CN115819595B - anti-LAG3 nano antibody and preparation method and application thereof - Google Patents

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 LAG3 nanometer antibody provided by the invention has an amino acid sequence of one of SEQ ID NO.7 and SEQ ID NO. 8; the preparation process is as follows: constructing LAG3 antigen to immunize alpaca to obtain alpaca PBMC cells; capturing RNA of the cells, reversely transcribing the cells into cDNA, carrying out PCR to obtain antibody gene fragments, and constructing the antibody gene fragments on a carrier; high-throughput expression is performed by a monoclonal mammalian cell high-throughput expression system; finally, ELISA and FACS detection are carried out, and sequencing verification is carried out, thus obtaining the DNA sequence. The preparation method provided by the invention is simple and convenient to operate, and the mammalian cell expression system induces the high-efficiency expression of the antibody, and the antibody can be processed and modified after translation, so that the activity of the antibody is more similar to that of a natural antibody.

Description

anti-LAG3 nano antibody and preparation method and application thereof

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

T cell surfaces have a number of important molecules that play an important role in the activation, proliferation and differentiation of T cells and in the functioning of effector functions. LAG3 is a newly discovered cell surface protein with immunosuppressive 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 monomeric, dimeric and higher order oligomers, independent of ligand. Because of structural homology to CD4, typical ligands for LAG3 are MHC class II molecules, other ligands may also interact with LAG3 through glycans, including galectin-3 (Gal-3) and liver sinus endothelial cell lectin (LSECtin), which are lectins with a carbohydrate-recognition domain. Gal-3 is a soluble galactose-binding lectin secreted by tumor cells and tumor stromal cells. LSECtin is expressed in the liver, but binding of LSECtin to LAG3 was also found on the melanoma cell surface to inhibit ifnγ production by antigen specific effector T cells. Fibrinogen-like protein 1 (FGL 1) is secreted by hepatocytes in the liver and is highly upregulated in tumors and is also considered to be LAG3 ligand with immunosuppressive activity. Therefore, intensive research into LAG3, research into anti-tumor and autoimmune diseases, 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 the immunotherapy of tumors.

The alpaca immune system produces two types of antibodies when detecting foreign invaders such as bacteria and viruses: the other is equivalent to one tenth of the size of a normal antibody, and these smaller antibodies are called single domain antibodies or nanobodies (i.e., light chain-deleted "heavy chain antibodies"), which have only a small fragment of the light chain deleted heavy chain antibody to bind antigen as normal IgG and the like, and have high specific strong affinity. Compared with the traditional antibody, the nano antibody has the advantages of higher affinity, higher water solubility, stable conformation, easiness in genetic engineering improvement, crossing of blood brain barrier and the like. In recent years, with the continuous and intensive research on nanobodies, the antibodies have been widely used in the fields of protein visual tracking, structural analysis, diagnosis and treatment of human and animal epidemic diseases, and the like. However, the production of conventional antibodies requires animal or fermentation cell culture, resulting in high production costs and complex production processes. The nano antibody has the advantages of easy genetic engineering improvement, 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 commonly 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, and the mammalian cell expression system induces the high-efficiency expression of the antibody, and the antibody can be processed and modified after translation, so that the activity of the antibody is more similar to that of a natural antibody.

In order to achieve the above purpose, the invention adopts the following technical scheme:

an anti-LAG3 nanobody comprising a framework region and a complementarity determining region; the complementarity determining region comprises CDR1, CDR2 and CDR3, wherein the sequence of the CDR1 of the complementarity determining region is SEQ ID NO.1, the sequence of the CDR2 of the complementarity determining region is SEQ ID NO.2, and the sequence of the CDR3 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 the following: SEQ ID No.7 (ANb-M120-4M-3 LP-106), SEQ ID No.8 (ANb-M120-4M-3 LP-319).

Preferably, the nucleotide sequence encoding the nanobody amino acid sequence is one of the following sequences: SEQ ID No.9 (ANb-M120-4M-3 LP-106), SEQ ID No.10 (ANb-M120-4M-3 LP-319).

The invention also provides a nucleic acid molecule vector comprising one of the nucleotide sequences of SEQ ID NO.9 and SEQ ID NO.10 as set forth 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 a HEK-293 cell or a CHO cell.

The invention also provides a preparation method of the anti-LAG3 nano antibody, which is characterized by comprising the following steps:

s1, expressing LAG3 immune antigen according to the protein sequence and gene sequence information of LAG 3;

s2, performing multiple immunization on alpaca by using the LAG3 antigen obtained in the step S1, and extracting alpaca PBMC cells;

s3, screening positive antibody secretion cells by using the alpaca PBMC cells obtained in the step S2 as raw materials through a single cell microfluidic technology, extracting RNA of the positive secretion cells, performing reverse transcription to obtain cDNA, obtaining a target gene fragment through PCR, and cloning the target gene fragment into a vector.

S4, inducing the LAG3 antibody to express in high flux through a mammalian cell high flux expression system, carrying out antigen binding detection through an antibody detection technology such as ELISA and FACS, and carrying out sequencing analysis to finally obtain the LAG 3-resistant nanobody.

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 preparing a reagent for detecting T cell surface protein.

The invention also provides application of the anti-LAG3 nano antibody in preparing a medicament for treating blood tumor and solid tumor.

Compared with the prior art, the invention has the technical advantages that: the preparation method of the anti-LAG3 nanobody provided by the invention effectively reduces development and production costs of target antibodies, shortens antibody expression time, and simultaneously uses 96-well cell culture plates for expression in the mammalian cell high-throughput expression system, increases throughput and improves expression efficiency of nanobodies.

Drawings

FIG. 1 shows serum titer measurements at different dilutions;

FIGS. 2-3 show the results of antigen binding to transient supernatant;

FIG. 4 shows the results of a cell binding assay;

FIG. 5 shows the results of the cell blocking assay.

Detailed Description

The present invention will be further explained with reference to 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 scope of the present invention. The specific techniques or conditions are not noted in this example and are practiced according to methods and apparatus conventional in the art; the reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example preparation method of 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 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-time immunization on alpaca by using the mixed solution of the modified antigen obtained in the step S1 and Freund' S adjuvant to obtain alpaca PBMC cells: priming alpaca with an emulsified mixture of 200 μg human LAG3/His protein (i.e. modified amino acid from step S1) and 200 μl of freund 'S complete adjuvant, boosting 3 times with 200 μg human LAG3/His protein and 200 μl of freund' S incomplete adjuvant on days 21, 42, and 63, and after 1 week of each immunization, blood sampling to detect Anti-LAG3/His serum titer; after 1 week of immunization 4, 50mL of blood was collected for screening and stock establishment.

The 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. Mu.g/mL, 100. Mu.L of serum obtained after four immunizations (control was immunized alpaca serum) was added at a 2-fold gradient dilution per well (i.e., 2-fold dilution per well to the previous 1-well), incubated at 37℃for 1.5h, washed 2 times, and 1 was added per well: 10000-diluted horseradish peroxidase-labeled anti-Alpaca IgG (H+L) secondary antibody, incubating at 37 ℃ for 1H, washing 5 times, and adding 100 mu L of TMSubstrate B, incubation at 37℃for 10min, 50. Mu.L of 0.1M H ₂ SO ₄ The reaction was stopped and 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 sample to be tested is judged to be positive in terms of the antiserum titer, the result is shown in figure 1, and the antiserum titer after 4 days is shown in figure 1 is 3200. Thus, the antigen can induce alpaca to produce high titer antisera specific to LAG3 protein.

S3, taking 50mL of blood sample obtained in the step S2 as a raw material, and carrying out functional antibody secretion cell sorting by using a microfluidic platform, wherein a droplet microfluidic technology can wrap cells and antigens in picoliter-level monodisperse oil droplets, so that the generation speed of thousands of monodisperse droplets per second can be reached, each micro droplet of a single cell is obtained independently, the environment independence between the cells is realized, and cross contamination is avoided; VHH is identified in oil drops through a fluorescence-labeled alpaca secondary antibody, and if the VHH antibody secreted by cells can identify a fluorescence-labeled antigen, FRET signals can be formed to be separated, and B cells which can secrete VHH and have binding activity are obtained through screening.

Extracting RNA of the obtained functional antibody secreting cells by using a Trizol method, reversing the RNA into cDNA by using oligo (dT) (TaKaRa-SMARTcribe Reverse Transcript as a reverse transcription kit), and obtaining a target fragment by PCR amplification; cloning the target gene into eukaryotic expression vector pcDNA3.1.

Extracting plasmids: selecting a monoclonal colony to a 96-hole deep hole plate, and shake culturing for 8 hours at 37 ℃; extracting plasmid DNA from the obtained bacterial liquid by high-flux plasmid extraction after centrifugation 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 solution I, and vibrating to uniformly suspend the bacterial body; then adding the solution II, gently and fully reversing the solution for 4 to 6 times, and uniformly mixing the solution to fully crack the bacterial liquid until a transparent solution is formed; finally adding the solution III, gently and fully reversing the solution for 6 to 8 times, and centrifuging for 10 minutes at 4000 r/min; after centrifugation, absorbing 800 mu L of supernatant from each hole into a 96-hole filter plate (the lower part of the filter plate is connected with a 96-hole deep hole plate), and centrifuging for 2min at 4000 r/min; collecting filtrate, adding 300 μl of isopropanol into each well, centrifuging at 4000r/min for 15min, and discarding supernatant; each of whichAdding 500 mu L of 70% absolute ethanol into the hole, centrifuging for 10min at 4000r/min, and discarding the supernatant; the 96-well deep-hole plate is placed for airing ethanol at room temperature, and 70 mu L of ddH is added into each well ₂ And O, shaking and mixing uniformly, and measuring the plasmid concentration.

Cell transfection and selection: the antibiotic-free medium DMEM was added to each of the 96-well cell culture plates at 75 μl per well. Adding 10 mu L of extracted plasmid into each well (corresponding to the position of the corresponding plasmid without stringing holes); a 10-plate 96-cell plate is transfected together, 75 mu L of DMEM diluted PEI transfection reagent (the volume ratio of the transfection reagent PEI to the DMEM culture medium is 1:75) is added into each of the 96-well cell culture plate with plasmids; the final mixed plasmid transfection reagent cell suspension was incubated for 15min at room temperature.

HEK-293 cell suspension was added drop-wise to a corresponding 96-well cell culture plate (containing plasmid transfection reagent cell suspension), 5X 10 per well ⁴ 100. Mu.L of cell suspension was added to each cell, 37℃and 5% CO ₂ Culturing in a cell culture box, and continuously culturing for 72 hours.

The transfected cell supernatant was tested for binding to LAG3 protein (i.e., antigen from step S1). Monoclonal ELISA results showed that the antibodies screened all bound to LAG3 eggs (as shown in figures 2-3, part of the results were displayed) and the sequences were sequenced and aligned to eliminate the repeat sequences. To further verify positive antibodies binding to LAG3-VHH protein in the library, cell supernatants were subjected to flow cytometry, FACS positive antibodies were purified for expression, then Cell binding assay (cell binding assay) was performed, and the screened antibodies were diluted at 4-fold gradient starting at 200nM and added to 2 x 10 pre-plating ⁵ For 1 hour at 4℃and twice with MACS buffer, adding a secondary antibody (gold anti-Human Fc, alexa Fluor 647), and for 30 minutes at 4℃and twice with MACS buffer, the results were shown in FIG. 4 (partial results show), which shows that the results of the four screens, as seen by Cell binding assay, finally gave Emax (maximum effect value) of VHH antibody, which was slightly better than the control antibody, while the results of FIG. 5 show that one of the blocking effects of the antibodies we screened was slightly better than the control antibody, and one slightly weaker than the control antibody.

Finally, it should be noted that the above-mentioned embodiments are merely illustrative of the principles, performances and effects of the present invention, and are not meant to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims (7)

1. An anti-LAG3 nanobody, wherein the nanobody comprises a framework region and a complementarity determining region; the complementarity determining region comprises CDR1, CDR2 and CDR3, wherein the sequence of the CDR1 of the complementarity determining region is SEQ ID NO.1, the sequence of the CDR2 of the complementarity determining region is SEQ ID NO.2, and the sequence of the CDR3 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, wherein the amino acid sequence of the anti-LAG3 nanobody is 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: SEQ ID NO.9, SEQ ID NO.10.

4. A nucleic acid molecule vector comprising one of the nucleotide sequences of SEQ ID No.9, SEQ ID No.10.

5. A host cell comprising the nucleic acid molecule vector of claim 4, wherein the host cell is a mammalian cell, and wherein the mammalian cell is a HEK-293 cell or a CHO cell.

6. A method for preparing the anti-LAG3 nanobody of 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, performing multiple immunization on alpaca by using the LAG3 antigen obtained in the step S1, and extracting alpaca PBMC cells;

s3, screening positive antibody secretion cells by using the alpaca PBMC cells obtained in the step S2 as raw materials through a single cell microfluidic technology, extracting RNA of the positive secretion cells, performing reverse transcription to obtain cDNA, obtaining a target gene fragment through PCR, and cloning the target gene fragment into a vector;

s4, inducing the LAG3 antibody to express in a high flux manner through a mammalian cell high flux expression system, carrying out antigen binding detection through an antibody detection technology ELISA and FACS method, and carrying out sequencing analysis to finally obtain the anti-LAG3 nanobody.

7. Use of an anti-LAG3 nanobody according to any of claims 1-3 in the preparation of a reagent for detecting T cell surface proteins.

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