CN115819595A - anti-LAG3 nano antibody and preparation method and application thereof - Google Patents
anti-LAG3 nano antibody and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of antibody preparation, and particularly relates to an anti-LAG3 nano antibody and a preparation method and application thereof. The amino acid sequence of the LAG3 nano antibody provided by the invention is one of SEQ ID NO.7 and SEQ ID NO. 8; the preparation process comprises the following steps: constructing LAG3 antigen to immunize alpaca to obtain alpaca PBMC cells; capturing RNA of cells, carrying out reverse transcription on the cells to obtain cDNA, carrying out PCR to obtain an antibody gene fragment, and constructing the antibody gene fragment on a carrier; high-throughput expression is carried out through a monoclonal mammalian cell high-throughput expression system; and finally, detecting by ELISA and FACS, and sequencing and verifying to obtain the nucleic acid sequence. The preparation method provided by the invention is simple and convenient to operate, the mammalian cell expression system induces the high-efficiency expression of the antibody, the processing and modification can be carried out after the translation, and the activity of the preparation method is closer to that of a natural antibody.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to an anti-LAG3 nano antibody and a preparation method and application thereof.
Background
The surface of T cells has many important molecules that play an important role in the activation, proliferation and differentiation of T cells and in the performance of effector functions. LAG3 is a newly discovered cell surface protein with immune suppression function in recent years, is a type I transmembrane protein and has four Ig-like domains. Biochemical analysis showed that LAG3 can be expressed on the cell surface in the form of monomers, dimers and higher order oligomers, independent of the ligand. Due to structural homology to CD4, the typical ligand of LAG3 is MHC class II, and other ligands can interact with LAG3 through glycans, including galectin-3 (Gal-3) and hepatic sinus endothelial lectin (LSECtin), which are all lectins with carbohydrate-recognition domains. Gal-3 is a soluble galactose-binding lectin, secreted by tumor cells and tumor stromal cells. LSECtin is expressed in the liver, but it was also found on the surface of melanoma cells that binding of LSECtin to LAG3 could inhibit IFN γ production by antigen-specific effector T cells. Fibrinogen-like protein 1 (FGL 1) is secreted by hepatocytes in the liver, is highly upregulated in tumors, and is also considered to be a LAG3 ligand with immunosuppressive activity. Therefore, extensive research into LAG3, and research into anti-tumor and autoimmune diseases, also has theoretical and practical significance. The anti-LAG3 antibody drug has wide application prospect as a novel immune checkpoint antibody drug and can be used for immunotherapy of tumors.
The immune system of alpaca produces two types of antibodies when it detects foreign invaders such as bacteria and viruses: one similar to human IgG and the other one tenth the size of a normal antibody, these smaller antibodies are called single domain antibodies or nanobodies (i.e., light chain-deleted "heavy chain antibodies"), and these light chain-deleted heavy chain antibodies have very small fragments that can bind antigen as well as normal IgG antibodies and have high specificity and affinity. Compared with the traditional antibody, the nano antibody has the advantages of higher affinity, higher water solubility, stable conformation, easy genetic engineering modification, blood brain barrier crossing and the like. In recent years, with the research on the nano antibody, the antibody has been widely applied in the fields of protein visualization tracing, structure analysis, diagnosis and treatment of epidemic diseases of human beings and animals, and the like. However, the production of conventional antibodies requires animal or fermentation cell culture, resulting in high production cost and complicated production process. The nano antibody has the advantages of easy genetic engineering modification, low production cost and simple production process. Therefore, the antibody can replace the traditional antibody and has wide market application prospect.
Disclosure of Invention
Aiming at the defects generally existing in the prior art, the invention provides an anti-LAG3 nano antibody and a preparation method and application thereof. The preparation method provided by the invention is simple and convenient to operate, the mammalian cell expression system induces the high-efficiency expression of the antibody, the processing and modification can be carried out after the translation, and the activity of the preparation method is closer to that of a natural antibody.
In order to achieve the purpose, the invention adopts the technical scheme that:
an anti-LAG3 nanobody comprising a framework region and a complementarity determining region; the complementarity determining region comprises CDR1, CDR2 and CDR3, wherein the CDR1 sequence of the complementarity determining region is SEQ ID NO.1, the CDR2 sequence of the complementarity determining region is SEQ ID NO.2, and the CDR3 sequence of the complementarity determining region is SEQ ID NO.3; or the CDR1 is SEQ ID NO.4, the CDR2 is SEQ ID NO.5, and the CDR3 is SEQ ID NO.6.
CDR1：DYFMS(SEQ ID NO.1)；
CDR2：DINDGGGSAFYADSVKG(SEQ ID NO.2)；
CDR3：GYISGDYYSLDY(SEQ ID NO.3)；
CDR1：SYYMS(SEQ ID NO.4)；
CDR2：DINAGGDSTYYSDSVKG(SEQ ID NO.5)；
CDR3：DVGYDGDYGLGAEYDY(SEQ ID NO.6)。
Preferably, the anti-LAG3 nanobody has an amino acid sequence selected from any one of: SEQ ID NO.7 (ANb 22-M120-4M-3 LP-106), SEQ ID NO.8 (ANb 22-M120-4M-3 LP-319).
Preferably, the nucleotide sequence encoding the amino acid sequence of the nanobody is one of the following sequences: SEQ ID NO.9 (ANb 22-M120-4M-3 LP-106), SEQ ID NO.10 (ANb 22-M120-4M-3 LP-319).
The invention also provides a nucleic acid molecule carrier, which comprises one of the nucleotide sequences of SEQ ID NO.9 and SEQ ID NO.10 in claim 3.
The invention also provides a host cell containing the nucleic acid molecule vector, wherein the host cell is a mammalian cell, and the mammalian cell is HEK-293 cell or CHO cell.
The invention also provides a preparation method of the anti-LAG3 nano antibody, which is characterized by comprising the following steps of:
s1, expressing LAG3 immune antigen according to the protein sequence and gene sequence information of LAG 3;
s2, after the alpaca is immunized for multiple times by the LAG3 antigen obtained in the step S1, PBMC (peripheral blood mononuclear cell) of the alpaca is extracted;
and S3, taking the alpaca PBMC obtained in the step S2 as a raw material, screening positive antibody secreting cells by a single cell microfluidic technology, extracting RNA of the positive secreting cells, performing reverse transcription to obtain cDNA, performing PCR to obtain a target gene fragment, and cloning the target gene fragment into a vector.
And S4, inducing high-throughput expression of the LAG3 antibody through a mammalian cell high-throughput expression system, carrying out antigen binding detection through antibody detection technologies such as ELISA and FACS and the like, and carrying out sequencing analysis to finally obtain the anti-LAG3 nano antibody.
The invention also provides an anti-LAG3 nano antibody prepared by the preparation method.
The invention also provides application of the anti-LAG3 nano antibody in preparation of a reagent for detecting T cell surface protein.
The invention also provides application of the anti-LAG3 nano antibody in preparation of a medicine for treating blood tumors and solid tumors.
Compared with the prior art, the invention has the following technical advantages: the preparation method of the anti-LAG3 nano antibody provided by the invention effectively reduces the development and production cost of a target antibody, shortens the expression time of the antibody, and simultaneously, the mammalian cell high-throughput expression system of the patent technology uses a 96-hole cell culture plate for expression, increases the throughput and improves the expression efficiency of the nano antibody.
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FIG. 1 shows the results of serum titer measurements at different dilutions;
FIGS. 2 to 3 show the results of the detection of antigen binding to transient supernatant;
FIG. 4 shows the results of the cell binding assay;
FIG. 5 shows the results of the cell blocking assay.
Detailed Description
The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only for explaining the present invention and are not intended to limit the present invention, and all technical solutions identical or similar to the present invention are within the protection scope of the present invention. The method and the device are operated according to the conventional technical method and the content of the instrument instruction, wherein the specific technology or condition is not indicated in the embodiment; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example a method for preparing LAG3 Nanobody
The preparation method of the LAG3 nano antibody comprises the following steps:
s1, expressing an immune antigen (which can be the complete sequence of LAG 3) according to the protein sequence and gene sequence information of the LAG3, and connecting His-tag at the C end of the immune antigen to obtain modified amino acid for subsequent purification and detection;
s2, performing four times of immunization on the alpaca by using the mixed solution of the modified antigen and Freund' S adjuvant obtained in the step S1 to obtain alpaca PBMC cells: priming alpacas with an emulsified mixture of 200. Mu.g of human LAG3/His protein (i.e., the modified amino acids obtained in step S1) and 200. Mu.L of Freund 'S complete adjuvant, boosting 3 times with 200. Mu.g of human LAG3/His protein and 200. Mu.L of Freund' S incomplete adjuvant on days 21, 42 and 63, and collecting blood to detect Anti-LAG3/His serum titer 1 week after each immunization; after 1 week of 4 th immunization, 50mL of blood was collected for screening and banking.
Anti-LAG3/His serum titers were detected by ELISA, as follows: the elisa plate was coated with LAG3/His protein at a concentration of 2 μ g/mL, 100 μ L of serum obtained after four immunizations (control was pre-immune alpaca serum) was added to each well in 2-fold gradient dilutions (i.e. diluted to 2-fold in the first 1 well per well), incubated for 1.5h at 37 ℃, washed 2 times, and 1:10000 diluted second antibody of horseradish peroxidase-labeled Goat anti-Alpaca IgG (H + L) is incubated at 37 ℃ for 1H, after 5 times of washing, 100 muL of TMB substrate is added, incubation at 37 ℃ is 10min,50 muL of 0.1M H 2 SO 4 The reaction was stopped and the OD450 nm was measured. When the OD450 value of the sample to be tested is more than 3 times of that of the negative control, the antiserum titer is judged to be positive, the result is shown in figure 1, and the antiserum titer after 4-immunization is 3200 in figure 1. It can be seen that the antigen can induce alpaca to produce high titer antiserum specific to LAG3 protein.
S3, taking the 50mL blood sample obtained in the step S2 as a raw material, and performing functional antibody secretory cell sorting by using a microfluidic platform, wherein the cells and the antigens can be wrapped in monodisperse oil drops at a picoliter level by using a droplet microfluidic technology, the generation speed of thousands of monodisperse oil drops per second can be achieved, each obtained micro-drop of single cells is independent, and the independent environment and the non-cross pollution between the cells are realized; VHH is identified by a fluorescence-labeled alpaca secondary antibody in oil drops, and if a VHH antibody secreted by cells can identify a fluorescence-labeled antigen, a FRET signal can be formed and sorted out, and B cells which can secrete VHH and have binding activity are screened.
Extracting RNA of the obtained functional antibody secreting cells by a Trizol method, inverting the RNA into cDNA (Reverse transcription kit TaKaRa-SMARTcrib Reverse Transcript) by using oligo (dT), and amplifying by PCR to obtain a target fragment; the target gene is cloned into eukaryotic expression vector pcDNA3.1.
And (3) plasmid extraction: picking the monoclonal colony to a 96-hole deep-hole plate, and carrying out shake table culture at 37 ℃ for 8h; after centrifugation, carrying out high-throughput plasmid extraction on the obtained bacterial liquid, extracting plasmid DNA according to an alkaline lysis method, placing a 96-hole deep-hole plate for culturing the bacterial liquid in a horizontal centrifuge, centrifuging, discarding the supernatant, adding the solution I, and shaking to uniformly suspend the thalli; adding the solution II, and mildly and fully reversing the solution from top to bottom for 4-6 times to uniformly mix so as to fully crack the bacterial liquid until a transparent solution is formed; finally adding the solution III, gently and fully reversing the upper part and the lower part for 6 to 8 times, and centrifuging the mixture for 10min at 4000 r/min; after centrifugation, sucking 800 mu L of supernatant into a 96-hole filter plate (the filter plate is connected with a 96-hole deep-hole plate below), and centrifuging at 4000r/min for 2min; collecting filtrate, adding 300 μ L isopropanol into each hole, centrifuging at 4000r/min for 15min, and discarding supernatant; adding 500 μ L70% anhydrous alcohol into each well, centrifuging at 4000r/min for 10min, and discarding the supernatant; standing 96-well deep-well plate, air drying ethanol at room temperature, adding 70 μ L ddH into each well 2 And O, shaking and mixing uniformly, and then measuring the plasmid concentration.
Cell transfection and selection: the antibiotic-free DMEM was added to 96-well cell culture plates at 75 μ L per well. Add 10. Mu.L of extracted plasmid into each well (no wells are required for corresponding plasmid positions); transfecting a 10-plate 96-cell plate together, and adding 75 mu L of DMEM diluted PEI transfection reagent into each hole of the 96-hole cell culture plate added with the plasmid (the volume ratio of the PEI transfection reagent to the DMEM culture medium is 1; the final mixed plasmid transfection reagent cell suspension was co-incubated for 15min at room temperature.
HEK-293 cell suspension was added dropwise to corresponding 96-well cell culture plates (containing plasmid transfection reagent cell suspension) at 5X 10 per well 4 Adding 100 μ L cell suspension to each cell, 5% CO at 37 deg.C 2 Culturing in a cell culture box for 72h continuously.
The transfected cell supernatants were tested for binding to LAG3 protein (i.e., the antigen of step S1). Monoclonal ELISA results showed that the selected antibodies all bound to LAG3 (partial results shown in FIGS. 2-3) and these sequences were includedAnd (4) performing sequencing alignment to eliminate repeated sequences. To further validate positive antibodies in the library that bind LAG3-VHH protein, cell supernatants were subjected to flow cytometry, FACS positive antibodies were purified for expression, followed by Cell binding assay, and the screened antibodies were diluted in 4-fold gradients starting at 200nM and added to a 2X 10 pre-applied layer 5 The cells are incubated at 4 ℃ for 1 hour, washed twice by MACS buffer, added with secondary antibodies (Goat anti-Human Fc, alexa Fluor 647), incubated at 4 ℃ for 30 minutes, washed twice by MACS buffer and detected by an up-flow cytometer, and the result is shown in figure 4 (partial result display), and the result shows that the Emax (maximum effect value) of the VHH antibody finally obtained by screening in figure IV is slightly better than that of the control antibody, and the result in figure 5 shows that the screened antibodies have slightly better blocking effect than that of the control antibody and slightly weaker than that of the control antibody.
Finally, it should be noted that the above-mentioned embodiments are only illustrative for the principle, performance and efficacy of the present invention, and are not meant to limit the present invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (8)
1. An anti-LAG3 nanobody, comprising a framework region and a complementarity determining region; the complementarity determining region comprises CDR1, CDR2 and CDR3, wherein the CDR1 sequence of the complementarity determining region is SEQ ID NO.1, the CDR2 sequence of the complementarity determining region is SEQ ID NO.2, and the CDR3 sequence of the complementarity determining region is SEQ ID NO.3; or the CDR1 is SEQ ID NO.4, the CDR2 is SEQ ID NO.5, and the CDR3 is SEQ ID NO.6.
2. The anti-LAG3 nanobody of claim 1, having an amino acid sequence selected from any one of: SEQ ID NO.7, SEQ ID NO.8.
3. The anti-LAG3 nanobody of claim 2, wherein the nucleotide sequence encoding the amino acid sequence of the anti-LAG3 nanobody is one of the following sequences: SEQ ID NO.9, SEQ ID NO.10.
4. A nucleic acid molecule vector, wherein the nucleic acid molecule vector comprises one of the nucleotide sequences of SEQ ID No.9 and SEQ ID No.10.
5. A host cell comprising the nucleic acid molecule vector of claim 4, wherein said host cell is a mammalian cell, and wherein said mammalian cell is a HEK-293 cell or a CHO cell.
6. A method of producing an anti-LAG3 nanobody according to any one of claims 1 to 3, comprising the steps of:
s1, expressing LAG3 immune antigen according to the protein sequence and gene sequence information of LAG 3;
s2, after the alpaca is immunized for multiple times by using the LAG3 antigen obtained in the step S1, the PBMC cells of the alpaca are extracted;
s3, taking the alpaca PBMC obtained in the step S2 as a raw material, screening positive antibody secreting cells by a single cell microfluidic technology, extracting RNA of the positive secreting cells, carrying out reverse transcription on the RNA into cDNA, obtaining a target gene fragment by PCR (polymerase chain reaction), and cloning the target gene fragment into a vector;
and S4, inducing high-throughput expression of the LAG3 antibody through a high-throughput expression system of mammalian cells, carrying out antigen binding detection through antibody detection technologies such as ELISA and FACS and the like, and carrying out sequencing analysis to finally obtain the anti-LAG3 nano antibody.
7. Use of the anti-LAG3 nanobody of any one of claims 1 to 3 for the preparation of a reagent for the detection of T cell surface proteins.
8. Use of the anti-LAG3 nanobody of any one of claims 1 to 3 for the preparation of a medicament for the treatment of hematological and solid tumors.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108101991A (en) * | 2012-07-02 | 2018-06-01 | 百时美施贵宝公司 | The optimization of antibody of bind lymphocytes activating gene -3 (LAG-3) and the purposes of the antibody |
| CN109475617A (en) * | 2017-02-22 | 2019-03-15 | 天境生物 | Anti-lag-3 antibodies and uses thereof |
| CN110621337A (en) * | 2017-05-10 | 2019-12-27 | 时迈药业 | anti-LAG 3 human monoclonal antibodies and uses thereof |
| US10946092B1 (en) * | 2020-06-05 | 2021-03-16 | Beijing Mabworks Biotech Co., Ltd. | Antibodies binding LAG3 and methods of treatment using them |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108101991A (en) * | 2012-07-02 | 2018-06-01 | 百时美施贵宝公司 | The optimization of antibody of bind lymphocytes activating gene -3 (LAG-3) and the purposes of the antibody |
| CN109475617A (en) * | 2017-02-22 | 2019-03-15 | 天境生物 | Anti-lag-3 antibodies and uses thereof |
| CN110621337A (en) * | 2017-05-10 | 2019-12-27 | 时迈药业 | anti-LAG 3 human monoclonal antibodies and uses thereof |
| US20210115135A1 (en) * | 2017-05-10 | 2021-04-22 | CentryMed Pharmaceutical Inc. | Human monoclonal antibodies against lag3 and uses thereof |
| US10946092B1 (en) * | 2020-06-05 | 2021-03-16 | Beijing Mabworks Biotech Co., Ltd. | Antibodies binding LAG3 and methods of treatment using them |
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