CN116003606A - TIGIT nano antibody and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of antibody preparation, and particularly relates to a TIGIT nano antibody, and a preparation method and application thereof. The amino acid sequence of the TIGIT nano antibody is one of SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11. The preparation process is as follows: constructing TIGIT antigen to immunize alpaca to obtain alpaca PBMC cells; capturing RNA of the cells, reversely transcribing the RNA into cDNA, carrying out PCR to obtain antibody gene fragments, and carrying out high-flux expression through a mammalian cell high-flux expression system; finally, the nano antibody of the TIGIT is obtained through ELISA and FACS detection and screening. The preparation method is simple and convenient, and the mammalian cell expression system induces the high-efficiency expression of the antibody, and the antibody can be processed and modified after translation, and the activity of the antibody is more similar to that of a natural antibody.

Description

TIGIT nano antibody and preparation method and application thereof

Technical Field

The invention belongs to the technical field of antibody preparation, and particularly relates to a TIGIT 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. TIGIT is a newly discovered cell surface protein with immunosuppressive function in recent years. Most of the presently known immune checkpoints are generally one-to-one or one-to-many relationship with their ligands, whereas TIGIT is different in that it maintains a "many-to-many" relationship with CD226, CD96, CD112, CD 155.

TIGIT and CD155 exist as homodimers, respectively, which bind to CD155 via intermolecular interactions. CD155 is expressed mainly on the surface of immune cells such as DC cells, T cells, B cells, macrophages, and the like, and also expressed in small amounts on the surface of non-immune cells such as kidney, lung, pancreas, and the like. CD112 is widely expressed on both hematopoietic and non-hematopoietic cell surfaces, but CD113 is only expressed on non-hematopoietic cell surfaces. Notably, CD155 and CD112 are highly expressed on the surface of many malignant tumors, such as colorectal cancer, melanoma. Therefore, intensive studies of TIGIT have also theoretical and practical significance for the study of anti-tumor and autoimmune diseases. The TIGIT antibody medicine has wide application prospect as a novel immune checkpoint antibody medicine, and can be used for the immune treatment 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

The invention provides a TIGIT nano antibody, a preparation method and application thereof. The preparation method provided by the invention effectively reduces development and production costs of the target antibody, shortens the expression time of the antibody, simultaneously uses 96-well cell culture plates for expression, increases the flux and improves the expression efficiency.

A nanobody of TIGIT comprising a framework region and a complementarity determining region, the complementarity determining region comprising CDR1, CDR2, CDR3, wherein the complementarity determining region CDR1 sequence is SEQ ID No.1, the complementarity determining region CDR2 sequence is SEQ ID No.2, the complementarity determining region CDR3 sequence is SEQ ID No.3; or the CDR1 is SEQ ID NO.1, the CDR2 is SEQ ID NO.4, and the CDR3 is SEQ ID NO.3; or the CDR1 is SEQ ID NO.1, the CDR2 is SEQ ID NO.5, and the CDR3 is SEQ ID NO.3; or the CDR1 is SEQ ID NO.1, the CDR2 is SEQ ID NO.6, and the CDR3 is SEQ ID NO.7. Specific sequence information is set forth in table 1.

TABLE 1 amino acid sequence of CDR region of TIGIT nanobody of the invention

Preferably, the TIGIT nanobody has an amino acid sequence selected from any one of the following: SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11.

Preferably, the nucleotide sequence encoding the nanobody amino acid sequence is one of the following sequences: one of SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO. 15.

The invention also provides a molecular expression vector, which comprises one of the nucleotide sequences of SEQ ID NO. 12-15.

The invention also provides a host cell of the molecular expression vector, and the host cell is a mammalian cell.

Preferably, the mammalian cell is one of HEK-293 cell and CHO cell.

The invention also provides a method for preparing the TIGIT nano antibody, which comprises the following preparation processes:

s1, expressing an immune antigen according to the protein sequence and gene sequence information of TIGIT, and connecting His-tag at the C end of the immune antigen to obtain a modified amino acid sequence;

s2, immunizing alpaca by using the modified amino acid sequence obtained in the step S1 to obtain alpaca PBMC cells;

s3, screening the alpaca PBMC cells obtained in the step S2 by a single cell microfluidic technology to obtain antibody secretion cells capable of secreting target antibodies, extracting RNA, reversely transcribing into cDNA, obtaining target gene fragments by PCR, and cloning the target gene fragments into eukaryotic expression vectors.

S4, the vector in the step S3 is induced to transfect the mammalian cells through a mammalian cell high-flux expression system to perform high-flux expression, and antibodies with high sensitivity and specificity are obtained through ELISA, FACS detection, cell binding assay detection screening and sequencing analysis.

The invention also provides application of the TIGIT nano antibody in preparing reagents for detecting T cell surface proteins.

The invention also provides application of the TIGIT nano antibody in preparing medicines for treating blood tumor and solid tumor.

Drawings

FIG. 1 shows serum titer measurements at different dilutions;

FIGS. 2-5 show the results of antigen binding assays with transient supernatants;

FIG. 6 shows the results of cell binding assays.

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 TIGIT nanobody

The preparation method of the TIGIT nano antibody comprises the following steps:

s1, expressing an immune antigen according to the protein sequence and gene sequence information of TIGIT, and connecting His-tag at the C end of the immune antigen for subsequent purification and detection to obtain a modified amino acid sequence;

s2, performing four immunization on alpaca by using the mixed solution of the modified amino acid sequence (antigen) obtained in the step S1 and Freund' S adjuvant to obtain alpaca PBMC cells: priming alpaca with 200 μg of the human TIGIT/His protein (i.e., the modified amino acid from step S1) in emulsified mixture with 200 μl of freund 'S complete adjuvant, boosting 3 times with human TIGIT/His protein and 200 μl of freund' S incomplete adjuvant on days 21, 42, 63, 1 week after each immunization, blood sampling to detect Anti-TIGIT/His serum titer; after 1 week of immunization 4, 50mL of blood was collected for screening and stock establishment.

Anti-TIGIT/His bloodThe titers were detected by ELISA as follows: the ELISA plate was coated with TIGIT/His protein at a concentration of 2. Mu.g/mL, and 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: 10000-diluted horseradish peroxidase-labeled anti-Alpaca IgG (H+L) secondary antibody, incubating for 1H at 37 ℃, washing 5 times, adding 100 μl TMB substrate, incubating for 10min at 37 ℃ and incubating for 50 μl 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 2 times of that of the negative control, the antiserum titer is judged to be positive, the result is shown in the figure 1, and the antiserum titer after 4 days is shown in the figure 1 to be 1600. Thus, the antigen can induce alpaca to produce high titer antisera specific to TIGIT 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 Trizol method, and reversing into cDNA by oligo (dT) (reverse transcription kit is TaKaRa-SMARTcribe Reverse Transcript); the target gene is obtained through PCR amplification, and then the target gene is cloned into a eukaryotic expression vector pcDNA3.1.

Plasmid extraction: the plasmid is extracted by adopting an alkaline lysis method, and the specific process is as follows: selecting a monoclonal colony to a 96-hole deep hole plate, shake shaking culture at 37 ℃ for 8 hours, placing the 96-hole deep hole plate of a culture bacterial liquid in a horizontal centrifuge, centrifuging at 4000r/min for 10min, discarding the supernatant, adding solution I, and shaking to uniformly suspend the bacterial body; then addThe solution II is gently and fully inverted for 4-6 times, and is uniformly mixed to lead the bacterial liquid to be fully cracked 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; adding 500 mu L of 70% absolute ethanol into each 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 respectively; a10-plate 96-well 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 to each well of the 96-well cell culture plate added with the plasmid, and the cells are 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 TIGIT protein (i.e., antigen of step S1). The results of the monoclonal ELISA showed (as shown in fig. 2-5, partial results showed) that these sequences were sequenced and aligned to eliminate the repeated sequences. To further verify positive antibodies binding TIGIT-VHH protein, cell supernatants were subjected to flow cytometry, FACS positive antibodies were expression purified and then subjected to Cell binding assay (cell binding assay), the screened antibodies were diluted at a 4-fold gradient, added to pre-plated cells, incubated at 4 ℃ for 1 hour, washed twice with MACS buffer, secondary antibodies were added, incubated at 4 ℃ for 30min, washed twice with MACS buffer, and detected by flow cytometry, the results shown in fig. 6 (partial results display), and the results showed that the Emax (maximum effect value) of the final VHH antibody obtained by Cell binding assay screening was slightly lower than that of the control antibody, probably due to fewer antigen binding sites of nanobodies.

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 (9)

1. A TIGIT nanobody, comprising a framework region and a complementarity determining region, wherein the complementarity determining region comprises CDR1, CDR2 and CDR3, wherein the complementarity determining region CDR1 sequence is SEQ ID No.1, the complementarity determining region CDR2 sequence is SEQ ID No.2, and the complementarity determining region CDR3 sequence is SEQ ID No.3; or the CDR1 is SEQ ID NO.1, the CDR2 is SEQ ID NO.4, and the CDR3 is SEQ ID NO.3; or the CDR1 is SEQ ID NO.1, the CDR2 is SEQ ID NO.5, and the CDR3 is SEQ ID NO.3; or the CDR1 is SEQ ID NO.1, the CDR2 is SEQ ID NO.6, and the CDR3 is SEQ ID NO.7.

2. The TIGIT nanobody of claim 1, wherein the TIGIT nanobody has an amino acid sequence selected from any one of: SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11.

3. The TIGIT nanobody of claim 2, wherein the nucleotide sequence encoding the TIGIT nanobody amino acid sequence is one of the following sequences: one of SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO. 15.

4. A molecular expression vector comprising one of the nucleotide sequences of SEQ ID nos. 12 to 15.

5. A host cell comprising the molecular expression vector of claim 4, wherein the host cell is a mammalian cell.

6. The host cell of claim 5, wherein the mammalian cell is one of a HEK-293 cell, CHO cell.

7. A method for preparing TIGIT nanobodies according to any of claims 1-3, characterized in that the preparation process is as follows:

s1, expressing an immune antigen according to the protein sequence and gene sequence information of TIGIT, and connecting His-tag at the C end of the immune antigen to obtain a modified amino acid sequence;

s2, immunizing alpaca by using the modified amino acid sequence obtained in the step S1 to obtain alpaca PBMC cells;

s3, screening the alpaca PBMC cells obtained in the step S2 by a single cell microfluidic technology to obtain antibody secretion cells capable of secreting target antibodies, extracting RNA, reversely transcribing into cDNA, obtaining target gene fragments by PCR, and cloning the target gene fragments into eukaryotic expression vectors;

s4, the vector in the step S3 is induced to transfect the mammalian cells through a mammalian cell high-flux expression system to perform high-flux expression, and antibodies with high sensitivity and specificity are obtained through ELISA, FACS detection, cell binding assay detection screening and sequencing analysis.

8. Use of TIGIT nanobody of claim 1 in the preparation of a reagent for detecting T cell surface protein.

9. Use of TIGIT nanobody of claim 1 in the preparation of a medicament for treating hematological tumors and solid tumors.

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