The extent of the framework region and CDR has been precisely defined, for example in Kabat (see Sequences of Proteins of Immunological Interest, E. Kabat et al., U.S. Department of Health and Human Services, (1983)) and Chothia. The framework region of an antibody, i.e., the combined framework region of the constituent light and heavy chains, serves to position and align the CDRs, which are primarily responsible for binding to the antigen.

[0018] As used herein, "framework" or "FR" regions mean regions of antibody variable domains excluding those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into adjacent regions (FR-H1, FR-H2, FR-H3, and FR-H4) separated by CDRs.

[0019] The variable regions VL / VH of the heavy and light chains can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: FR-H1 CDR1 FR-H2 CDR2 FR-H3 CDR3 FR-H4.

[0020] The binding protein of the present application includes the framework regions FR-H1, FR-H2, FR-H3 and FR-H4 whose amino acid sequences are shown below in sequence:

[0021] FR-H1: QVQLQESGGGSVQAGGSLRLSCVASGSRFH;

[0022] FR-H2: WFRQPPGKAREFVA;

[0023] FR-H3:RFTISRDNAKNTVLLQMNNLKPEDTADYYCAA;

[0024] FR-H4:WGQGTQVTVSS.

[0025] The binding protein of the present application includes a complementarity determining region CDR-VH1, a complementarity determining region CDR-VH2, and a complementarity determining region CDR-VH3.

[0026] Among them, the amino acid sequence of the complementary determining region CDR-VH1 is: DYTM-X1:

[0027] In CDR-VH1 example 1, X1 is A,

[0028] In CDR-VH1 Example 2, X1 is H,

[0029] In CDR-VH1 example 3, X1 is S,

[0030] In CDR-VH1 example 4, X1 is T,

[0031] In CDR-VH1 example 5, X1 is N.

[0032] Among them, the amino acid sequence of the complementary determining region CDR-VH2 is: AVSWSGS-X2-T-X3-YADSV-X4-G, X2 is F, T, N, S or R, X3 is K, Y, F, H or D, and X4 is K, R, E, A or Q. Examples of CDR-VH2 are listed in Table 1.

[0033] Table 1

[0034] CDR-VH2 Example 1 A-V-S-W-S-G-S-F-T-K-Y-A-D-S-V-K-G CDR-VH2 Example 2 A-V-S-W-S-G-S-T-T-Y-Y-A-D-S-V-R-G CDR-VH2 Example 3 A-V-S-W-S-G-S-N-T-F-Y-A-D-S-V-E-G CDR-VH2 Example 4 A-V-S-W-S-G-S-S-T-H-Y-A-D-S-V-A-G CDR-VH2 Example 5 A-V-S-W-S-G-S-R-T-D-Y-A-D-S-V-Q-G

[0035] Among them, the complementary determining region CDR-VH3 is GSTGPLA-X5-RTSPGDYD-X6, X5 is A, Q, G, T or V, and X6 is R, Y, S, F or H. Examples of CDR-VH3 are listed in Table 2.

[0036] Table 2

[0037] CDR-VH3 Example 1 G-S-T-G-P-L-A-A-R-T-S-P-G-D-Y-D-R CDR-VH3 Example 2 G-S-T-G-P-L-A-Q-R-T-S-P-G-D-Y-D-Y CDR-VH3 Example 3 G-S-T-G-P-L-A-G-R-T-S-P-G-D-Y-D-S CDR-VH3 Example 4 G-S-T-G-P-L-A-T-R-T-S-P-G-D-Y-D-F CDR-VH3 Example 5 G-S-T-G-P-L-A-V-R-T-S-P-G-D-Y-D-H

[0038] The sequence after combining FR-H1 CDR1 FR-H2 CDR2 FR-H3 CDR3 FR-H4 is:

[0039] QVQLQESGGGSVQAGGSLRLSCVASGSRFH DYTMX1 WFRQPPGKAREFVA AVSWSGSX2TX3YADSVX4G RFTISRDNAKNTVLLQMNNLKPEDTADYYCAA GSTGPL AX5RTSPGDYDX6WGQGTQVTVSS

[0040] The amino acid sequence of Example 1 obtained is shown in SEQ NO. 1: QVQLQESGGGSVQAGGSLRLSCVASGSRFH DYTMA WFRQPPGKAREFVA AVSWSGSFTKYADSVKG RFTISRDNAKNTVLLQMNNLKPEDTADYYCAA GSTGPLAARTSPGDYDR WGQGTQVTVSS

[0041] The amino acid sequence of Example 2 obtained is shown in SEQ NO2: QVQLQESGGGSVQAGGSLRLSCVASGSRFH DYTMH WFRQPPGKAREFVA AVSWSGSTTYYADSVRG RFTISRDNAKNTVLLQMNNLKPEDTADYYCAAGSTGPLAQRTSPGDYDY WGQGTQVTVSS

[0042] The amino acid sequence of Example 3 obtained is shown in SEQ NO 3: QVQLQESGGGSVQAGGSLRLSCVASGSRFH DYTMS WFRQPPGKAREFVA AVSWSGSNTFYADSVEG RFTISRDNAKNTVLLQMNNLKPEDTADYYCAAGSTGPLAGRTSPGDYDS WGQGTQVTVSS

[0043] The amino acid sequence of Example 4 obtained is shown in SEQ NO 4: QVQLQESGGGSVQAGGSLRLSCVASGSRFH DYTMT WFRQPPGKAREFVA AVSWSGSSTHYADSVAG RFTISRDNAKNTVLLQMNNLKPEDTADYYCAAGSTGPLATRTSPGDYDF WGQGTQVTVSS

[0044] The amino acid sequence of Example 5 obtained is shown in SEQ NO5: QVQLQESGGGSVQAGGSLRLSCVASGSRFH DYTMN WFRQPPGKAREFVA AVSWSGSRTDYADSVQG RFTISRDNAKNTVLLQMNNLKPEDTADYYCAAGSTGPLAVRTSPGDYDH WGQGTQVTVSS

[0045] 1. The following is an exemplary method for preparing anti-AAV-9 recombinant antibodies.

[0046] Get variable region genes:

[0047] (1) Designing heavy and light chain extraction primers, and using the primers to extract the heavy and light chain genes of the antibody from the phage strain with the best detection effect;

[0048] (2) Primers:

[0049] F:ttggagattttcaacgtgaaa

[0050] R:tgtcgtctttccagacgttagtaaa

[0051] (3) PCR reaction

[0052] 1) Take the PCR tube, prepare the PCR reaction solution according to the following reaction system, and perform the PCR reaction

[0053] 2) PCR reaction system

[0054]

[0055]

[0056] To ensure the accuracy of the reaction solution preparation, the MasterMix should be prepared according to (number of reactions + 0.5) times the reaction volume, and then dispensed into each reaction tube, and finally the template sample was added. The PCR cycle conditions refer to the table below.

[0057] 3) PCR Cycling Conditions

[0058] (4) Gel recovery: The antibody heavy and light chain genes amplified by PCR were recovered by agarose gel electrophoresis and inserted into the pCDNA3.1 expression vector.

[0059] (5) Transformation and plasmid extraction

[0060] Transform the expression plasmid into competent cells:

[0061] 1) Take out the prepared competent cells and thaw them on ice;

[0062] 2) 100 μl competent cells correspond to 5 μg plasmid DNA, then use a pipette to mix evenly and place on ice for 30 minutes;

[0063] 3) Heat shock in a 42°C water bath for 90 seconds, followed by an ice bath for 3 minutes;

[0064] 4) Add 900 μl of antibiotic-free LB medium and incubate on a shaker at 37°C for 45 minutes to allow the bacteria to recover;

[0065] 5) Take an appropriate volume of the revived bacteria and spread it evenly on an LB plate containing antibiotics, invert the LB plate, and culture it at 37°C for 12 to 16 hours to complete the transformation.

[0066] 6) Perform plasmid extraction on the transformed plasmid

[0067] (6) Cellular expression

[0068] HEK293 cells were selected and subcultured:

[0069] 1) Take out HEK293 cells from the liquid nitrogen tank or take out HEK293 cells transported on dry ice for cell recovery and culture;

[0070] 2) Count the cells first, add the cell suspension into the culture medium according to the required ratio, and start subculture;

[0071] 3) The cell density after subculture was controlled at 0.3×10 6 / ml, subcultured once every 4 days; the cell survival rate was controlled above 95% until the entire subculture process was completed.

[0072] (7) Transient transfection of HEK293 cells:

[0073] 1) Prepare two 15 ml sterile centrifuge tubes, add 5 ml KPM and 100 μg sterile plasmid DNA to one of the sterile centrifuge tubes, and gently pipette to mix; add 5 ml KPM and 500 μl TA-293 transfection reagent to the other sterile centrifuge tube, and gently pipette to mix;

[0074] 2) Transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the sterile plasmid DNA, gently pipette to mix, and let stand at room temperature for 10 minutes to prepare the plasmid-vector complex;

[0075] 3) Remove the HEK293 cells from the CO2 constant temperature shaker, add the plasmid-vector complex in step (F2) while shaking, and then return to the CO2 constant temperature shaker for shaking culture; add an appropriate amount of antibiotics after 3 hours.

[0076] (8) Recombinant antibody purification:

[0077] Affinity purification was performed using a protein A affinity chromatography column.

[0078] The antibodies were subjected to immune serum titer determination and primary antibody Elisa titer determination, wherein the results of Example 1 are shown in the following table:

[0079] Immune serum titer determination

[0080]

[0081] Original antibody Elisa titer determination

[0082]

[0083]

[0084] The results of the immune serum titer determination and the original antibody Elisa titer determination reflect that the content and activity of the specific antibodies in the serum are good, which indirectly indicates that the binding protein in the present application is highly active. Similarly, the content and activity of the specific antibodies in Examples 2-5 are good, and the binding protein in the present application is highly active.

[0085] 2. X1, X2, X3, X4, X5, and X6 mentioned above are all sites to be mutated related to antibody affinity. The present invention performs affinity detection and analysis on the mutants of Examples 1-5. The specific detection scheme is as follows:

[0086] The indirect enzyme immunoassay was used to dilute the recombinant antigen to 3ug / ml, 1.5ug / ml, 0.75ug / ml, and 0.375ug / ml for microplate coating, 100uL per well, overnight at 4°C; the next day, wash twice with washing solution and pat dry; add blocking solution (20% BSA + 80% PBS), 120uL per well, 37°C, 1h, pat dry; the antibody was diluted 2-fold from 1000ng / ml to 0.9 77ng / ml sample, 100uL / well, 37℃, 30min; wash 5 times with washing solution, pat dry; add goat anti-mouse-HRP, 100uL per well, 37℃, 30min; wash 5 times with washing solution, pat dry; add colorimetric solution A (50uL / well), add colorimetric solution B (50uL / well), 10min; add stop solution, 50uL / well; read OD value at 450nm (reference 630nm) on the microplate reader. Obtain the OD value corresponding to different antibody concentrations under different coating concentrations. At the same coating concentration, with the antibody concentration as the horizontal axis and the OD value as the vertical axis, a logarithmic plot was made, and the antibody concentration at 50% of the maximum OD value was calculated according to the fitting equation; the reciprocal of the affinity constant was calculated by substituting the formula: K = (n-1) / (2×(n×Ab`-Ab)), where Ab and Ab` represent the antibody concentration at 50% of the maximum OD value at the corresponding coating concentration (Ag, Ag`), respectively, and n = Ag / Ag`; every two coating concentrations can be combined to calculate a K value, and finally six K values ​​can be obtained, and the average value is taken, and the reciprocal is the affinity constant KD.

[0087] After testing, the mutated antibody sequences of Examples 1-5 all had an affinity of KD≤8.14×10 9 mol / L.

Claims

1. A binding protein comprising an AAV-9 antigen binding domain, characterized in that The antigen binding domain includes a complementarity determining region CDR-VH1, a complementarity determining region CDR-VH2, and a complementarity determining region CDR-VH3; The amino acid sequence of the complementary determining region CDR-VH1 is DYTM-X1, and X1 is any one of A, H, S, T or N.

2. The binding protein comprising an AAV-9 antigen binding domain according to claim 1, characterized in that The amino acid sequence of the complementary determining region CDR-VH2 is AVSWSGS-X2-T-X3-YADSV-X4-G, wherein X2 is any one of F, T, N, S or R, X3 is any one of K, Y, F, H or D, and X4 is any one of K, R, E, A or Q.

3. The binding protein comprising an AAV-9 antigen binding domain according to claim 1, characterized in that: The amino acid sequence of the complementary determining region CDR-VH3 is GSTGPLA-X5-RTSPGDYD-X6, wherein X5 is any one of A, Q, G, T or V, and X6 is any one of R, Y, S, F or H.

4. Use of a binding protein comprising an AAV-9 antigen binding domain as described in any one of claims 1 to 3 for highly specific binding to AAV9 virus particles / AAV9 vectors, characterized in that: It has a KD≤8.14×10 8 affinity.

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