Lgi1 monoclonal antibody and application thereof

Abstract

The application relates to the technical field of biological medicine, in particular to a LGI1 monoclonal antibody and application thereof. The LGI1 monoclonal antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 9 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 10. The humanized LGI1 monoclonal antibody has better specificity, sensitivity and other performances, and provides a new functional reagent for the treatment of brain diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to an LGI1 monoclonal antibody and its applications. Background Technology

[0002] LGI1 is a neuronal secretory protein closely related to brain development, neuronal excitability, and synaptic transmission. LGI1 interacts with presynaptic ADAM23 and postsynaptic ADAM22 to form a complex structure, and together with the CASPR2 protein, it participates in regulating the function of voltage-gated potassium channels (VGKCs).

[0003] Monoclonal antibodies target a single antigenic epitope, exhibiting higher specificity and sensitivity. Using monoclonal antibodies can eliminate expression differences between hybridoma cells, significantly improving the stability, specificity, and sensitivity of immunoassays. Simultaneously, monoclonal antibodies play a crucial role in studying cellular antigenic markers, purifying soluble antigens, further investigating antibody structure and function, and antibody drug development. Therefore, obtaining monoclonal antibodies to proteins is an important tool for conducting protein function research. Since its invention in 1975, hybridoma technology has been widely used in the preparation of monoclonal antibodies, becoming a powerful tool for their acquisition.

[0004] Hybridoma technology has long been a traditional tool for obtaining monoclonal antibodies, but its overall process is quite complex, including: selection of immunogens, animal immunization, preparation of fusion cells, screening of hybridomas, production of monoclonal antibodies, and antibody validation and application. Traditional hybridoma technology has a long cycle, including: preparation of recombinant antigens (4-7 weeks), animal immunization (6-8 weeks), cell fusion and screening (4-8 weeks), and antibody production (1-2 weeks). Existing hybridoma technologies are also costly.

[0005] In recent years, single-cell sequencing has become a popular research tool for disease research. By combining B cells and single-cell sequencing technology, B cell sequencing technology can be used to obtain novel human LGI antibodies. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an LGI1 monoclonal antibody.

[0007] In a first aspect, the present invention provides an LGI1 monoclonal antibody comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:9, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:10.

[0008] Preferably, the amino acid sequence of SEQ ID NO:1 in the heavy chain variable region is as follows: positions 1-135 from the N-terminus of SEQ ID NO:9, and the amino acid sequence of SEQ ID NO:5 in the light chain variable region is as follows: positions 1-107 from the N-terminus of SEQ ID NO:10.

[0009] Preferably, the heavy chain variable region includes HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3, and HCDR3 as shown in SEQ ID NO:3.

[0010] Preferably, the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region are as follows: positions 30-35, 49-66, and 98-124 from the N-terminus of SEQ ID NO:9.

[0011] Preferably, the light chain variable region includes LCDR1 as shown in SEQ ID NO:6, LCDR2 as shown in SEQ ID NO:7, and LCDR3 as shown in SEQ ID NO:8.

[0012] Preferably, the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are as shown in positions 24-34, 50-55, and 89-96 from the N-terminus of SEQ ID NO:10.

[0013] In a second aspect of the invention, a nucleic acid encoding the aforementioned LGI1 monoclonal antibody is provided.

[0014] Preferably, the expression vector contains the above-mentioned nucleic acid.

[0015] In a third aspect of the invention, the LGI1 monoclonal antibody described above is used in the preparation of articles for detecting LGI1 monoclonal antibodies, and in the preparation of articles for quality control of LGI1 antigen-transfected cells.

[0016] In a fourth aspect of the invention, there is an LGI1 monoclonal antibody detection kit, the kit comprising the LGI1 monoclonal antibody as described above as a positive standard.

[0017] The present invention has the following beneficial effects:

[0018] 1. This invention directly extracts antibody sequences from LGI1 antibody-positive patients, omitting the time required for traditional immune stimulation to produce antibodies, and the resulting antibody is a human antibody. The human LGI1 monoclonal antibody of this invention has better specificity and sensitivity. Attached Figure Description

[0019] Figure 1 The LGI1 antibody screening fluorescence pattern of Example 1 is shown;

[0020] Figure 2 The fluorescence pattern of the LGI1 expression antibody in Example 2 is shown.

[0021] Figure 3 The expression diagram of the LGI1-4 monoclonal antibody in Example 3 is shown;

[0022] Figure 4 The following is a fluorescence image showing the specific recognition of LGI1 by the LGI1-4 monoclonal antibody in Example 4;

[0023] Figure 5 The following image shows the immunofluorescence of the LGI1-4 monoclonal antibody and rat brain tissue from Example 5;

[0024] Figure 6 The results of the immunoprecipitation of the LGI1-4 monoclonal antibody in Example 6 are shown. Detailed Implementation

[0025] The present disclosure will now be discussed with reference to several exemplary embodiments. It should be understood that these embodiments are discussed only to enable those skilled in the art to better understand and thus implement the present disclosure, and are not intended to imply any limitation on the scope of the disclosure.

[0026] Example 1

[0027] 1. Screening of patients with positive LGI1 antibodies:

[0028] Patient inclusion: Encephalitis patients with positive anti-LGI1 antibodies in cerebrospinal fluid were included, and cerebrospinal fluid cells were obtained. Patients had signed informed consent forms.

[0029] The patient's cerebrospinal fluid first undergoes an immune reaction of antigen and antibody to verify that it indeed contains antibodies against the LGI1 antigen. Currently, the main method for detecting anti-LGI1 antibodies is immunofluorescence (IF).

[0030] 1.1 Preparation of cell matrix for anti-LGI1 antibody detection:

[0031] (1) HEK293T cells were seeded into 48-well plates and cultured in 37°C, 5% CO2 incubator using 10% FBS+DMEM cell culture medium.

[0032] (2) When the cell density reaches 40%-50%, use PEI MAX 40K to co-transfect LGI1 and ADAM23 plasmids. After 6 hours of transfection, replace with fresh culture medium to remove the transfection reagent. After 24-48 hours of transfection, the cell density reaches 80%-90%.

[0033] (3) Cell fixation: Fix cells with 1-4% paraformaldehyde at room temperature, wash with 200μL PBS 2-3 times, and store in a sterile environment for later use.

[0034] 1.2 Immunofluorescence assay:

[0035] (1) Take about 150-200 μl of the patient’s cerebrospinal fluid and add it to a 48-well plate transfected with LGI1 antigen expression, and incubate at 37°C for 1 hour in the dark.

[0036] (2) Wash 5 times with PBS;

[0037] (3) Add the secondary antibody conjugated with Alexa Fluor 488 Anti-human IgG and incubate for 1 hour;

[0038] (4) Wash 5 times with PBS;

[0039] (5) Observe and photograph fluorescence under a 20X objective lens of a fluorescence microscope.

[0040] like Figure 1 As shown, the cerebrospinal fluid of the experimental patients contained anti-LGI1 antibodies, and after transfection with LGI1 cells, a significant green fluorescent signal was produced.

[0041] This invention directly extracts antibody sequences from LGI1 antibody-positive patients, eliminating the time required for traditional immune stimulation to produce antibodies, and the resulting antibodies are human-derived antibodies. Human LGI1 protein has better specificity and sensitivity.

[0042] Example 2

[0043] 2. Screening of human anti-LGI1 antibodies

[0044] 2.1 Cerebrospinal fluid samples that tested positive for LGI1 antibodies were sent to the company for single-cell sequencing to obtain a certain number of light and heavy chain paired antibody sequences. Based on the characteristics of the antibody amino acid sequence and single-cell distribution, different types of antibodies, such as IgG1, IgG2, or IgG3, were selected for further expression and validation.

[0045] The six selected paired heavy and light chains were modified by completing the human IgG1κ / λ pair and optimizing the codons. The resulting gene was then inserted into the eukaryotic expression vector pTT5 (synthesized and subcloned by General Biotechnology) to obtain the heavy and light chain expression plasmid. HEK293T was then transfected into six-well plates to produce antibodies.

[0046] The specific steps are as follows:

[0047] (1) HEK293T cells were seeded into 6-well plates at a rate of 1 x 10⁶ cells / well, 2 mL of DMEM cell culture medium was added, mixed well, and incubated overnight at 37°C in a CO₂ incubator.

[0048] (2) When the cell density is around 60-70%, perform transfection: Add 2 μg of paired antibody light and heavy chain expression plasmids (light and heavy chain plasmid ratio 1:2) and 6 μL of PEI MAX 40K (1 mg / mL) to 250 μL OptiMEM, mix well, and let stand at room temperature for 10-15 minutes.

[0049] (3) Change the medium for each well of cells, add 1 mL of OptiMEM, then add the transfection reagent from step 2), mix gently, and incubate at 37°C in a CO2 incubator for 4 hours. Then change the medium to OptiMEM containing 3% FBS and continue incubation. Change the medium every 3 days. Take the culture medium twice and use a Pall 30K centrifuge tube to concentrate it by ultracentrifugation to obtain the crude product of anti-LGI1 antibody.

[0050] (4) Take about 150-200 μl of the crude product of anti-LGI1 antibody and add it to a 48-well plate transfected with LGI1 antigen expression, and incubate at 37°C for 1 hour in the dark.

[0051] (5) Wash 5 times with PBS;

[0052] (6) Add the secondary antibody conjugated with Alexa Fluor 488 Anti-human IgG and incubate for 1 hour;

[0053] (7) Wash 5 times with PBS;

[0054] (8) Observe and photograph fluorescence under a 20X objective lens of a fluorescence microscope.

[0055] like Figure 2 As shown, the crude product contains a specific anti-LGI1 antibody, which produces a distinct green fluorescent signal after reacting with transfected LGI1 cells.

[0056] 2.2 Of the six expressed antibodies, one (LGI1-4) produced a significant green fluorescence signal after transfection of LGI1 cells, such as... Figure 2 As shown.

[0057] LGI1-4 is the human anti-LGI1 antibody of this invention. IMGT IgBlast alignment analysis revealed that the heavy chain variable region (LGI1-4-VH) of LGI1-4 is: SEQ ID NO:1, HCDR1 is SEQ ID NO:2, HCDR2 is SEQ ID NO:3, HCDR3 is SEQ ID NO:4; the light chain variable region (LGI1-4-VL) of LGI1-4 is: SEQ ID NO:5, LCDR1 is SEQ ID NO:6, LCDR2 is SEQ ID NO:7, LCDR3 is SEQ ID NO:8; the complete heavy chain of LGI1-4 is LGI1-4-H: SEQ ID NO:9; and the complete light chain of LGI1-4 is LGI1-4-L: SEQ ID NO:10.

[0058] The CDRs of the heavy and light chains of LGI1 BCR-11, as analyzed by IMGT IgBlast, are shown in Table 1. For detailed amino acid sequences, please refer to the sequence listing below.

[0059] Table 1. CDRs of heavy and light chains of LGI1 BCR-11

[0060]

[0061] Example 3

[0062] 3. Expression and purification of human LGI1 antibody

[0063] The screened human LGI1 antibody LGI1-4 was formally expressed and purified.

[0064] 3.1 LGI1-4 antibody expression:

[0065] (1) FreeStyle TM CHO-S cells were cultured to a density of 2.5-3.0 x 10⁻⁶. 6 Cells / mL, ready for transfection;

[0066] (2) Add the human anti-LGI1 antibody expression plasmid (heavy and light chain plasmids, transfected at a ratio of 1:2) to 3 mL of OptiMEM and mix well;

[0067] (3) Take 120 μL of PEI MAX 40K (1 mg / mL) and add it to the above OptiMEM. Mix well and let stand at room temperature for 10-15 minutes.

[0068] (4) Add the well-mixed plasmid and PEI mixture to 30 mL of FreeStyle TM Transfection was performed in CHO-S cells by shaking culture at 37°C.

[0069] (5) 24 hours after transfection, add VPA and glucose solution to the final concentrations of 1 mM and 2 g / L, respectively;

[0070] (6) Continue shaking culture for 4 days, and then collect the culture.

[0071] 3.2 Purification of LGI1-4 antibody:

[0072] (1) The collected FreeStyle TM CHO-S cell culture was centrifuged at 15,000 rpm and 4°C for 10 minutes, and the supernatant was collected to remove dead cells and cell debris.

[0073] (2) Add the appropriate volume of Protein A beads to the supernatant according to the instructions for use of Protein A beads, and rotate the supernatant and Protein A beads to combine and incubate.

[0074] (3) After incubation for 1 hour, rinse Protein A beads with Wash buffer 3-5 times to remove impurities that are weakly bound to beads or antibodies.

[0075] (4) Then, the antibody bound to Protein A beads was eluted with acidic glycine buffer (pH 3.0-3.5), and the elution buffer was neutralized with 1 / 10 volume of 1M Tris-HCl pH 9.0.

[0076] (5) After Protein A affinity chromatography, the eluent was concentrated to about 0.5 mL using an Amicon Ultra 4 mL ultrafiltration tube. The second step of molecular sieve chromatography was performed using a BioCore SEC-300 column. The protein peak flow-through was collected and concentrated by ultrafiltration to obtain about 2 mL of LGI1-4 antibody.

[0077] (6) Take 2 μg of LGI1-4 antibody and add it to SDS-loading buffer containing / without mercaptoethanol (β-ME) to prepare samples. Use SurePAGE to analyze the samples. TM Bis-Tris SDS-PAGE 4-12% precast gel, electrophoresis at 100V constant voltage for 1 hour, remove the gel plate, stain with Coomassie Brilliant Blue staining solution for 20 minutes, and you can see the protein band of LGI1-4 antibody.

[0078] Molecular sieve diagram and Coomassie brilliant blue staining diagram of expressed and purified LGI1-4 antibody are shown below. Figure 3 As shown, M represents the protein marker, 1 is the LGI1-4 antibody under reduced conditions, and 2 is the LGI1-4 antibody under non-reduced conditions.

[0079] The approximately 2 ml of LGI1-4 antibody obtained in step (5) of purification was measured using a nanodrop A280, and the protein concentration was found to be 2.78 mg / mL. Based on this, it can be deduced that 30 ml of FreeStyle... TM CHO-S cells expressed approximately 4.5 mg of antibody. Based on the above measurements and calculations, it can be concluded that in FreeStyle... TM The expression level of LGI1-4 antibody in CHO-S cells is approximately 150 mg / L.

[0080] Example 4

[0081] 4. Specificity of LGI1-4 antibody recognition

[0082] 4.1 LGI1 is a neuronal secretory protein that is closely related to brain development, neuronal excitability, and synaptic transmission. LGI1 interacts with presynaptic ADAM23 and postsynaptic ADAM22 to form a complex structure. Therefore, the LGI1 protein requires the presence of ADAM23 or ADAM22 to remain on the cell membrane.

[0083] The LGI1-4 antibody can effectively recognize the LGI1 complex expressed by cells (LGI1+ADAM23 plasmid co-transformation). The present invention further demonstrates through cell immunofluorescence experiments that the LGI1 antibody specifically recognizes the LGI1 antigen, rather than LGI1 receptor proteins such as ADAM23.

[0084] The specific experimental steps are as follows:

[0085] (1) HEK293T cells were seeded into 48-well plates and cultured in 37°C, 5% CO2 incubator using 10% FBS+DMEM cell culture medium.

[0086] (2) When the cell density reaches 40%-50%, use PEI MAX 40K to transfect the LGI1+ADAM23 plasmid and the ADAM23 plasmid alone. After 6 hours of transfection, replace with fresh culture medium to remove the transfection reagent. After 24-48 hours of transfection, the cell density reaches 80%-90%.

[0087] (3) Cell fixation: Fix cells with 1-4% paraformaldehyde at room temperature, wash with 200μL PBS 2-3 times, and store in a sterile environment for later use.

[0088] 4.2 Immunofluorescence assay:

[0089] (1) Take about 200 μl of LGI1-4 antibody (1:1000 dilution) and add it to a 48-well plate transfected with LGI1+ADAM23 co-expression or ADAM23 expression alone, and incubate at 37°C for 1 hour in the dark.

[0090] (2) Wash 5 times with PBS;

[0091] (3) Add the secondary antibody conjugated with Alexa Fluor 488 Anti-human IgG and incubate for 1 hour;

[0092] (4) Wash 5 times with PBS;

[0093] (5) Observe and photograph fluorescence under a 20X objective lens of a fluorescence microscope.

[0094] The expressed and purified LGI1-4 antibody produced a significant green fluorescence signal in cells co-expressing LGI1+ADAM23, but showed no immune response or positive signal in cells transfected solely with ADAM23 antigen. Figure 4 As shown, this indicates that the LGI1-4 antibody specifically recognizes the LGI1 antigen, rather than the co-transferred ADAM23 antigen.

[0095] Example 5

[0096] 5. LGI1-4 monoclonal antibody for IHC

[0097] 5.1 The LGI1-4 antibody can effectively recognize plasmid-transfected cells expressing LGI1. In this example, rat brain tissue was used to detect that the LGI1-4 antibody can recognize the rat LGI1 protein.

[0098] The specific experimental steps are as follows:

[0099] (1) Frozen rat brain tissue sections were taken out of the -80℃ freezer and thawed;

[0100] (2) Wash rat brain tissue sections with PBS for 5 minutes;

[0101] (3) The rat brain tissue sections were permeabilized in 0.2% Triton for 20 minutes;

[0102] (4) The rat brain tissue sections were blocked in 10% BSA for 1 hour;

[0103] (5) Rat brain tissue sections were incubated overnight at 4°C with 100 μl of anti-LGI1 antibody dilution solution (antibody concentration 1 μg / ml, diluted with PBS containing 2% BSA);

[0104] (6) Wash rat brain tissue sections three times with PBS;

[0105] (7) Add the anti-human fluorescent secondary antibody Alexa Fluor 488 Anti-human IgG and incubate at room temperature for 1 hour;

[0106] (8) Wash rat brain tissue sections three times with PBS;

[0107] (9) Add DAPI dropwise and incubate in the dark for 15 minutes;

[0108] (10) Mount the slides with anti-fluorescence quenching mounting solution and dry them at 37°C for storage;

[0109] (11) Rat brain tissue sections were observed under a 10X fluorescence microscope and fluorescence photographs were taken.

[0110] Immunofluorescence image of the reaction between LGI1-4 monoclonal antibody and rat brain tissue sections is shown below. Figure 5 As shown, the LGI1-4 monoclonal antibody detected significant positive signals in rat brain tissue sections, primarily in the hippocampus and cortex. The left image shows the immunofluorescence of the rat cortex, and the right image shows the immunofluorescence of the rat hippocampus.

[0111] Therefore, through tissue immunofluorescence experiments, the LGI1-4 monoclonal antibody was able to recognize the LGI1 protein expressed at the synapses of rat cortical and hippocampal neurons.

[0112] Example 6

[0113] 6. LGI1-4 monoclonal antibody used in immunoprecipitation experiments

[0114] 6.1 The LGI1-4 monoclonal antibody according to the present invention can effectively recognize plasmid-transfected cells expressing human LGI1 and LGI1 protein in rat brain tissue. The present invention further uses HEK293T cell lysates transiently transfected with Flag-LGI1 to detect whether the LGI1-4 monoclonal antibody according to the present invention can be used for immunoprecipitation experiments.

[0115] The specific experimental steps are as follows:

[0116] (1) Transiently transfect the combination of Flag-LGI1+ADAM23 plasmids or the Flag-LGI1 plasmid alone into HEK293T cells in 6cm dishes to express membrane-anchored LGI1-ADAM23 protein complex or free LGI1 protein.

[0117] (2) One day after transfection, HEK293T cells were washed with 1 ml of PBS 1-2 times;

[0118] (3) After rinsing, HEK293T cells were collected in EP tubes using 1 ml of NP40 protein lysis buffer (containing 0.4% NP40), lysed by rotation at 4°C for 1 h, and centrifuged to remove cell debris or insoluble substances to obtain supernatant.

[0119] (4) Pre-incubate the cell lysate samples with unloaded Dynabeads Protein G magnetic beads for 1 hour to remove non-specific proteins that adhere to the magnetic beads;

[0120] (5) Use LGI1-4 monoclonal antibody to incubate pretreated cell lysate samples at 4 degrees for 6-12 hours;

[0121] (6) Add Dynabeads Protein G magnetic beads and continue incubation for 2-4 hours;

[0122] (7) After thorough washing, discard the supernatant, add protein loading buffer to the bound Protein G magnetic beads, heat at 70 degrees for 10 minutes, and collect the protein loading buffer as IP sample.

[0123] (8) Perform SDS-PAGE gel electrophoresis on the initial cell lysate and IP sample at a constant voltage of 120V for 1 hour;

[0124] (9) Remove the gel plate, stain with Coomassie Brilliant Blue staining solution for 20 minutes, rinse and soak several times with water, and you can see the protein bands in the IP.

[0125] Figure 6 The image shows the results of the immunoprecipitation of the LGI1-4 monoclonal antibody using the present invention.

[0126] like Figure 6 As shown, the LGI1-4 monoclonal antibody according to the present invention can recognize and bind to the LGI1 protein. A clear protein band is generated at the corresponding 60KD position of LGI1. This indicates that the LGI1-4 monoclonal antibody can bind to the native LGI1 protein and can be used for immunoprecipitation experiments.

[0127] This invention compares the results of cell immunofluorescence reaction, animal immunofluorescence reaction, and immunoprecipitation reaction of LGI1 antibody, commercial antibody 1 (PTG, Cat No: 12483-1-AP), and commercial antibody 2 (Invitrogen, Cat No: MA5-45677), as shown in Table 2:

[0128] Table 2 Comparison of fluorescence and precipitation reactions between the antibodies in the examples and commercial antibodies.

[0129] Cellular immunofluorescence reaction Animal immunofluorescence reaction Immunoprecipitation reaction Example: LGI1 antibody √ √ √ Commercial Antibody 1 X X X Commercial Antibody 2 √ X X

[0130] As shown in Table 2, the LGI1 antibody of the present invention has superior specificity and sensitivity compared with commercial antibody 1 and commercial antibody 2.

[0131] This invention effectively combines B-cell sequencing technology with clinical samples to rapidly obtain human anti-LGI antibodies via B-cell sequencing, and the results have been successfully verified. These antibodies can be used for immunofluorescence and immunohistochemical experiments of the LGI1 protein. The method of this invention is characterized by its speed (4-6 weeks) and low cost (one-third the price of traditional methods).

[0132] Those skilled in the art will understand that the above embodiments are specific examples of implementing this disclosure, and in practical applications, various changes can be made in form and detail without departing from the scope of this disclosure.

Claims

1. A monoclonal antibody against LGI1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:

5.

2. The monoclonal antibody against LGI1 according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody includes HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3, and HCDR3 as shown in SEQ ID NO:

4.

3. A monoclonal antibody against LGI1 according to claim 1 or claim 2, characterized in that, The amino acid sequence of the light chain variable region of the antibody includes LCDR1 as shown in SEQ ID NO:6, LCDR2 as shown in SEQ ID NO:7, and LCDR3 as shown in SEQ ID NO:

8.

4. A nucleic acid encoding a monoclonal antibody against LGI1 as described in any one of claims 1-3.

5. An expression vector containing the nucleic acid as described in claim 4.

6. The use of the anti-LGI1 monoclonal antibody as described in any one of claims 1-3 in the preparation of a kit for detecting LGI1 antigen.

7. A monoclonal antibody detection kit for anti-LGI1, the kit comprising an anti-LGI1 monoclonal antibody as a positive standard as described in any one of claims 1-3.

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