An anti-GLP-1R nanobody, its preparation method and its application

Abstract

This invention belongs to the field of molecular biology technology, specifically relating to an anti-GLP-1R nanobody, its preparation method, and its application. This invention provides an anti-GLP-1R nanobody and its construction method. The method is as follows: a GLP-1R overexpression plasmid and an overexpression cell line are constructed. Alpacas are immunized with the plasmid and cells to obtain PBMC cells. Total RNA is extracted, reverse transcribed into cDNA, and the antibody gene fragment is amplified by PCR and ligated into a phage expression vector. The constructed vector is electroporated into SS320 competent cells to obtain a GLP-1R immune antibody library. After one round of cell panning and Cell ELISA detection, antibodies binding to GLP-1R are obtained. The antibody sequence is sequenced, and the antibody protein is obtained using mammalian cells. FACS detection is performed using CHOK1-GLP-1R cells to finally obtain the GLP-1R-binding antibody. The method of this invention is simple to operate, and the antibody activity obtained using the mammalian cell expression system is closer to that of natural antibodies.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to an anti-GLP-1R nanobody, its preparation method, and its application. Background Technology

[0002] Diabetes is considered the fastest-growing health problem globally, affecting approximately 10% of adults worldwide. About 90% of diabetes cases are type 2 diabetes (T2DM). T2DM is caused by dysfunction of pancreatic beta cells and the resulting insulin deficiency.

[0003] Most current medications for type 2 diabetes mellitus (T2DM) do not target beta cells, therefore their effectiveness gradually diminishes during diabetes treatment. Metformin is a common treatment for type 2 diabetes. When a single medication is insufficient, sulfonylureas, alpha-glucosidase inhibitors, thiazolidinediones, and insulin injections can be added. However, these medications may cause side effects such as hypoglycemia, weight gain, and cardiovascular risks.

[0004] Studies have found that insulinotropic peptides, naturally secreted in the gut, can promote insulin secretion, offering a new direction for the treatment of type 2 diabetes mellitus (T2DM). Insulinotropic peptides, targeting β-cells and possessing potent hypoglycemic effects and significant safety profiles, have become the preferred injectable therapy for T2DM. Insulinotropic peptides include glucagon-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). Although both GLP-1 and GIP promote insulin secretion, the use of GIP alone to treat T2DM remains controversial due to its efficacy. GLP-1, as an endogenous agonist of GLP-1R, has appetite-suppressing, insulin-secreting, and weight-loss effects, making it a promising natural anti-diabetic product. However, in vivo, GLP-1 is immediately cleaved by dipeptidyl peptidase 4 (DPP-4) at its N-terminus, the second amino acid (alanine), after secretion. This leads to rapid degradation of GLP-1 in the body, limiting its therapeutic application. Although DPP-4 inhibitors and GLP-1 analogs have been used to treat type 2 diabetes mellitus (T2DM) and have shown good efficacy, GLP-1R agonists are more favored because they provide better control of weight and cardiovascular disease compared to inhibitors and analogs, and reduce the risk of hypoglycemia compared to insulin and sulfonylureas.

[0005] Therefore, developing an anti-GLP-1R antibody drug as a treatment for type 2 diabetes mellitus (T2DM) has broad application prospects. Anti-GLP-1R antibody drugs can also be used to treat obesity and other conditions. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide an anti-GLP-1R nanobody, its preparation method and its application.

[0007] The technical solution of this invention to solve the technical problem is as follows:

[0008] In a first aspect of the invention, an anti-GLP-1R nanobody is provided, the anti-GLP-1R nanobody comprising a heavy chain variable region, the heavy chain variable region comprising a framework region and a complementarity-determining region; the complementarity-determining region comprising CDR1, CDR2, and CDR3, the amino acid sequence of the complementarity-determining region being selected from any one of the following two groups:

[0009] 1) The amino acid sequence of CDR1 is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR3 is shown in SEQ ID NO.3.

[0010] 2) The amino acid sequences of CDR1-3 are at least 90% identical to the amino acid sequences of SEQ ID NO.1-3.

[0011] Preferably, the heavy chain variable region of the anti-GLP-1R nanobody has an amino acid sequence as shown in SEQ ID NO: 4.

[0012] In a second aspect of the invention, a nucleic acid encoding the anti-GLP-1R nanobody as described in the first aspect is provided. Preferably, the heavy chain variable region nucleic acid sequence of the anti-GLP-1R nanobody is shown in SEQ ID NO. 5.

[0013] In a third aspect of the invention, a vector is provided containing the nucleic acid as described in the second aspect. Preferably, the vector is -pComb3XSS.

[0014] In a fourth aspect of the invention, a host cell is provided, comprising the vector as described in the third aspect. Preferably, the host cell is TG1 or SS320; more preferably, the host cell is an SS320 host cell.

[0015] In a fifth aspect of the present invention, a method for preparing anti-GLP-1R nanobodies as described in the first aspect is provided, comprising the following steps: constructing a GLP-1R overexpression plasmid and an overexpression cell line; immunizing alpacas with the plasmid and cells to obtain PBMC cells; extracting total RNA; reverse transcribing it into cDNA; amplifying an antibody gene fragment by PCR and ligating the fragment into a phage expression vector; electroporating the constructed vector into SS320 competent cells to finally obtain a GLP-1R immune antibody library; obtaining antibodies binding to GLP-1R through one round of cell panning and Cell ELISA detection; sequencing the antibody sequence; obtaining the antibody protein through mammalian cells; performing FACS detection using CHOK1-GLP-1R cells to finally obtain antibodies binding to GLP-1R.

[0016] In this invention, the primers used for alpaca immunization, antibody gene fragment amplification, screening methods, and the construction of GLP-1R high-expression cell lines are obtained through optimization of routine operations.

[0017] In a sixth aspect of the invention, a detection reagent or kit is provided, which contains nanobodies as described in the first aspect or biomaterials as described in the second, third or fourth aspects.

[0018] In a seventh aspect of the invention, the use of the anti-GLP-1R nanobody as described in the first aspect is provided in the preparation of medicaments for treating diabetes and obesity, or in the preparation of reagents or medicaments for diagnosing, preventing or treating GLP-1R-related diseases or hypoglycemia-related diseases.

[0019] In an eighth aspect of the invention, the use of the anti-GLP-1R nanobody as described in the first aspect in the preparation of GLP-1R detection reagents is provided.

[0020] In a ninth aspect of the invention, the use of the anti-GLP-1R nanobody as described in the first aspect in the preparation of a product that binds to GLP-1R is provided.

[0021] The present invention has the following technical effects:

[0022] 1) The antibody of the present invention has the characteristics of small molecular weight, strong binding ability to antigen GLP-1R, high specificity, simple structure and easy engineering.

[0023] 2) The antibody obtained by this invention is efficiently expressed by a mammalian protein expression system, and its three-dimensional structure and chemical modification are closer to its state in mammals, and its activity is closer to that of natural antibodies. Attached Figure Description

[0024] Figure 1 These are the results of 7 immune serum titers tests.

[0025] Figure 2 These are the results of the first round of PCR electrophoresis.

[0026] Figure 3 These are the results of the second round of PCR electrophoresis.

[0027] Figure 4 The results are from the antibody binding assay to CHOK1-GLP-1R cells. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0029] The reagents or instruments used, for which the manufacturer is not specified, are all conventional products that can be purchased through legitimate channels.

[0030] Example 1

[0031] In this embodiment, the method for preparing GLP-1R nanobodies includes the following steps:

[0032] 1) Based on the gene and protein sequence information of GLP-1R (NCBI Reference Sequence: NM_002062.5→NP_002053.3), GLP-1R overexpression plasmid and CHOK1-GLP-1R cells were constructed; the construction of the overexpression plasmid and overexpression cell line were carried out in accordance with the conventional procedures in this field.

[0033] 2) Use the plasmid or cells obtained in step 1) to immunize alpacas seven times, with the first two immunizations being plasmid-based and the last five being cell-based immunizations, to obtain alpaca PBMC cells. The specific immunization method is as follows: Alpacas are immunized for the first and second times with a GLP-1R overexpression plasmid. On days 28, 42, 56, 70, and 84, immunization is performed using CHOK1-GLP-1R cells with GERBU adjuvant. One week after each immunization, blood is collected to detect the anti-GLP-1R serum titer. The anti-GLP-1R serum titer is detected by FACS. When the MFI value of the test sample is more than three times that of the negative control, the antiserum titer is considered positive. The results are as follows. Figure 1 As shown, the serum MFI value after the seventh immunization was more than 3 times that of the negative control; 50 mL of blood was collected one week after the seventh immunization for screening and library construction.

[0034] 3) Dilute the 50ml blood collected in step 2) twice with PBS, separate PBMCs using lymphocyte separation medium (Ficoll reagent), and then dissolve them with Trizol to obtain a cell concentration of 10. 7 / ml of PBMC cells, stored at -80 degrees Celsius.

[0035] 4) RNA was extracted from PBMCs using the Trizol method. The RNA was then reverse transcribed into cDNA using oligo(dT) (using the TaKaRa-SMARTcribe Reverse Transcript kit). Nested PCR was performed twice to obtain the nanobody gene fragment. Figure 2 These are the results of the first round of PCR; the target fragment is around 750bp. Figure 3 The results of the second round of PCR show that the target band is approximately 400 bp. The obtained nanobody gene fragment was inserted into a phage expression vector to obtain a phage vector containing the antibody fragment. This vector was then electroporated into SS320 electroporation competent cells, and the cells were collected to obtain a GLP-1R immune phage antibody library.

[0036] 5) Panning of the GLP-1R phage antibody library: The obtained library bacteria are packaged into phages, and their titers are measured; this is the input phage. An appropriate amount of input phage is incubated with negative screening cells, i.e., CHOK1 cells. After incubation, it is incubated with GLP-1R protein. Finally, the phage is eluted with trypsin, infects SS320 cells, plated, and single clones are selected for culture and clone ELISA.

[0037] 6) Clone ELISA: CHOK1-GLP-1R cells were inoculated overnight at 37°C into 96-well ELISA plates. The next day, 1% BSA was added for blocking at room temperature for 1 hour. The cells were then washed three times with 0.05% PBST. The cultured monoclonal bacterial culture was centrifuged and incubated at room temperature for 1 hour. The cells were washed three times with 0.05% PBST. The diluted secondary antibody anti-M13 (1:10000) was added and incubated at room temperature for 40 minutes. The cells were washed six times with 0.05% PBST. TMB chromogenic solution was added and chromogenic solution was added. The cells were incubated for 5-10 minutes. The stop solution was added to stop the reaction. The OD450 value was measured using an ELISA reader. Clones with an OD450 value greater than 3 times that of the negative control were considered positive clones and expressed in mammalian cells after sequencing.

[0038] 7) Cell binding assay: cell binding (FACS). Cultured CHOK1-GLP-1R cells were prepared to a cell count of 2E+05 cells / ml using MACS buffer. 50 μL of cell suspension was added to each well of a 96-well plate. The prepared antibody (obtained after sequencing in step 6 via mammalian cell expression and purification) was serially diluted 4-fold with MACS buffer. 50 μL of antibody dilution was added to each well of the 96-well plate, and the mixture was thoroughly combined. Positive and negative controls were added simultaneously (positive control: PC-Anti-hGLP-1R antibody; negative controls: NC-Anti-HEL-VHH-IgG1 Isotype and NC-Anti-HEL hIgG1-isotype). The cells were incubated at 4°C in the dark for 60 min. After washing three times with MACS buffer, the cells were resuspended with fluorescent secondary antibody and incubated at 4°C in the dark for 40 min. After washing three times with MACS buffer, the cells were resuspended with 180 μL of MACS buffer. The cells were then analyzed by flow cytometry. Results are shown in [Figure number missing]. Figure 4 The results showed that NC-Anti-HEL-VHH-IgG1 Isotype and NC-Anti-HEL hIgG1-isotype did not bind significantly to CHOK1-GLP-1R cells, while PC-Anti-hGLP-1R antibody and antibody numbered ANb1336-7M-21G9 bound significantly to CHOK1-GLP-1R cells.

[0039] The antibody numbered ANb1336-7M-21G9 has the following amino acid sequence in its heavy chain variable region: SEQ ID NO.4; the CDR1 sequence in the heavy chain variable region: SEQ ID NO.1; the CDR2 sequence: SEQ ID NO.2; the CDR3 sequence: SEQ ID NO.3; and the nucleotide sequence in the heavy chain variable region: SEQ ID NO.5.

[0040] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. An anti-GLP-1R nanobody, wherein the anti-GLP-1R nanobody comprises a heavy chain variable region, the heavy chain variable region comprising a framework region and a complementarity-determining region; the complementarity-determining region comprising CDR1, CDR2, and CDR3, characterized in that, The amino acid sequence of CDR1 is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

3.

2. The anti-GLP-1R nanobody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-GLP-1R nanobody is shown in SEQ ID NO:

4.

3. A nucleic acid encoding the anti-GLP-1R nanobody as described in claim 1 or 2.

4. The nucleic acid according to claim 3, characterized in that, The heavy chain variable region nucleic acid sequence of the anti-GLP-1R nanobody is shown in SEQ ID NO.

5.

5. A carrier, characterized in that, It contains the nucleic acid as described in claim 3.

6. A host cell, characterized in that, It contains the carrier as described in claim 5.

7. A detection reagent or kit, characterized in that, Contains the nanobody as described in claim 1 or 2.

8. The use of the anti-GLP-1R nanobody as described in claim 1 or 2 in the preparation of GLP-1R detection reagents.

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