CN113651891B - Application of diabody in preparation of medicine for treating congenital alpha-muscular dystrophy - Google Patents
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Abstract
The invention provides application of a bispecific antibody in preparing a medicament for treating congenital alpha-muscular dystrophy, belonging to the technical field of biological medicine. The bispecific antibody comprises: (1) At least one antibody or antigen-binding fragment thereof that specifically binds DAG 1; (2) At least one antibody or antigen-binding fragment thereof that specifically binds laminin 211. The dual antibody is used for preparing a joint or a medicine for treating congenital muscular dystrophy; or in the preparation of a reagent or a kit for detecting DAG1 and/or laminin211 molecules. The invention develops the antibody with good specificity and physical property aiming at the beta-DAG 1 for the first time, and the double-characteristic antibody constructed by the invention has good drug property.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of a bispecific antibody in preparation of a medicine for treating congenital alpha-muscular dystrophy.
Background
Alpha-dystrogycapopathy is a subtype of congenital muscular dystrophy that itself again exhibits heterogeneity and complexity both clinically and genetically. The biochemical hallmark of this disease type is a reduction or absence of specific O-linked glycosylation on the alpha-dystroglycan (aDG) protein. Resulting in reduced binding of aDG to ligands, including Laminin211 (Laminin 211), agrin, and basoglycan outside of muscle, nerve cells, etc., which leads to clinical abnormalities in the muscle, central nervous system and eyes of the patient. Patients with severe subtype a-dystrogyca pathies, such as Walker-Warburg syndrome (WWS), muscle-eye-brain disease (MEB) and Fushan Congenital Muscular Dystrophy (FCMD), develop systemic muscle strength decline and cerebral nerve and muscle development deformity shortly after birth. While the other subtype, the limb-girdle muscular dystrophy (LGMD) patient, has normal mobility after birth, but starts to shrink after 3-4 years of muscle development, and becomes weak by the teens, and starts to need a wheelchair around 20 years old.
The genetic heterogeneity of alpha-dystrogycapopathy and the rarity of each subtype present significant challenges in developing therapeutic approaches. The current standard therapy is steroid hormone therapy. However, hormone therapy only relieves inflammation caused by muscle injury, and does not help control disease progression. Thus, α -dystroglivcanopathy patients have a highly unmet medical need.
Novel therapies for α -dystrogiycapropathy include antisense nucleic acid therapy (anti-sense RNA) AAV gene therapy methods and enzyme replacement therapies, all of which are at an early stage and have the disadvantage that only one of the gene defects of α -dystrogiycapropathy patients can be treated. Whereas α -dystrogyca nopath is currently known to be caused by at least more than 18 genes. Thus, current therapies are not directed to all patients with alpha-dystroglivcanopathy.
aDG, which binds to fibronectin 211 in the basal layer of skeletal muscle via matriglycan, is the basic structure for muscle cell generation and transmission of mechanical force. Thus, conceptually, repairing the link between fibronectin 211 and the muscle cell membrane with a functional mimetic can be used as a treatment (i.e., protein replacement) for this patient population.
Local injection of purified matriglycan O-glycosylation aDG into the muscle of the LARGE-myd mice demonstrated protection of the myomembrane from laser-induced damage, suggesting that protein substitution may be feasible. However, when matrix glycans are glycosylated aDG by systemic injection, there is little change in muscle and thus finding new functional mimics may be another approach.
In normal muscle tissue, the DGC protein complex binds to fibronectin 211 in the basal layer of skeletal muscle via matriglycan, enabling muscle power conduction (see fig. 1). Sanofi has proposed the use of antibody compositions directed against Laminin-2 and beta-DAG 1 to link myofascial and fibronectin. And in the Large-myd mouse model, a certain muscle strength repairing effect was seen (see fig. 2).
However, since the development of antibodies to the β -DAG1 target is highly challenging, the dual antibody of Sanofi has non-specific tissue binding, aggregation and poor PK/PD, and thus is not drug-resistant, unable to bring clinical benefit to the patient, and Sanofi has stopped the development of this project.
While Sanofi proposes a viable solution, their assumptions cannot be confirmed in human experiments due to the difficulty in antibody development. We performed validation experiments on their antibodies. Indeed, sanofi is directed against β -DAG1 antibodies, has a low affinity, and there is no specific binding, whereas our development is directed against β -DAG1 antibodies that exhibit high specificity, and a higher affinity.
The Sanofi dual anti-beta DAG1& anti-Laminin211 antibody adopts a DVD-IG form. This diabody presents poor biophysical stability. There is therefore an urgent need for a drug that has good biophysical properties and does not undergo degradation or aggregation.
Disclosure of Invention
In order to solve the technical problems, the invention provides application of a bispecific antibody in preparing a medicament for treating congenital alpha-muscular dystrophy. The invention develops the antibody with good specificity and physical property aiming at the beta-DAG 1 for the first time, and the double-characteristic antibody constructed by the invention has good drug property.
A bispecific antibody comprising:
(1) At least one antibody or antigen-binding fragment thereof that specifically binds DAG 1; the method comprises the steps of,
(2) At least one antibody or antigen-binding fragment thereof that specifically binds laminin 211.
In one embodiment of the invention, the at least one antibody or antigen binding fragment thereof that specifically binds DAG1 is: specifically binds to an epitope in human β -DAG 1domain hβ -DAG 1; the at least one antibody or antigen binding fragment thereof that specifically binds laminin211 is: specifically binds to at least one epitope in murine laminin G-like 5 domain mLG-5, human laminin G-like 5 domain hLG-5, murine laminin G-like 4 and 5 domains mLG-4/5, human laminin G-like 4 and 5 domains hLG-4/5.
In one embodiment of the invention, the method of constructing the bispecific antibody is: ligating a Laminin-211lg5VHH antibody that specifically binds Laminin211 to the Fc terminus of a β -DAG1 antibody that specifically binds DAG 1; the beta-DAG 1 antibody contains a heavy chain variable region with an amino acid sequence shown in any one of SEQ ID NO.1-SEQ ID NO.7 and a light chain variable region with an amino acid sequence shown in any one of SEQ ID NO.08-SEQ ID NO. 14; the amino acid sequence of the Laimin-211 LG5VHH antibody is shown in any one of SEQ ID NO.15-SEQ ID NO.19.
In one embodiment of the invention, the amino acid sequence of mβ -DAG1 is: uniProt Q62165, 652-893; the amino acid sequence of the hβ -DAG1 is as follows: uniProt Q14118, 654-895.
In one embodiment of the present invention, the amino acid sequence of mLG-5 is: uniProt Q60675, 2932-3118; the amino acid sequence of hLG-5 is as follows: uniProt P24043, 2936-3122; the amino acid sequence of mLG-4/5 is as follows: uniProt Q60675, 2725-3118; the amino acid sequence of hLG-4/5 is as follows: uniProtP24043, 2729-3122.
A nucleic acid encoding said bispecific antibody.
A vector of the nucleic acid.
A host cell expressing the vector.
A pharmaceutical composition comprising said bispecific antibody and at least one pharmaceutically acceptable excipient, diluent or carrier.
The use of said bispecific antibody, said use comprising: (a) Preparing a linker or a medicament for treating congenital muscular dystrophy; or (b) preparing a reagent or kit for detecting DAG1 and/or laminin211 molecules.
Compared with the prior art, the technical scheme of the invention has the following advantages:
the invention develops an antibody with high specificity and high affinity (see figure 3) aiming at beta-DAG 1 for the first time and good physical characteristics; the double-characteristic antibody constructed by the invention has good drug property, and has specific binding to beta-DAG 1& Laminin-211, and the double-characteristic antibody molecule has the potential of treating Alpha-Dystroglyceropy patients in the future.
The double antibody of the invention shows good biophysical characteristics on a PAGE gel and a HPLC size exclusion column, and does not have degradation or aggregation.
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings, in which
FIG. 1 is a schematic diagram of the binding of myomembrane protein DGC and fibronectin 211 to muscle function in muscle tissue, wherein (a) is a schematic diagram of the binding of myomembrane protein DGC on the membrane of muscle cell to muscle function in normal muscle tissue via matriglycan and fibronectin 211 in the basal layer of skeletal muscle; (b) Is that the linkage between the muscle membrane protein DGC and fibronectin 211 is reduced or absent after the DGC glycosylation is lost in Dystroglycation patients, resulting in the loss of muscle function.
FIG. 2 is a schematic representation of the ligation of myomembrane and fibronectin by antibody composition diabodies of Laimin-2 and beta-DAG 1.
FIG. 3 is a graph showing non-specific binding of β -DAG1 antibodies to CHO cells.
FIG. 4 is a graph showing that the P38G1& P57A1 antibodies of the invention specifically bind to human and mouse β -DAG1 protein, P79D02 is a negative antibody, CHO control mother cells, (CHO) -hDAG1, CHO cells express human DAG1 protein, (CHO) -mDAG1, CHO cells express mouse DAG1 protein.
FIG. 5 is a schematic diagram of a portion of a protein expression plasmid; wherein A is a schematic diagram of a plasmid expressing His tag protein, and B is a schematic diagram of a plasmid expressing FC fusion protein; c is a plasmid for constructing a stable cell line, and the basic skeleton structure of the plasmid is PCDNA1.
FIG. 6 is a schematic illustration of single B cell microfluidic packaging; wherein, (a) single B cells of the microfluidic chip are split, a schematic diagram (B) is shown by splitting, and a microfluidic chip pipeline is obtained under a microscope.
FIG. 7 is a graph showing the conditions of clones P79E02, P79F03, P79C112, P80D05, P80A07, P38G1 and 57A1 showing the mDAG1 and hDAG1 of the specific junction CHO cell surface.
FIG. 8 is a graph showing the results of antigen binding kinetics of clone P38G 1.
FIG. 9 is a graph showing the results of antigen binding kinetics of clone P57A 1.
FIG. 10 is a graph of the ability of VHH hits PL60B1, PL60B2, PL60B3, PL60C1, PL60C2, PL60F1, PL60F3 to bind CHO, CHO-hLG5& CHO-mLG5 at different concentrations.
FIG. 11 is an identification of affinity with Carterra for antibodies hits PL60B1, PL60B2, PL60B3, PL60C1, PL60C2, PL60F1, PL60F 3.
FIG. 12 shows the binding results of PL60B1 (same as PL60B 3), PL60F3, PL60C1, PL60B2 to a Laminin family protein at various concentrations.
FIG. 13 is an Anti-beta DAG1 and Anti-LG5 diabody structure of the present invention.
FIG. 14 is an identification of beta DAG1 on cells to which the diabody and the parent monoclonal antibody of the present invention bind.
FIG. 15 is the identification of LG5 on cells bound by the diabody and parent monoclonal antibody of the invention.
FIGS. 16 and 17 are the results of affinity tests for the diabodies and monoclonal antibodies of the invention.
FIG. 18 shows the case of electrophoretic separation of the diabodies of the invention on non-reducing and reducing PAGE gels.
FIG. 19 is a HPLC separation and purification dual-antibody liquid chromatogram of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
Examples
1 antibody discovery: anti-beta DAG1 and anti-LG4/5 specific antibodies
1.1 protein preparation
For expression of the murine or human β -DAG extracellular domain (beta-DAG ECD) (UniProt Q62165, amino acids 652-746), a plasmid was constructed according to the present invention (FIGS. 5-A, 5-B) using PCDNA1 as the backbone, CMV promoter and enhancer.
The Expi-lipofectamine was complexed with a DNA vector and added to the suspension cell culture. On day 2, enhancers and feed were added to the culture according to the manufacturer's protocol.
Cells were incubated with 8% CO in shaking culture 2 Incubate for 7 days at 37℃and then remove cells by centrifugation. The supernatant was sterilized by passing through a 0.22 μm filter and mixed with an equal volume of DPBS without magnesium or calcium.
The supernatant from the Fc fusion protein was loaded onto a 1mL protein A Sepharose column at 1mL/min, and the column was then washed with 50mM NaAc pH 5.5. The fusion protein was then eluted with 50mM NaAc pH3.0 and collected in 0.5mL fractions, the high protein concentration fractions were pooled and diafiltered with DPBS.
Supernatants from non-Fc proteins were diluted 10-fold with DPBS and then concentrated using tangential flow dialysis. The concentrate was loaded onto a 2ml ni column, washed with PBS buffer containing 25mM imidazole, and then eluted with PBS buffer containing 300mM imidazole. Fractions containing high protein were pooled and diafiltered with DPBS, then purified by separation using a Nickel column.
Expression of laminin G-like 5 domains (mLG-5 or hLG-5) constructs similar to the above methods, and mLG-5 (see UniProt Q60675, amino acids 2932-3118;SEQ ID NO:292) or hLG-5 (UniProt P24043, amino acids 2936-3122;SEQ ID NO:293). This construct was used to transfect the Expi293F cells with an expfectamine reagent (Thermo Fisher). After 7 days of expression, the cells were isolated and purified using a Nickel column.
1.2 cell line establishment
Stable cell lines with surface expression of human or murine β -DAG were created by codon optimized constructs, the construct vector DNA was linearized with Fsp I restriction enzymes (FIG. 5-C), with myc tags at the N-terminus and extracellular and endogenous transmembrane domains of β -DAG (mouse Unit Prot Q62165, amino acids 652-893; human Unit Prot Q14118, amino acids 654-895). Adherent human embryonic kidney cells (HEK) and adherent chinese hamster ovary cells (CHO-K1) were transfected with lipofectamine (Thermo Fisher) and cells were selected with geneticin (Gibco). Surviving cells were serially diluted to obtain single cell clones, and surface expression of β -DG was confirmed by anti-myc flow cytometry.
Stable cell lines with surface expression of human or murine LG-5 were created by codon-optimized constructs comprising an N-terminal myc tag, gly/Ser linker, LG-5 (mouse Unit Prot Q60675, amino acids 2932-3118; human Unit P24043, amino acids 2936-3122) and Tfr1 transmembrane domain for mammalian expression. Adherent chinese hamster ovary cells (CHO-K1) were transfected with lipofectamine (Thermo Fisher) and cells were selected with geneticin (Gibco). Surviving cells were serially diluted to obtain single cell clones, and surface expression of β -DG was confirmed by anti-myc flow cytometry.
Stable cell lines with human or murine LG-4/5 surface expression were created by codon-optimized constructs comprising the N-terminal murine FC tag, LG-4/5 (mouse UniProt Q60675, amino acids 2725-3118; human UniProtP24043, amino acids 2729-3122). Adherent human embryonic kidney cells (HEK) were transfected with lipofectamine (Thermo Fisher) and cells were selected using geneticin (Gibco). Surviving cells were serially diluted to obtain single cell clones, and surface expression of β -DG was confirmed by anti-murine FC flow cytometry.
1.3 mouse immunization
Gene gun DNA immunization was performed using the Helios gene gun system (Biorad, cat#:1652-431). Plasmid expression vector (FIG. 5-C) DNA (Biorad 1652263) was coated with gold particles as an adjuvant for a total of 4-6 injections into mice. Protein immunization the antigen solution was emulsified with Freund's or RIBI adjuvant. Balb/c mice were immunized and then boosted 2 times per week with these proteins. Serum antibody levels were measured approximately every 7 days after 2 weeks, and when sufficient antibody titers were achieved, mice were screened for antibodies using the ablnk platform after spleen and bone marrow B cells were purified.
The Llama alpaca was emulsified with hLG-5 protein in combination with Freund's adjuvant. Alpaca immunization was performed once a month. Serum antibody levels were measured after about 3 months, and when sufficient antibody titers were reached, alpaca spleen B cells were purified and screened for antibodies using the AbLINK platform.
1.4 microfluidic sub-packaging of single B cells
The invention uses a glass emulsion droplet microfluidic chip with custom design to split B cells and capture antibody heavy and light chain mRNA. The microfluidic chip had two fluorocarbon oil (dolomite) input channels, one was the input channel for the cell suspension mixture, and one oligo-dT magnetic bead (NEB) input channel (20 mM Tris pH 7.5, 0.5M NaCl, 1mM EDTA, 0.5% Tween-20 and 20mM DTT) in cell lysis buffer at 1.25 mg/mL. The third channel is an oil mixture to package single B cells and magnetic beads (see fig. 6). The B cells were collected in a chilled 15mL microcentrifuge tube after the emulsion droplets were lysed, and the mRNA-capturing magnetic beads were subsequently purified and recovered.
1.5 Single B cell antibody heavy and light chain pairing PCR amplification and Yeast Natural VH-VL SCFV-FC library establishment
mRNA-bound magnetic beads were resuspended in RT-PCR mixture and with proprietary mineral oil and surfactant, and independent approximately 10nL size microdroplet RT-PCR mixture was formed using the technology specific to the attic ladder containing one-step RT-PCR buffer, (2.0 mM MgSO) 4 Super-Script III reverse transcriptase and Platinum Taq (Thermo Fisher Scientific), plus PCR primers for IgK C region, igG C region and all VH regions. The overlap region linking the heavy and light chain PCR products is a Gly-Ser linker sequence. The PCR amplification product VL-GS-VH was loaded into 1% agarose gel for electrophoretic separation, and then purified and recovered using Qiagen Gel PurificationKit.
The purified PCR amplification product VL-GS-VH was subjected to PCR again with an adapter at both ends, and then separated and purified on an agarose gel.
Yeast cells (ATCC) were electroporated (Bio-Rad Gene pulser II;0.54kV,25uF, resistance set to infinity) and homologous recombination occurred after introduction of the PCR product and linearized pYD vector into the yeast cells. Transformed cells were amplified and induced with galactose to generate a yeast scFv-FC antibody display library.
Alpaca VHH antibody mRNA was directly PCR amplified with VHH primers and introduced into yeast cells to create an antibody library.
1.6 screening of Yeast library and antibody hits production
The antigens mbeta-DG, hbeta-DG, mLG-5, hLG-5 were first biotinylated and bound to magnetic beads using EZ-Link Micro Sulfo-NHS-LC-Biotinylation kit (Thermo Fisher Scientific). The antigen-specific yeasts are first enriched with magnetic beads, and then the enriched yeasts are stained with biotinylated antigen (at a concentration of 10nM-1 μm). Recovered clones were inoculated on SD-CAA plates with kanamycin, streptomycin, and penicillium (Teknova) and amplified on a flow instrument BD FacsAria II (for double positive cell (FITC/PE) isolation) the amplified yeasts were then subjected to a second round (sometimes a third round) of FACS flow analysis for isolation.
1.7ELISA binding assay
Yeast clones with the desired characteristics were inoculated into semi-solid YPD medium for growth, amplified, transferred to 2mL deep 96-well plates (12111077,Thermo Fisher Scientific) after 3 days and cultured.
Purified antigens (mbeta-DG, mLG-5, hLG-5, mLG-4/5 or hLG-4/5) were coated at 1. Mu.g/mL on Nunc MaxiSorp 96 well ELISA plates (Thermo Scientific). Monoclonal antibody supernatants from 96-well plate clones were added to each well and positive or negative binding was detected using anti-mouse, HRP-labeled secondary antibodies (Jackson Immunoresearch).
1.8 identification of antigen binding kinetics of Carterra antibodies
Antibody antigen affinity the binding kinetics of anti-DAG 1 or LG5 antibodies were measured using a 96 x96 array-based SPR imaging system (cartera USA). For the anti-human Fc capture method: 5ug/mL of anti-human Fc (So μthern Bio, # 2047-01) was immobilized on a Chip-HC200M-Polycarboxylate-200nm coating thickness Chip (Cartera Bio, cat #HC 200M) or Chip-HC30M on-Polycarboxylate-30 nm coating thickness Chip (Cartera Bio, cat #HC 30M) by amine-based coupling. For direct coupling, the antibodies are directly coupled to the chip. The supernatant or purified form of the anti-DAG 1 or LG5 antibody is captured and passed over DAG1 Ag or LG5 Ag. Ag 2-3 min was injected, run buffer at 25 ℃ and dissociation was monitored for 5 or 10 min. This regenerates the surface between binding cycles using 10mM glycine HCl pH 1.7. The sensorgrams were subtracted from the blank and analyzed using the manufacturer-supplied software using the 1:1langmuir binding model. For direct coupling, the antibodies are directly coupled to the chip.
Antibody competition by cartera LSA
Cartera LSA was used to epitope map a panel of anti-LG5 antibodies. Purified monoclonal hybridoma antibodies were diluted at 10 μg/mL in 10mM sodium acetate buffer pH 4.5. Antibodies were directly immobilized on HC200M chips using amine coupling using a continuous flow microscope spotter to create an antibody array. For the profiling analysis, IBIS MX96SPRi was used to evaluate binding to immobilized antibodies. The experiments were performed in running buffer at 25 ℃. Antigen was injected at 100nM for 4 min, followed by antibody injection at 10 μg/mL for 4 min. The surface was regenerated between cycles using 10mM glycine pH 2.0. The data were processed and analyzed in the cartera KIT software tool with point-to-point reference and double reference data, and then fitted into a simple lang Miao Erbang model using global ka, kd and Rmax values for each point. The quality of the fit was determined by examining the residuals and processing the data using cartera.
1.9 antibody variable region sequencing
Yeast cell plasmids were purified using Qiagen miniprep and sequenced using Genscript Sanger to obtain variable region antibody sequences, the results are shown in tables 3 and 5.
2 double antibody construction plasmid
beta-DAG 1 antibody clone PL79F3 and Laminin domain LG antibody clone P60C01 were used for bispecific antibodies, the antibody expression plasmid vector is shown in FIG. 5-C, with FC linked at the C-terminus. The FC-terminus of the PL97F3 antibody was linked to the VHH clone P60C01 using a GS Linker. The structure of the antibody is shown in FIG. 13.
3 results
3.1 antibody identification of anti-beta DAG1 and anti-LG5
To screen and select yeasts that produce antibodies specific for β -DAG1, LG-5, the specificity of the antibodies for binding antigen was assessed using ELISA, FACS analysis and cartera kinetic analysis. The hits assay of β -DG by Carterra shows that a series of antibodies have binding affinity for β -DG, or LG-5 (see tables 1 and 2) showing an affinity of 10 for β -DAG1 antibody -9 M to 10 -10 M. Antibody affinity against Laminin-211LG5 was at 10 -10 M to 10 -11 M. The clones identified were sequenced using Sanger and the beta-DAG 1 and LG-5 antibody sequences are shown in tables 3 and 4.
TABLE 1 binding kinetics of beta-DAG antibody clones
| CloneID | k a (M -1 s -1 ) | k d (s -1 ) | KD |
| PL57A1 | 2.40E+05 | 1.20E-04 | 5.00E-10 |
| PL57B1 | 1.30E+04 | 1.00E-05 | 7.69E-10 |
| PL57C1 | 1.50E+04 | 2.30E-04 | 1.53E-08 |
| PL57D1 | 3.00E+04 | 2.50E-04 | 8.33E-09 |
| PL57D2 | 6.20E+04 | 9.50E-04 | 1.53E-08 |
| PL57E2 | 9.20E+05 | 2.20E-03 | 2.39E-09 |
| P38G1 | 2.00E+04 | 1.00E-05 | 5.00E-10 |
| PL57A1 | 3.00E+05 | 2.70E-04 | 9.00E-10 |
| PL79D2 | 1.90E+04 | 1.90E-05 | 1.00E-09 |
| PL79E2 | 1.90E+04 | 1.90E-05 | 1.00E-09 |
| PL79F3 | 2.70E+04 | 2.10E-05 | 7.78E-10 |
| PL79C12 | 1.90E+04 | 1.10E-05 | 5.79E-10 |
| PL80D5 | 1.70E+04 | 1.10E-05 | 6.47E-10 |
| PL80A7 | 1.60E+04 | 1.20E-05 | 7.50E-10 |
TABLE 2 binding kinetics of Laimin-211 antibody clones
| CloneID | k a (M -1 s -1 ) | k d (s -1 ) | KD |
| PL60C2 | 4.00E+05 | 1.80E-04 | 4.50E-10 |
| PL60B3 | 2.60E+05 | 1.70E-05 | 6.54E-11 |
| PL60F3 | 2.40E+05 | 2.60E-05 | 1.08E-10 |
| PL60B2 | 2.60E+05 | 3.60E-05 | 1.38E-10 |
| PL60C1 | 2.60E+05 | 4.10E-05 | 1.58E-10 |
Lanmin-211 domain LG5 was immobilized as substrate to a Carterra well plate and antibody as Inslow.
TABLE 3 cloning of VH and VL amino acid sequences by beta DAG antibodies
TABLE 4 amino acid sequence of the clone of the Laminin-211LG5VHH antibody
TABLE 5 beta-DAG 1 and Laminin-211 bispecific VH, VL and VHH amino acid sequences
The CHO cell lines expressed hβdag1, mβ -dag1 were used to determine the binding and specificity of hits, (fig. 7 clones P79E02, P79F03, P79C112, P80D05, P80a07, P38G1 and P57A1 showed specific binding to mDAG1 and hDAG1 on CHO cell surfaces, but no CHO cells, showing high specificity and human-mouse cross-reactivity the cartera SPR affinity assay showed that P38G1 and P57A1 binding to hβdag1 were all on the sub nanomolar scale (see fig. 8 and 9).
Identification of LG-5 antibodies
CHO cell line expression hLG, mLG5 was used to determine binding and specificity of VHH hits (fig. 10) clones PL60B1, PL60B2, PL60B3, PL60C1, PL60C2, PL60F1, PL60F3 exhibited specific binding to CHO cell surface mLG and hLG5, whereas CHO cells were not bound, showing a high degree of specificity and human-mouse cross-reactivity. Cartera SPR affinity assay showed that binding force of PL60B1 (same as PL60B 3), PL60F3, PL60C1, PL60B2 binding hLg5 was all 10 -10 To 10 -11 Level of(see FIG. 11), and shows high specificity within the Laminin family of proteins (see FIG. 12).
3.2 construction and identification of double antibodies
The construction scheme of the double antibody is shown in FIG. 13, in which VHH anti-Laminin-211 clone P60C01 is connected to the FC end of beta-DAG 1 antibody PL79F3, and the antibody sequence is shown in Table 5. The diabodies and monoclonal antibodies have very close affinity (see fig. 16 and 17) to bind βdag1 at the cellular level and the Laminin-211LG5 region very close to the monoclonal antibodies (see fig. 14 and 15). In non-reducing agarose electrophoresis (see NR in FIG. 18), the diabody was an approximately 220KD band, and no degradation or separation of the heavy and light chains of the antibody was observed. On reductive agarose electrophoresis (RD in FIG. 18), disulfide bonds between the heavy and light chains were broken, and the antibody was split into two fragments, heavy and light, with a heavy chain of about 65KD and a light chain of about 25KD. Also no degradation of the heavy and light chains of the antibody was observed, indicating good biophysical properties of the antibody. The analysis of HPLC size exclusion in FIG. 19 also shows that the diabodies of the present invention have only one peak, indicating that the antibodies do not undergo degradation or aggregation and have good stability.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
SEQUENCE LISTING
<110> Suzhou day ladder biological medicine Co., ltd
Application of <120> diabody in preparing medicine for treating congenital alpha-muscular dystrophy
<130> 19
<160> 19
<170> PatentIn version 3.3
<210> 1
<211> 113
<212> PRT
<213> (Synthesis)
<400> 1
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Ile
20 25 30
Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Glu Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Gly Gly Asn Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser
100 105 110
Ser
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Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
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Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Ile
20 25 30
Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Glu Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Gly Gly Asn Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser
100 105 110
Ser
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Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
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Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Ile
20 25 30
Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
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Gly Arg Ile Asp Pro Glu Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe
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Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
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Gly Gly Asn Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser
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Ser
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Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
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Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Ile
20 25 30
Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Glu Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
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Gly Gly Asn Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser
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Ser
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Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
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Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Ile
20 25 30
Tyr Ile His Trp Met Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
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Gly Arg Ile Asp Pro Glu Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe
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Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
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Gly Gly Asn Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser
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Ser
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Gln Ile Gln Leu Leu Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
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Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Ile
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Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
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Gly Arg Ile Asp Pro Glu Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Val Gly Asn Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser
100 105 110
Ser
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Gln Ile Gln Leu Leu Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
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Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Ile
20 25 30
Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Glu Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Gly Gly Asn Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser
100 105 110
Ser
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Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
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Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
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Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
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<212> PRT
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Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asp Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile His Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
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<212> PRT
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Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Val His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
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<212> PRT
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Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Gly Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
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Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
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Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
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Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Arg Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Gln Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
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Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
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Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Pro
35 40 45
Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Glu
100 105 110
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<212> PRT
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Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly
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Asp Gln Ala Ser Ile Ser Cys Arg Pro Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Glu
100 105 110
<210> 15
<211> 116
<212> PRT
<213> (Synthesis)
<400> 15
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ala Gln Ala Gly Gly
1 5 10 15
Pro Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Asn Ala Ile Asn
20 25 30
Thr Met Glu Trp His Arg Gln Pro Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Arg Met Ser Ser Ser Gly Gly Arg Tyr Tyr Val Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Leu
85 90 95
Tyr Gly Asp Ile Asn Met Arg Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
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<211> 116
<212> PRT
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Gly Ile Asn
20 25 30
Thr Met Glu Trp His Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Arg Met Ser Ser Ser Gly Gly Arg Tyr Tyr Val Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Val
85 90 95
Tyr Gly Asp Ile Asn Met Arg Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Gly
115
<210> 17
<211> 116
<212> PRT
<213> (Synthesis)
<400> 17
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ala Gln Ala Gly Gly
1 5 10 15
Phe Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Asn Ala Ile Asn
20 25 30
Thr Met Glu Trp His Arg Gln Pro Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Arg Met Ser Ser Ser Gly Gly Arg Tyr Tyr Val Asp Ser Val Lys
50 55 60
Gly Arg Ser Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Leu
85 90 95
Tyr Gly Asp Ile Asn Met Arg Asp Tyr Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser
115
<210> 18
<211> 116
<212> PRT
<213> (Synthesis)
<400> 18
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ala Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Asn Ala Ile Asn
20 25 30
Thr Met Glu Trp His Arg Gln Pro Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Arg Met Ser Ser Ser Gly Gly Arg Tyr Tyr Val Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Cys Cys Leu
85 90 95
Tyr Gly Asp Ile Asn Met Arg Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 19
<211> 116
<212> PRT
<213> (Synthesis)
<400> 19
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ala Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Asn Ala Ile Asn
20 25 30
Thr Met Glu Trp His Arg Gln Pro Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Arg Met Ser Ser Ser Gly Gly Arg Tyr Tyr Val Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Leu
85 90 95
Tyr Gly Asp Ile Asn Met Arg Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Gly
115
Claims (6)
1. A bispecific antibody comprising a β -DAG1 antibody and a Laminin-211LG5VHH antibody, wherein the Laminin-211LG5VHH antibody that specifically binds Laminin211 is linked to the Fc terminus of the β -DAG1 antibody that specifically binds DAG1, wherein:
the beta-DAG 1 antibody contains a heavy chain variable region with an amino acid sequence shown as SEQ ID NO.4 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 11; the amino acid sequence of the Laimin-211 LG5VHH antibody is shown as SEQ ID NO.19.
2. A nucleic acid encoding the bispecific antibody of claim 1.
3. A vector comprising the nucleic acid of claim 2.
4. A host cell expressing the vector of claim 3.
5. A pharmaceutical composition comprising the bispecific antibody of claim 1 and at least one pharmaceutically acceptable excipient, diluent or carrier.
6. The use of a bispecific antibody of claim 1, wherein the use comprises: (a) Preparing a linker or a medicament for treating congenital muscular dystrophy; or (b) preparing a reagent or kit for detecting DAG1 and/or laminin211 molecules.
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| CN108495685A (en) * | 2015-10-13 | 2018-09-04 | 马里兰大学巴尔的摩分校 | For the immunotherapy based on yeast of clostridium difficile infection |
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| WO2019179391A1 (en) * | 2018-03-19 | 2019-09-26 | Wuxi Biologics (Shanghai) Co., Ltd. | Novel bispecific pd-1/ctla-4 antibody molecules |
| WO2019179390A1 (en) * | 2018-03-19 | 2019-09-26 | Wuxi Biologics (Shanghai) Co., Ltd. | Novel bispecific pd-1/egfr antibody molecules |
| WO2021115456A1 (en) * | 2019-12-11 | 2021-06-17 | Wuxi Biologics (Shanghai) Co., Ltd. | BI-FUNCTIONAL ANTIBODY AGAINST PD-L1 AND TGFβ |
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