An anti-LAG3 antibody and its application

Abstract

The present invention relates to an anti-LAG3 antibody and uses thereof. The amino acid sequence of the anti-LAG3 antibody of the present invention is shown in SEQ ID NO: 6 or SEQ ID NO: 7. The anti-LAG3 antibody of the present invention has high affinity and can improve the sensitivity and specificity of LAG3 antigen detection. The anti-LAG3 antibody of the present invention is a nanobody derived from alpacas. Compared with traditional antibodies, the anti-LAG3 antibody of the present invention has a smaller molecular weight, is more stable, is easier to produce, has a high yield, is low in cost, and is easy to scale up. It will provide more and better options for the development of highly sensitive LAG3 antigen detection reagents or detection kits.

Description

Technical Field

[0001] The present invention belongs to the field of antibody technology, and specifically relates to an anti-LAG3 antibody and applications thereof. Background Art

[0002] Lymphocyte-activation gene 3 (LAG-3) is a type I transmembrane protein belonging to the immunoglobulin (Ig) superfamily, primarily expressed on the surface of activated T cells and natural killer (NK) cells. As a key immune checkpoint molecule, LAG3 plays a crucial role in cancer immunotherapy. As the value of LAG3 antigen in clinical diagnosis is increasingly recognized, improving the sensitivity and specificity of LAG3 antigen detection is a current research priority.

[0003] Anti-LAG3 antibodies can specifically bind to the LAG3 antigen and are required in a variety of rapid LAG3 antigen detection methods or test kits. For example, in immunohistochemistry (IHC), anti-LAG3 antibodies are used to specifically stain LAG3 protein in tissue sections to observe its distribution in cells and tissues. In immunoblotting (WB), anti-LAG3 antibodies are used to detect the presence and expression level of LAG3 protein in samples. The specific binding of LAG3 protein to the antibody is a key step in LAG3 protein detection. In enzyme-linked immunosorbent assay (ELISA), anti-LAG3 antibodies are immobilized on an enzyme-labeled plate, bind to LAG3 protein in the sample, and then detected using an enzyme-labeled secondary antibody. This method requires the use of anti-LAG3 antibodies to achieve specific capture of the LAG3 protein.

[0004] Antibody affinity refers to the strength of the binding between the antibody and the antigen and is a key factor in determining the sensitivity and specificity of rapid antigen detection kits. High-affinity anti-LAG3 antibodies can improve the sensitivity and specificity of LAG3 antigen immunoassays.

[0005] The development of more high-affinity anti-LAG3 antibodies will provide more options for efficient immunoassays for LAG3 antigens. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a high-affinity anti-LAG3 antibody for LAG3 antigen detection.

[0007] To solve the above problems, the present invention provides an anti-LAG3 antibody in a first aspect, wherein the amino acid sequence of the anti-LAG3 antibody is shown in SEQ ID NO: 6 or SEQ ID NO: 7.

[0008] In an embodiment of the present invention, the nucleotide sequence of the anti-LAG3 antibody is shown as SEQ ID NO: 1 or SEQ ID NO: 2.

[0009] The present invention constructs an antibody library using serum from alpacas immunized with the hLAG3 antigen. Using reverse-transcribed RNA to synthesize cDNA as a template, a first-round PCR was performed using a pair of self-designed primers. Using the 700bp fragment from the first-round PCR as a template, a second-round PCR amplification of the VH2 and VH3 fragments was performed using eight pairs of self-designed degenerate primers. The 500bp PCR product was electroporated to construct a library, yielding 205 clones. After three rounds of library screening, 96 monoclonal colonies were obtained. ELISA panning yielded 18 positive clones, which were expressed in a mammalian expression system to produce monoclonal antibodies. Antibody binding comparison experiments revealed the two anti-LAG3 antibodies with the highest affinity. Experiments have shown that both anti-LAG3 antibodies can be used as primary antibodies for rapid LAG3 antigen detection (e.g., immunoblotting), effectively detecting positive samples. The anti-LAG3 antibody of the present invention is a nanobody derived from alpacas, comprising only the heavy chain variable region and having a molecular weight significantly smaller than that of traditional antibodies. Compared with traditional antibodies, the anti-LAG3 antibody of the present invention has a smaller molecular weight, is more stable, has a higher affinity, is easier to produce, has a high yield, is low-cost, and is easy to scale up. It has great prospects in the development of highly sensitive detection reagents or detection kits.

[0010] According to the present invention, when referring to an amino acid sequence, unless otherwise specified, the amino acid sequence referred to includes not only sequences that are exactly the same as the sequence but also sequences that can be easily thought of and expected to have equivalent effects based on the sequence by those skilled in the art (for example, those skilled in the art should know that sequences with high identity (for example, more than 90%, particularly more than 96%, and especially more than 98%) usually have equivalent effects).

[0011] A second aspect of the present invention provides an hFc-tagged anti-LAG3 antibody, comprising the above-mentioned anti-LAG3 antibody and an hFc tag, wherein the hFc tag is linked to the C-terminus of the anti-LAG3 antibody.

[0012] In an embodiment of the present invention, hFc-tagged anti-LAG3 antibodies are produced by a mammalian expression system.

[0013] A third aspect of the present invention provides the use of the aforementioned anti-LAG3 antibody or hFc-tagged anti-LAG3 antibody in the preparation of a LAG3 antigen detection reagent or kit. The anti-LAG3 antibody of the present invention has high affinity and can improve the sensitivity and specificity of LAG3 antigen detection.

[0014] In a fourth aspect, the present invention provides a LAG3 antigen detection reagent comprising the hFc-labeled anti-LAG3 antibody and a PBS buffer solution having a pH of 7.4 to 7.6.

[0015] In one embodiment of the present invention, the LAG3 antigen detection reagent is constructed by transfecting HEK293 cells with the mammalian expression vector pCDNA3.1 to express an hFc-tagged anti-LAG3 antibody, which is then purified using a Protein G column. The concentration of the LAG3 antigen detection reagent is 0.4-0.9 mg / mL, and the molecular weight of the hFc-tagged anti-LAG3 antibody is approximately 40 kDa.

[0016] In a fifth aspect, the present invention provides a LAG3 antigen detection kit, comprising the above-mentioned anti-LAG3 antibody or the above-mentioned hFc-labeled anti-LAG3 antibody or the above-mentioned LAG3 antigen detection reagent.

[0017] In an embodiment of the present invention, the LAG3 antigen detection kit is an immunohistochemistry detection kit, an immunoprecipitation detection kit, an enzyme-linked immunosorbent assay kit, or an immunoblotting detection kit.

[0018] In a sixth aspect, the present invention provides an immunoblot detection kit for LAG3 antigen detection, comprising a primary antibody and a secondary antibody, wherein the primary antibody is the hFc-labeled anti-LAG3 antibody described above, and the secondary antibody is a horseradish peroxidase-labeled goat anti-human antibody.

[0019] In a seventh aspect, the present invention provides a recombinant vector comprising a nucleotide sequence encoding the anti-LAG3 antibody or a nucleotide sequence encoding the hFc-tagged anti-LAG3 antibody.

[0020] The expression vector used in the recombinant vector of the present invention is a mammalian cell expression vector, preferably a plasmid vector or a viral vector. In a preferred embodiment of the present invention, the expression vector used in the recombinant vector is pCDNA3.1.

[0021] In an eighth aspect, the present invention provides a recombinant cell comprising the above-mentioned recombinant vector.

[0022] The host cells of the recombinant cells of the present invention are mammalian cells, including but not limited to human embryonic kidney cells (HEK293 cells), HeLa cells, or A549 cells.

[0023] Due to the adoption of the above technical solution, the present invention has the following advantages compared with the prior art:

[0024] The anti-LAG3 antibodies of the present invention have high affinity and can improve the sensitivity and specificity of LAG3 antigen detection. The anti-LAG3 antibodies of the present invention are alpaca-derived nanobodies. Compared with traditional antibodies, the anti-LAG3 antibodies of the present invention have a smaller molecular weight, are more stable, are easier to produce, have high yields, are low-cost, and are easy to scale up. They will provide more and better options for the development of highly sensitive LAG3 antigen detection reagents or kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the electrophoresis diagram of hLAG antigen;

[0026] Figure 2 is the electrophoresis diagram of RNA extracted from whole blood of alpaca after the fourth immunization;

[0027] Figure 3 This is the electrophoresis diagram of the first round of PCR products when constructing the antibody library;

[0028] Figure 4 This is the electrophoresis diagram of the second round of PCR products when constructing the antibody library;

[0029] Figure 5 It is the four-parameter fitting equation curve in affinity detection;

[0030] Figure 6 is the electropherogram of hFc-tagged LAG3-A-16;

[0031] Figure 7 This is the electrophoresis diagram of the immunoblotting detection of LAG3 antigen in Example 1;

[0032] Figure 8 is the electropherogram of hFc-tagged LAG3-A-21;

[0033] Figure 9 This is the electrophoresis diagram of the LAG3 antigen immunoblotting detection in Example 2. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further illustrated below with reference to specific embodiments and comparative examples.

[0035] In the present invention, it should be noted that, unless otherwise specified or contradicted by the context, the terms or expressions used herein should be read in conjunction with the entire content of this document and as understood by those of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0036] "Antigen" refers to an immunogenic substance, such as a protein or polypeptide.

[0037] "Antibody" refers to a substance, such as a protein or polypeptide, that specifically binds to an antigen.

[0038] "Nanobody" (Nb) is a new type of antibody derived from camelids (such as camels and alpacas) and sharks. It has a unique structure, containing only the heavy chain variable region (VHH) and lacking the light chain. Its molecular weight is only 1 / 10 of that of traditional antibodies, making it the smallest known unit that can bind to antigens.

[0039] "Affinity" refers to the binding strength between the antigen-binding site of an antibody and the epitope on the antigen, reflecting the degree of specific binding between the two.

[0040] "Specificity" refers to the ability of an antibody to recognize and bind to a specific antigen, that is, an antibody can only specifically bind to an antigen epitope with a specific chemical structure.

[0041] "Immunity" refers to the body's ability to identify and eliminate antigenic foreign substances and maintain its own physiological balance and stability.

[0042] "Serum" is the pale yellow transparent liquid separated from plasma after blood coagulation.

[0043] "Serum titer" refers to the concentration or activity of specific antibodies in serum. A higher titer indicates a higher concentration or activity of antibodies in the serum.

[0044] ELISA (Enzyme-Linked Immunosorbent Assay) is a highly sensitive detection technique based on antigen-antibody specific reactions. It involves immobilizing an antigen or antibody on a solid support, allowing the enzyme-labeled antibody or antigen to bind to the target substance, and then using the enzyme to catalyze the substrate for color development to quantitatively or qualitatively analyze the target substance.

[0045] An "antibody library" is a technique that uses genetic engineering to clone the variable region genes of antibody heavy and light chains into plasmids or phages for expression. Using antigen screening, clones carrying specific antibody genes can be obtained from the antibody library, thereby obtaining specific antibodies.

[0046] The "mammalian expression system" is a technology platform used to express exogenous proteins in mammalian cells. It is widely used in biopharmaceuticals, antibody production, vaccine development, and basic research.

[0047] The hFc tag is a fusion tag designed based on the Fc fragment of human immunoglobulin G (IgG). The Fc fragment is the constant region of an antibody and has the ability to bind to Fc receptors.

[0048] "Western Blot" (WB) is a detection method that combines high-resolution gel electrophoresis and immunochemical analysis techniques. Proteins are separated by molecular weight through polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins are then transferred to a solid phase carrier (such as a nitrocellulose membrane or PVDF membrane). Subsequently, specific antibodies are used to bind to the target protein, and then detection is performed using an enzyme- or fluorescent-labeled secondary antibody. Finally, the presence and expression level of the target protein are observed and analyzed through methods such as chemiluminescence (ECL), fluorescence display, or staining.

[0049] The technical solutions of the present invention are further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0050] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, cell culture, recombinant DNA technology, and related techniques in the art.

[0051] The implementation conditions in the present invention can be further adjusted according to specific requirements. The implementation conditions not specified are generally those in routine experiments. Instruments, raw materials and reagents not otherwise specified can be obtained commercially.

[0052] The development and preparation methods of the high-affinity anti-LAG3 antibodies for rapid detection of LAG3 antigens of the present invention are as follows:

[0053] 1. Preparation of Antigen

[0054] The human LAG3 (hLAG3) gene sequence was obtained from NCBI's GenBank, and recombinant human LAG3 protein (hLAG antigen) was prepared using a eukaryotic expression system. SDS-PAGE was used for quality control. The results were as follows: Figure 1 As shown, the molecular weight of hLAG antigen is approximately 60KDa.

[0055] 2. Immunize alpacas

[0056] Select adult, healthy alpacas for the immunization experiment:

[0057] First immunization: Take 1 mg of hLAG antigen, resuspend it with 1 mL of PBS buffer, add 1 mL of Freund's complete reagent (Sigma, F5881) and mix and emulsify. The completely emulsified mixture is used as the first immune injection solution and injected subcutaneously on both sides near the alpaca's cervical lymph nodes, with 2 injections on each side, and about 0.5 mL of immune injection solution at each point.

[0058] Second Immunization: Two weeks later, 1 mg of hLAG antigen was resuspended in 1 mL of PBS buffer and emulsified with 1 mL of Freund's complete reagent (Sigma, F5881). This emulsified mixture served as the second immunization injection and was injected subcutaneously into the alpaca's neck near the lymph nodes, at two sites on each side, with approximately 0.5 mL of the injection administered at each site. Seven days after the second immunization, blood was collected from the alpaca's neck vein for subsequent antibody titer testing.

[0059] Third Immunization: Four weeks later, 1 mg of hLAG antigen was resuspended in 1 mL of PBS buffer and emulsified with 1 mL of Freund's incomplete reagent (Sigma, F5506). The fully emulsified mixture was used as the immune injection solution and injected subcutaneously into the alpaca's neck near the lymph nodes, with approximately 0.5 mL of the solution injected at each site. Seven days after the third immunization, blood was collected from the alpaca's neck vein for subsequent antibody titer testing.

[0060] Fourth immunization: Six weeks after the third immunization, 1 mg of hLAG antigen was resuspended in 1 mL of PBS buffer and emulsified with 1 mL of Freund's incomplete reagent (Sigma, F5506). The fully emulsified mixture was used as the immune injection solution and injected subcutaneously into the alpaca's neck near the lymph nodes, with approximately 0.5 mL of the solution injected at each site. Seven days after the third immunization, blood was collected from the alpaca's neck vein for subsequent antibody titer testing.

[0061] ELISA was used to detect the titer of hLAG3 antigen-immunized alpaca serum. The detection method is as follows:

[0062] (1) Coating: Dilute the hLAG antigen to 1 μg / mL with CBS buffer and add 100 μL / well to a high-affinity ELISA plate (BeaverBio). Seal the reaction wells with plastic wrap and incubate at 4°C overnight. Wash the plate three times with PBST (PBS buffer containing 0.05% Tween-20) and pat dry.

[0063] (2) Blocking: Fill the reaction wells with 3% skim milk powder blocking solution, seal with plastic wrap, and leave at room temperature for 1 h. Wash the plate 3 times with PBST and pat dry.

[0064] (3) Sample addition: Use CBS buffer to dilute the post-immune serum and negative serum to 1:1000, 1:3000, 1:9000, 1:27000, 1:81000, and 1:243000, respectively. Then, take 100 μL of each and add it to the blocked wells. Cover with plastic wrap and let it stand at room temperature for 1 h. Wash the plate 3 times with PBST and pat dry.

[0065] (4) Secondary antibody: Add rabbit anti-alpaca IgG antibody (Rabbit Anti-Alpaca IgG, HRP, Abbott Bio), dilute to 1:20000, 100 μL / well, seal with plastic wrap, and place at room temperature for 1 h. Wash the plate three times with PBST and pat dry.

[0066] (5) TMB color development: Add TMB color development solution, 100 μL / well, color development for 15 min, and add stop solution, 50 μL / well.

[0067] (6) Reading: Place the microplate into a microplate reader for reading. Read the absorbance at 450 nm. The results are shown in Table 1.

[0068] Table 1

[0069]

[0070] Table 1 shows that the titer of alpaca serum after the fourth immunization was >27,000, indicating that a large amount of antibodies had been produced and could be used to construct an antibody library.

[0071] 3. Construction of Antibody Library

[0072] (1) RNA was extracted from alpaca whole blood after the fourth immunization using the TRIZOL method. The electrophoresis results are shown in Figure 2 Lane 1 is the marker, lane 2 is the anti-LAG3 antibody positive control sample, and lane 3 is the RNA sample extracted from immune alpaca blood.

[0073] (2) RNA was reverse transcribed into cDNA using a reverse transcription kit (Novozyme, R312-02).

[0074] (3) Amplification of VH2 and VH3 fragments: Using reverse transcribed cDNA as a template, a pair of primers was designed and synthesized and the first round of PCR was performed. The electrophoresis results were shown in Figure 3 , where lane 1 is the marker and lane 2 is the first round PCR product; using the 700bp fragment of the first round PCR as a template, 8 pairs of degenerate primers were designed and synthesized for the second PCR. The electrophoresis results of the 4 pairs of degenerate primers amplifying the VH2 fragment are shown in Figure 4 The electrophoresis results of the VH3 fragments of the four pairs of degenerate primers are shown in lanes 1 to 4 in Figure A. Figure 4 Lanes 6 to 9 and Figure 4 In the middle panel B, the PCR product is a band of about 500 bp, and the PCR product was recovered by gel cutting.

[0075] The PCR amplification system is as follows:

[0076]

[0077]

[0078] The PCR amplification procedure is as follows:

[0079]

[0080] (4) Digest the target fragment and perform T4 ligation.

[0081] Recover the second-round amplified fragment using a gel extraction kit. Digest the recovered fragment and the phagemid vector (pComb3X vector) with SfiI enzyme. Purify the digested fragment using a gel extraction column. Recover the pComb3X linear vector using a gel extraction kit. Mix the target fragment (digested fragment) and the pComb3X linear vector (digested vector) and ligate using T4 ligase overnight at 4°C.

[0082] The enzyme digestion system (taking 50 μL of enzyme digestion system as an example) is as follows:

[0083]

[0084] The above enzyme digestion system was mixed and placed in a 50°C PCR instrument for reaction for 60 minutes, and then the gel was cut and recovered.

[0085] Ligate the vector recovered from the gel with the target fragment at 4°C overnight. The reaction system (taking 600 μL as an example) is as follows:

[0086]

[0087]

[0088] (5) Electroporation to construct library and calculate library capacity

[0089] The purified ligation product was mixed with XL1-Blue competent medium in an ice bath and aliquoted into 80 μL / cup electroporation cuvettes. Electroporation was performed at 1800V. After electroporation, the mixture was transferred to 2YT-ATG medium containing ampicillin and tetracycline. The volume was then fixed to 200 mL using 2YT-ATG medium. The fixed-volume library was recovered by shaking and incubating at 37°C, 250 rpm, for 1 hour. This constituted the antibody bacterial library. From each library, 100 μL of the bacterial solution was diluted 10,000-fold using 2YT medium (100 μL of the bacterial solution + 900 μL of 2YT medium). 100 μL of the diluted bacterial solution was then spread onto 2YT-ATG semi-solid medium plates and incubated overnight at 37°C. The following day, single colonies were counted and the number of transformants was calculated.

[0090] Library capacity: 200 mL of original bacterial solution was diluted 1000-fold, and then 100 μL was inoculated to obtain 205 clones. The total storage capacity of VH2 and VH3 was calculated to be 4.1E+08.

[0091] After the bacterial solution was cultured at 37°C and 250rpm for 1 hour, VCSM13 helper phage was added and allowed to infect for half an hour, and then further cultured at 37°C and 200rpm for 1 hour. Finally, the bacterial precipitate was collected by centrifugation and resuspended in 100mL of 2YT-ATK medium containing ampicillin, tetracycline and kanamycin, and amplified overnight at 30°C and 225rpm. The supernatant was collected the next day, and 4% PEG8000 and 3% NaCl were used for ice bath and centrifugation to obtain phage precipitate. The obtained phage precipitate was resuspended and dissolved in PBS (pH 7.4) and sterilized by filtration using a 0.2μm syringe filter. 10μL of the filtered library was aspirated and diluted 10 times using the 2YT medium 100-fold dilution method (10μL+990μL). 8 After doubling, take 10 μL of the dilution and infect 100 μL of XL1-blue. After 30 minutes, spread the solution onto a 2YT-ATG semi-solid medium plate. The next day, count the number of monoclonal colonies to calculate the concentration of the amplified phage library. After filtering the library, add DMSO to 7% of the total volume, mix thoroughly, and freeze at -80°C until ready for use.

[0092] (6) Sequencing and identification of the antibody library.

[0093] 20 single clones were randomly selected from the library plate for sequencing, 19 of which were correct clones, 1 of which had a stop codon sequence, and the sequence accuracy rate was 95%.

[0094] 4. Antibody library screening

[0095] (1) hLAG antigen was diluted to 5 μg / mL with PBS buffer and CBS buffer, respectively, and added to a high-affinity ELISA plate (BeaverBio) at a rate of 100 μL / well. The plate was incubated at 4°C overnight. The next day, 3% skim milk powder was added at a rate of 340 μL / well and incubated at room temperature for 1 hour. The blocking buffer was discarded and the plate was washed.

[0096] (2) The above-constructed library (about 5×10 12 PFU) and incubated with hLAG antigen at 37°C for 2 hours.

[0097] (3) Wash the plate with PBST.

[0098] (4) Elution was performed with glycine-HCl buffer, and then neutralized with Tris-HCl buffer to pH 7.4.

[0099] (5) The eluate was mixed with E. coli XL1-Blue and incubated at 37°C with shaking.

[0100] (6) After adding phage, culture at 30°C overnight and collect the supernatant the next day. This is the first round of antibody library, recorded as LAG3-A-1st (high-affinity ELISA plate coated with PBS buffer containing hLAG) and LAG3-B-1st (high-affinity ELISA plate coated with CBS buffer containing hLAG).

[0101] (7) Repeat (1)-(6) to obtain the second round of antibody libraries, which are named LAG3-A-2nd and LAG3-B-2nd respectively.

[0102] (8) The antibody 2nd library was mixed with Escherichia coli XL1-Blue, incubated at 37°C with shaking, and after adding phage, cultured at 30°C overnight. The supernatant was collected the next day, which was the third round of amplification library, correspondingly recorded as LAG3-A-3rd and LAG3-B-3rd.

[0103] The third-round amplified library was assayed by ELISA, and the results are shown in Table 2. The ELISA assay follows the same protocol as that for hLAG3 antigen-immunized alpaca serum, with the exception that the serum sample was replaced with the third-round amplified library sample during the loading step, and the secondary antibody was replaced with M13-HRP (0.2 μg / mL) instead of rabbit anti-alpaca IgG. An ELISA without antigen coating served as a control.

[0104] Table 2

[0105]

[0106] According to Table 2, the library in the third round of amplification was positive and could be used for monoclonal detection.

[0107] The 96 monoclonal colonies from the third round of positive library were used for ELISA detection of phage expression. The ELISA detection method was based on the titer method of alpaca serum immunized with hLAG3 antigen. The differences were that the hLAG antigen concentration was adjusted to 0.5 μg / mL or no hLAG antigen was added in the coating step, the serum sample was replaced with a 10× diluted phage expression sample in the loading step, and the secondary antibody was changed from rabbit anti-alpaca IgG antibody to M13-HRP (Albertson Biotechnology, concentration 0.2 μg / mL).

[0108] The OD value results under the hLAG antigen coating (LAG3 0.5 μg / mL) detection conditions are shown in Table 3, and the OD value results under the control detection conditions without hLAG antigen coating (LAG3 blank) are shown in Table 4.

[0109] Table 3

[0110]

[0111]

[0112] Table 4

[0113] 1 2 3 4 5 6 7 8 9 10 11 12 A 0.0302 0.0242 0.0221 0.0371 0.0275 0.0529 0.0325 0.0301 0.0356 0.0246 0.023 0.0442 B 0.0359 0.0182 0.0388 0.0177 0.0206 0.0327 0.0272 0.0232 0.014 0.0313 0.0264 0.0399 C 0.019 0.0225 0.0159 0.0143 0.0148 0.0339 0.0304 0.0237 0.0237 0.0179 0.0244 0.0478 D 0.0274 0.0969 0.0164 0.0304 0.0192 0.0401 0.0251 0.0269 0.0269 0.026 0.0258 0.0487 E 0.0228 0.0225 0.0158 0.0267 0.0201 0.0312 0.0331 0.0268 0.0307 0.0234 0.0288 0.0296 F 0.1612 0.0173 0.0238 0.0243 0.0225 0.0336 0.0317 0.0307 0.0263 0.0219 0.0311 0.0464 G 0.0546 0.0354 0.0957 0.0293 0.0414 0.0416 0.0407 0.0277 0.0259 0.0352 0.0314 0.0431 H 0.0411 0.0589 0.0315 0.0267 0.0305 0.0398 0.0394 0.0329 0.0367 0.0351 0.0358 0.021

[0114] The clones were retested by ELISA, and the results are shown in Table 5.

[0115] Table 5

[0116] mark Orifice plate LAG3 0.5 μg / mL LAG3blank LAG3-A-1 C01 1.5719 0.0329 LAG3-A-2 B03 1.5826 0.0478 LAG3-A-3 D03 1.4863 0.0399 LAG3-A-6 B04 1.6409 0.0237 LAG3-A-16 C04 1.6069 0.0275 LAG3-A-21 G04 1.4213 0.0339 LAG3-A-28 E05 1.3728 0.0268 LAG3-A-39 H05 1.3802 0.0246 LAG3-A-41 C06 1.6022 0.0307 LAG3-A-43 A07 1.6796 0.0356 LAG3-A-50 G07 1.4017 0.0258 LAG3-A-59 B08 1.4403 0.0325 LAG3-A-61 G09 1.6409 0.0277 LAG3-A-65 C10 1.6124 0.0331 LAG3-A-66 F10 1.8154 0.0293 LAG3-A-69 G10 1.5959 0.0546 LAG3-A-73 F11 1.9886 0.0158 LAG3-A-82 D12 1.6876 0.0314

[0117] The positive clones in Table 5 were sequenced.

[0118] 5. Antibody Preparation

[0119] An anti-LAG3 antibody with an hFc tag was prepared using a mammalian expression system. The hFc tag is derived from the Fc region of human immunoglobulin G (IgG), and its amino acid sequence is:

[0120] PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIE RTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO: 11).

[0121] A mammalian expression vector, PCDNA3.1-LAG-A-3hFc, was constructed. HEK293 cells were subcultured in 293 serum-free CD medium. PCDNA3.1-LAG-A-3hFc was mixed with transfection reagent TF2 and added to the HEK293 cells. 293 serum-free feed medium was added on days 1, 3, and 5 after transfection. Shake flask culture conditions included 5% CO2, 37°C, and a shaker at 175 rpm.

[0122] The cell supernatant was centrifuged, filtered (0.22 μm), and then passed through a Protein G column for protein purification. The steps are as follows:

[0123] (1) Wash with 5 column volumes of deionized water to remove air and 20% ethanol;

[0124] (2) Equilibrate the column with 5 to 10 column volumes of buffer (PBS, pH 7.4);

[0125] (3) The sample was passed through the purification column at a rate of 0.5 ml / min;

[0126] (4) Equilibrate the column with the above buffer;

[0127] (5) Elution was performed with glycine solution, and the target protein (fusion protein of hFc tag and anti-LAG3 antibody) in the eluate was detected by SDS-PAGE gel, i.e., anti-LAG3 antibody with hFc tag, with a molecular weight of approximately 40KD.

[0128] The hFc-tagged anti-LAG3 antibody prepared above was used to detect monoclonal antibody binding by ELISA. The test method is as follows:

[0129] (1) Coating: Dilute the hLAG antigen to 2 μg / mL with CBS buffer, then dilute it in a 2-fold gradient, add 100 μL / well to the ELISA plate, seal the reaction wells with plastic wrap, incubate at 4°C overnight, wash the plate twice with PBST, and pat dry.

[0130] (2) Blocking: Fill the reaction wells with 5% skim milk powder blocking solution, seal with plastic wrap, and leave at room temperature for 1 h. Wash the plate twice with PBST and pat dry.

[0131] (3) Sample addition: Add 1 μg / mL of hFc-tagged anti-LAG3 antibody, 100 μL / well, incubate at room temperature for 1 h, wash the plate 4 times with PBST, and pat dry.

[0132] (4) Secondary antibody: Add rabbit anti-alpaca IgG antibody (Rabbit Anti-Alpaca IgG, HRP, Abbott Bio), dilute to 1:20000, 100 μL / well, seal with plastic wrap, and place at room temperature for 1 h. Wash the plate 4 times with PBST and pat dry.

[0133] (5) Color development: Add TMB color development solution, 100 μL / well, develop at room temperature for 10 min, and add stop solution, 50 μL / well.

[0134] (6) Reading: Place the plate into the microplate reader for reading, and read the absorbance at 450 nm.

[0135] The ELISA test results of the top five anti-LAG3 antibodies with the highest binding ability are shown in Table 6.

[0136] Table 6

[0137]

[0138] With log1 / (x) as the horizontal axis (x: hLAG antigen concentration, μg / mL) and OD value as the vertical axis, the four-parameter fitting equation curve is as follows Figure 5 As shown, calculate EC 50The EC50 value of LAG3-A-16 was 0.0001567 μg / mL, and the EC50 value of LAG3-A-21 was 0.0001567 μg / mL. 50 The value is 0.0001785 μg / mL, and the EC corresponding to LAG3-A-66 50 The value is 0.03007 μg / mL, and the EC corresponding to LAG3-A-73 50 The value is 0.003468 μg / mL, and the EC corresponding to LAG3-A-82 50 The value is 0.001016μg / mL.

[0139] Combined EC50 values, Table 7 and Figure 5 The affinity of monoclonal antibodies for antigen binding was ranked from high to low as follows: LAG3-A-16 > LAG3-A-21 > LAG3-A-82 > LAG3-A-73 > LAG3-A-66. LAG3-A-16 and LAG3-A-21 had comparable affinities, and their EC50 values ​​differed significantly from those of other antibodies, being approximately 1 to 2 orders of magnitude lower.

[0140] The nucleotide and amino acid sequences of the screened high-affinity anti-LAG3 antibodies are shown in Table 7.

[0141] Table 7

[0142]

[0143]

[0144]

[0145] Example 1

[0146] This embodiment provides a LAG3 antigen Western blot detection kit and a LAG3 antigen Western blot detection method.

[0147] The LAG3 antigen immunoblotting detection kit includes a primary antibody, a secondary antibody, a 5% skim milk powder blocking buffer, a PBST washing solution, a nitrocellulose membrane, a loading buffer (2× Laemmli sample buffer), a protein standard (marker), and a chemiluminescent substrate (ECL). The primary antibody is an anti-LAG3 antibody with an hFc tag. In this example, the hFc-tagged LAG3-A-16 antibody was prepared using the mammalian system described above. The electrophoresis quality control test results are shown in Figure 6 The secondary antibody was goat anti-human-HRP at a concentration of 1 mg / mL.

[0148] The detection method is:

[0149] (1) Add an equal volume of 2× Laemmli sample buffer to the cell lysate sample and boil at 100°C for 5 min to denature the protein.

[0150] (2) Load the protein onto the SDS-PAGE gel and run electrophoresis at 100V for 1 hour. Remove the gel from the electrophoresis tank and soak it in transfer buffer. Place the transfer device in the transfer tank, add transfer buffer, start the power supply, and transfer the membrane. After transfer, wash the membrane twice with PBST, each time for 5 minutes, and block the membrane with 5% skim milk powder blocking solution. Incubate at room temperature for 1 hour.

[0151] (3) Pour off the 5% skim milk powder blocking solution, add diluted primary antibody, incubate at room temperature for 1 hour, wash the membrane three times with PBST, each time for 10 minutes, add diluted secondary antibody, incubate at room temperature for 1 hour, and wash the membrane three times with PBST, each time for 10 minutes.

[0152] (4) Add chemiluminescent substrate (ECL) and develop.

[0153] Positive samples were lysates of 293T cells that overexpressed LAG3, and negative samples were lysates of 293T cells that did not express LAG3. Figure 7 , a clear band appeared at 60KD in the positive sample, while no clear band was found in the negative sample, indicating that the LAG3 antigen was detected in the positive sample.

[0154] Example 2

[0155] This example provides another LAG3 antigen immunoblotting detection kit, except that the primary antibody is replaced with LAG3-A-21 with hFc tag prepared in the mammalian system described above (the electrophoresis quality control test results are shown in Figure 8 , concentration is 0.56 mg / mL) and the rest are the same as in Example 1. The positive samples and negative samples are tested according to the method of Example 1. The test results are shown in FIG. Figure 9 , a clear band appeared at 60KD in the positive sample, while no clear band was found in the negative sample, indicating that the LAG3 antigen was detected in the positive sample.

[0156] The above detailed description of the present invention is intended to enable those skilled in the art to understand the contents of the present invention and implement them, but it does not limit the scope of protection of the present invention. The present invention is not limited to the above embodiments. Any equivalent changes or modifications made based on the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. An anti-LAG3 antibody, characterized in that The amino acid sequence of the anti-LAG3 antibody is shown in SEQ ID NO: 6 or SEQ ID NO:

7.

2. The nucleic acid encoding the anti-LAG3 antibody according to claim 1, characterized in that The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

3. An hFc-labeled anti-LAG3 antibody, characterized in that The anti-LAG3 antibody comprises the anti-LAG3 antibody according to claim 1 and an hFc tag, wherein the hFc tag is connected to the C-terminus of the anti-LAG3 antibody.

4. Use of the anti-LAG3 antibody according to claim 1 or the hFc-labeled anti-LAG3 antibody according to claim 3 in the preparation of a LAG3 antigen detection reagent or detection kit.

5. A LAG3 antigen detection reagent, characterized in that: The method comprises the hFc-labeled anti-LAG3 antibody according to claim 3 and a PBS buffer solution with a pH value of 7.4-7.

6.

6. A LAG3 antigen detection kit, characterized in that: It comprises the anti-LAG3 antibody according to claim 1, the hFc-labeled anti-LAG3 antibody according to claim 3, or the LAG3 antigen detection reagent according to claim 4.

7. The LAG3 antigen detection kit according to claim 6, characterized in that The LAG3 antigen detection kit is an immunohistochemistry detection kit, an immunoprecipitation detection kit, an enzyme-linked immunosorbent assay kit or an immunoblotting detection kit.

8. An immunoblotting detection kit for LAG3 antigen detection, characterized in that: The immunoblotting detection kit comprises a primary antibody and a secondary antibody, wherein the primary antibody is the hFc-labeled anti-LAG3 antibody according to claim 3, and the secondary antibody is a horseradish peroxidase-labeled goat anti-human antibody.

9. A recombinant vector, characterized in that The recombinant vector comprises a nucleotide sequence encoding the anti-LAG3 antibody of claim 1 or a nucleotide sequence encoding the hFc-tagged anti-LAG3 antibody of claim 3.

10. A recombinant cell, characterized in that The recombinant cell comprises the recombinant vector according to claim 9.

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