CN113583124A

CN113583124A - Anti-progastrin release peptide monoclonal antibody and preparation method thereof

Info

Publication number: CN113583124A
Application number: CN202110665746.4A
Authority: CN
Inventors: 周小林; 张伟; 郭玉凤
Original assignee: China Institute for Radiation Protection
Current assignee: China Institute for Radiation Protection
Priority date: 2021-06-16
Filing date: 2021-06-16
Publication date: 2021-11-02
Anticipated expiration: 2041-06-16
Also published as: CN113583124B

Abstract

The invention belongs to the technical field of antibody drugs, and relates to an anti-progastrin release peptide monoclonal antibody and a preparation method thereof. The amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the monoclonal antibody are respectively shown in SEQ ID NO. 5-10. The monoclonal antibody for resisting progastrin release peptide and the preparation method thereof can be used for better preparing the monoclonal antibody for resisting progastrin release peptide, and the prepared monoclonal antibody for resisting progastrin release peptide can be better used for SCLC targeted diagnosis and treatment.

Description

Anti-progastrin release peptide monoclonal antibody and preparation method thereof

Technical Field

The invention belongs to the technical field of antibody drugs, and relates to an anti-progastrin release peptide monoclonal antibody and a preparation method thereof.

Background

Lung Cancer (Lung Cancer) is a malignant tumor with high morbidity and mortality, and Small Cell Lung Cancer (SCLC) is a Lung Cancer with high malignancy and low cure rate, and accounts for about 15% -20% of Lung Cancer. SCLC is characterized by short tumor doubling time, poor prognosis, lowest differentiation degree, easy transfer and 5-year survival rate of only 1-7%. However, SCLC is sensitive to radiotherapy and chemotherapy, if comprehensive treatment means such as systemic chemotherapy and local radiotherapy are adopted in early detection, the 3-year survival rate can reach more than 30%, so that the early diagnosis and treatment of SCLC are very important.

Progastrin Releasing Peptide (PGRP) is a reliable, sensitive, specific tumor marker for SCLC and is relatively stable in blood. PGRP can be detected before the physical diagnosis is not clear, the positive rate is up to 76%, and the PGRP is used for the early clinical diagnosis of SCLC. PGRP consists of a signal Peptide, Gastrin Releasing Peptide (GRP), a cleavage site, a constant region, and a variable carboxy terminus.

With the development of genetic engineering technology, antibody drugs are continuously used for early diagnosis and treatment of malignant tumors, and good economic and social benefits are obtained. The development of the anti-progastrin releasing peptide antibody is one of SCLC targeting diagnosis and treatment directions, and can lay a certain technical and material foundation for the diagnosis and treatment of SCLC patients.

Disclosure of Invention

The primary object of the present invention is to provide an anti-progastrin-releasing peptide monoclonal antibody that can be better used for SCLC targeted therapy.

To achieve this object, in a basic embodiment, the present invention provides an anti-progastrin releasing peptide monoclonal antibody having amino acid sequences of HCDR1 (heavy chain complementarity determining region 1), HCDR2 (heavy chain complementarity determining region 2), HCDR3 (heavy chain complementarity determining region 3), LCDR1 (light chain complementarity determining region 1), LCDR2 (light chain complementarity determining region 2), and LCDR3 (light chain complementarity determining region 3) as set forth in SEQ ID nos. 5 to 10, respectively.

In a preferred embodiment, the present invention provides a monoclonal antibody against progastrin-releasing peptide, wherein the heavy chain variable region V of said monoclonal antibody_HThe amino acid sequence of (A) is shown in SEQ ID NO.3, and the variable region V of the light chain is shown in_LAmmonia (D) in the presence of ammoniaThe amino acid sequence is shown as SEQ ID NO. 4.

In a preferred embodiment, the present invention provides a monoclonal antibody against progastrin releasing peptide, wherein said monoclonal antibody is a mouse monoclonal antibody.

The second objective of the present invention is to provide a method for preparing the monoclonal antibody against progastrin release peptide, so as to be able to better prepare the monoclonal antibody against progastrin release peptide, and the prepared monoclonal antibody against progastrin release peptide can be better used for SCLC targeted therapy.

To achieve this object, in a basic embodiment, the present invention provides a method for producing the above-described monoclonal antibody against progastrin releasing peptide, the method comprising the steps of:

(1) immunizing mouse with mouse progastrin releasing peptide or its segment and taking spleen cell;

(2) fusing the splenocytes obtained in the step (1) with myeloma cells, and screening out hybridoma cells through selective culture and ELISA detection;

(3) cloning and stably culturing the hybridoma cells screened in the step (2) to obtain a monoclonal cell strain expressing the anti-progastrin release peptide antibody;

(4) and (4) injecting the monoclonal cell strain obtained in the step (3) into the abdominal cavity of the mouse, and extracting ascites to purify and identify the monoclonal antibody.

In a preferred embodiment, the present invention provides a method for producing the above-mentioned monoclonal antibody against progastrin-releasing peptide, wherein in step (1),

the mouse progastrin releasing peptide or the fragment thereof is a constant region amino acid peptide segment of the mouse progastrin releasing peptide,

the mouse is a Balb/c mouse.

In a preferred embodiment, the present invention provides a method for producing the above monoclonal antibody against progastrin-releasing peptide, wherein in step (2), the myeloma cells are SP2/0 cells, and the medium used for selective culture is HAT selective medium.

In a preferred embodiment, the present invention provides a method for producing the above-described monoclonal antibody against progastrin-releasing peptide, wherein in step (4), the monoclonal antibody is purified using a protein a-sepharose affinity chromatography column.

In a preferred embodiment, the present invention provides a method for producing the above-described monoclonal antibody against progastrin releasing peptide, wherein the production method further comprises the steps of:

(5) extracting total RNA from the monoclonal cell strain obtained in the step (3), carrying out reverse transcription on the total RNA to obtain cDNA, amplifying variable regions of heavy chains and light chains of the antibody, respectively connecting the variable regions to a vector, transforming the variable regions to competent cells, and then sequencing the quality-improved particles.

In a more preferred embodiment, the present invention provides a method for producing the above-mentioned monoclonal antibody against progastrin-releasing peptide, wherein in step (5), the vector used for the ligation is pMD 19-T and the competent cell used is DH5 α.

The third purpose of the present invention is to provide the use of the anti-progastrin releasing peptide monoclonal antibody described above for preparing a reagent or a medicament for diagnosing or treating lung cancer, so as to be better used for the targeted diagnosis and treatment of lung cancer, especially SCLC.

To achieve this object, in a basic embodiment, the present invention provides the use of the above-described anti-progastrin-releasing peptide monoclonal antibody for the preparation of an agent or medicament for the diagnosis or treatment of lung cancer.

The invention has the advantages that the monoclonal antibody for resisting progastrin release peptide and the preparation method thereof can be used for better preparing the monoclonal antibody for resisting progastrin release peptide, and the prepared monoclonal antibody for resisting progastrin release peptide can be better used for SCLC targeted diagnosis and treatment.

The invention obtains the anti-progastrin release peptide monoclonal antibody and the light chain variable region V thereof through immunizing mice, cell fusion, cloning culture and a series of cell screening experiments_LAnd heavy chain variable region V_HAnd (4) sequencing. Experiments prove that the monoclonal antibody has specificity, and a new method is provided for the targeted therapy of tumors.

The invention obtains the sequence of the antibody, can directly obtain a large amount of antibodies through later-stage recombinant expression, does not need animal experiments, and has high efficiency and low cost.

Drawings

FIG. 1 is the ultraviolet absorption spectrum of the washed and eluted protein A-agarose affinity chromatography products of the examples, wherein I is the washing peak of the hetero protein and II is the elution peak of the monoclonal antibody.

FIG. 2 is an SDS-PAGE identification of the protein A-agarose anti-progastrin release peptide monoclonal antibody before and after ascites purification in the example, wherein 1 is the peritoneal fluid, 2-4 are the wash proteins (2 is the peak I acrolein, 3 and 4 are the peak I descending), 5-8 are the monoclonal antibodies (5 is the peak II ascending, 6 and 7 are the peak II acrolein, 8 is the peak II descending, and the two protein bands are the antibody heavy chain and light chain, respectively), and M is the protein molecular weight standard.

FIG. 3 is a graph showing the purity of SDS-PAGE in the fold-dilution of the monoclonal antibodies purified in the examples, wherein 1: 1X; 2: 10X; 3: 20X; 4: 40X; 5: 80X; 6: 160X; 7: 320 x.

Detailed Description

The following description will further describe embodiments of the present invention with reference to the accompanying drawings.

Example 1: preparation of monoclonal antibody against progastrin-releasing peptide

1. Immunogens (antigens)

The constant region amino acid peptide segment of the murine progastrin releasing peptide (rPGRP) was prepared according to the method disclosed in the section of the specific embodiment of the Chinese patent application CN 200510065889.2.

2. Immunization of mice

Balb/c mice were immunized with rPGRP at a dose of 100 ug/mouse. Mixing the antigen and the same amount of Freund's complete adjuvant, emulsifying, and injecting into abdominal cavity to immunize mouse; after two weeks, the same dose of antigen and the same amount of Freund's incomplete adjuvant are taken, mixed and emulsified evenly, and injected into the immunized mouse at subcutaneous multiple points and abdominal cavity on the back; after two weeks, the same dose of antigen is dissolved in physiological saline for tail vein injection to strengthen immunity; the same dose of intravenous injection is taken three days before the fusion.

3. Hybridoma cell preparation

(1) Preparation of feeder cells

The Balb/c mice are killed after cervical vertebra removal, soaked in 75% alcohol solution for 5 minutes, placed in an ultra-clean workbench, and fixed on an anatomical platen with the abdomen facing upwards. The abdominal skin is cut open to fully expose the peritoneum; sucking 8ml of the basic culture solution by using an injector, injecting the basic culture solution into the abdominal cavity of the mouse, fixing the injector to enable the needle head to be remained in the abdominal cavity, and slightly massaging the abdomen for 1-2 minutes by holding an alcohol cotton ball by hand; pumping back the liquid in the abdominal cavity by using an original syringe, injecting the liquid into a centrifugal tube, centrifuging at 1000rpm for 10 minutes, and removing the supernatant; suspending and mixing the precipitated cells with 5ml of culture medium, counting the cells, and adjusting the cell concentration to 2 × 10 according to the counting result⁵cells/ml. Adding the cell suspension into 96-well culture plate, 0.1ml per well, in 5% CO₂Culturing at 37 deg.C. Prepared the day before use.

(2) Cell fusion

Spleen cell and myeloma cell suspensions were prepared from the basic culture medium and counted by the leukocyte counting method. Respectively taking out and containing 1 x 10⁸Spleen cells and 2-3X 10⁷Mixing the suspension of myeloma cells into the same centrifuge tube, centrifuging to paste the cells, adding 0.7ml of 50% PEG solution to fuse the cells, diluting with 25ml of incomplete culture solution after 90s, and centrifuging to remove PEG. Adding the cells into HAT culture solution 10ml, inoculating into 96-well culture plate 0.1ml per well, and placing in a culture container containing 5% CO₂Cultured in an incubator at 37 ℃. After successful fusion, hybridoma cells are subjected to cloning culture to screen cell strains secreting specific monoclonal antibodies. The hybridoma cells were cultured for two weeks by changing the HAT selective medium to 1/2-2/3 every 3 days, starting on day 4 after the fusion, and changing the HT medium every 2 days. Then transferred into a common culture solution containing 5% CO₂The culture was continued in an incubator at 37 ℃. Hybridoma cells expressing an anti-progastrin-releasing peptide antibody were selected by ELISA and cultured for cloning.

4. Cloning culture and stable culture of monoclonal cell strain

The positive well cells were counted accurately, diluted to a cell concentration of 10cells/ml, and 0.1ml was added to each well of a 96-well plate to give an average of 1 cell per well. And (5) timely carrying out liquid change and detection, and taking the positive hole of a single clone for cloning culture again. And when all the produced cell clone holes are positive, a cell strain secreting the monoclonal antibody can be selected. Culturing the screened secretable monoclonal antibody cell strains for 3 months, changing the liquid once according to the growth condition of the cells for 2-3 days, detecting the OD value of the monoclonal antibody in the supernatant, detecting once every week, abandoning the decrease of the OD value, and finally obtaining the cell strains F-B6, E-B5, F-B3, D-D3 and E-C6 which can stably express the anti-progastrin release peptide monoclonal antibody.

5. Preparation and purification of ascites

(1) Preparation of ascites

One week before hybridoma inoculation, mice were injected with 0.5ml of primary liquid paraffin intraperitoneally. Centrifuging at 1000rpm for 10min, collecting cultured hybridoma cells, removing supernatant, suspending and mixing with serum-free culture medium, adjusting cell number to 1-2 × 10⁵cells/ml, 0.5ml per mouse was injected intraperitoneally. Collecting ascites, inoculating for 1-2 weeks to show that the abdomen of the mouse is obviously enlarged, sterilizing the skin of the lower abdomen, extracting the ascites by using a sterilized syringe, extracting 3-5ml of ascites once for each mouse at intervals of 1-2 days, after the ascites is regenerated and accumulated, extracting again by the same method, centrifuging the extracted ascites for 15 minutes at 3000rpm, collecting supernatant, measuring titer, subpackaging and freezing for later use.

(2) Purification of ascites

Centrifuging prepared mouse ascites of about 1.5ml at 10000g for 10min, removing jelly precipitate, and diluting the sample to 2ml with PBS of 0.02mol/L for use; fixing the protein A-agarose affinity chromatography column, washing out alcohol preservative by 25ml double distilled water, then passing through the column by 10ml of 0.1mol/L sodium citrate buffer solution (pH 4.6), then washing the column by 20ml of 0.02mol/L phosphate buffer solution (pH 7.0), and balancing the column by 3mol/L NaCl and 0.02mol/L PBS 25 ml; 2ml of the prepared ascites sample was slowly injected into the column; washing with 25ml of 0.5mol/L NaCl, 0.02mol/L PBS and 25ml of 0.02mol/L phosphate buffer solution (pH 7.0) successively to remove unbound protein, and collecting respective effluent for detection; eluting the bound monoclonal antibody with 30ml of glycine-hydrochloric acid buffer solution with pH 2.4, collecting the eluate, 2ml per tube, and adjusting the eluate to neutrality (pH 7.0) with Tris-HCl buffer solution with pH 9.0 as soon as possible (the flow rate in the above purification process is 2 ml/min); the affinity chromatography sample wash and elution process was monitored at 280nm and recorded using uv absorption.

As shown in FIG. 1, the ascites sample was separated into 2 UV absorption peaks after purification, i.e., 2 protein fractions which could be separated.

The fractions were collected in steps, the immunocompetence was determined by indirect ELISA and detected by SDS-PAGE. The results are shown in table 1, the specific activity of component II is significantly higher than that of component I and ascites, indicating that it contains a large amount of antibodies, which are isolated and purified monoclonal antibodies; the component I has extremely low antibody content and is other hybrid proteins, and simultaneously shows that the specific activity of the purified antibody is obviously higher than that of the antibody before purification. SDS-PAGE results are shown in FIG. 2, bands 2-4 (peak I) contain a large amount of hetero-proteins; the two distinct protein bands of bands 5-8 (peak II) combined with the ELISA assay results indicated that peak II was a purified monoclonal antibody, and that the two protein bands were the antibody heavy and light chains, respectively.

TABLE 1 ELISA test results (OD values) for different fractions before and after purification

Negative control: 0.125, positive control: 1.243, cutoff: 0.125 × 2.1 ═ 0.2625

The negative control is 0.01mol/L PBS; the positive control is known antibody positive culture supernatant.

Example 2: identification of monoclonal antibodies prepared in example 1

(1) The titers of the monoclonal antibodies were measured by an indirect enzyme-linked immunosorbent (ELISA) method, and the specific activities were calculated from the concentrations/titers of the purified antibodies, with the results shown in table 2, indicating that the specific activities of the purified antibodies were all higher than those before purification.

(2) The protein concentration was measured by the ultraviolet absorption method, and the protein recovery rate was calculated, and the results are shown in table 3, which indicates that the ascites monoclonal antibody mass concentration was 5.21mg/ml on average, and the protein recovery rate was 98.56% on average.

(3) The purified monoclonal antibody is diluted by a multiple ratio and then subjected to SDS-PAGE detection to identify the purity of the purified antibody, and the result is shown in FIG. 3, which shows that relatively pure antibody protein is successfully purified, and the light chain and the heavy chain of the antibody after melting can be clearly observed.

(4) anti-HBs IgG negative control myeloma cell culture supernatant₁The subclass of MAb was positive control, and the subclass of monoclonal antibody was identified according to the test kit for mouse monoclonal antibody from Sigma, USA, and the results are shown in Table 4, which indicates that MAb cell lines F-B6, E-A6, F-B3 and E-C6 are IgG_2bD-D3 is IgG₁。

(5) Determining cross reaction between monoclonal antibody and hepatitis B surface antigen (HBSAg) by indirect ELISA method, wherein HBSAg negative and positive control are hepatitis B surface antibody negative and positive sample, rPGRP negative is cell primary culture solution, positive is known antibody positive culture supernatant, and PGRP_(42～53)、PGRP_(68～82)The positive control is the serum of the immunized mouse, the result is shown in Table 5, which shows that the 5 monoclonal antibodies have no cross reaction with the hepatitis B surface antigen, and the E-C6 antibody can react with PGRP_(68～82)Reacting the synthetic peptide, D-D3 with PGRP_(42～53)Antigen reaction, remaining 3 strains with PGRP_(42～53)And PGRP_(68～82)No reaction takes place.

TABLE 2 comparison of specific Activity before and after purification of monoclonal antibodies

TABLE 3 recovery and monoclonal antibody content after purification of ascites

Table 4 monoclonal antibody subclass identification results (OD value) (n ═ 5)

Table 5 monoclonal antibody specificity detection results (OD value) (n ═ 5)

Example 3: sequence analysis of monoclonal antibody against progastrin-releasing peptide

1. Acquisition of Gene sequence of monoclonal antibody against progastrin-releasing peptide

(1) RNA extraction and cDNA transformation

Culturing fresh E-B5 hybridoma cells, and centrifuging to collect 10⁷The above cells; extracting total RNA of cells by using Trizol reagent; mixing 9. mu.L of total RNA, 2.5. mu.L of oligo (dT) 12-18 primer (10mM), and 5. mu.L of dNTPs, incubating at 70 ℃ for 5 minutes, and then standing on ice for 5 minutes, or performing denaturation operation according to the reverse transcriptase (BPI) used; subsequently, 5. mu.L of RT buffer (5X), 2.5. mu.L of DTT (0.1M) and 1. mu.L of reverse transcriptase were added, and the reaction was carried out at 42 ℃ for 1 hour; the reaction was terminated by incubation at 70 ℃ for 15 minutes, and the obtained cDNA was stored at-20 ℃.

(2) Gene acquisition

Performing PCR amplification on the obtained first strand cDNA: adding 25pmol of each primer into a 50 mu L reaction system, adding dNTP and Buffer according to a conventional method, and adding 1 mu L of cDNA template; the procedure is as follows: 95 ℃ for 3min, 95 ℃ for 30s, annealing temperature of 55 ℃ for 60s, and annealing temperature of 72 ℃ for 40s, 25 cycles, and 72 ℃ for 3 min.

2. Gene sequencing of monoclonal antibody against progastrin-releasing peptide

(1) Connection of

Mu.l of Vector (pMD 19-T Vector, TaKaRa), 3. mu.l of recovered product, 4. mu.l of ligase mixture (TaKaRa), mixed well and reacted at 16 ℃ for 2 h.

(2) Transformation of

Taking out the competent cells (DH5 alpha) preserved at-80 ℃, and slowly thawing on ice; adding the ligation product into the competent cells, uniformly mixing, and standing on ice for 30 min; heat shock at 42 ℃ for 90 s; after ice-bath for 2min, 800. mu.l of non-resistant LB medium was added; culturing at 37 deg.C for 45 min; centrifuging at 8000rpm for 1min, discarding most of supernatant, leaving about 50-100 μ l, resuspending thallus, selecting LB plate with corresponding resistance, and coating; air-dried and cultured in an incubator at 37 ℃ for overnight in an inverted state.

(3) Identification

Single colony shake bacteria from the plate, 1 mul bacterial liquid as template, M13F/R as primer, PCR verification (program: 95 ℃ 3min, 95 ℃ 30s, annealing temperature 55 ℃ 60s, 72 ℃ 40s, 25 cycles, 72 ℃ 3 min).

(4) Sequencing

The sequencing of the recombinant plasmid is completed by Beijing Huada protein research and development center, and the sequencing result is as follows:

(1) expression of monoclonal antibody heavy chain variable region V_HAnd light chain variable region V_LThe nucleotide sequences of (A) are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.

(2) Heavy chain variable region V of monoclonal antibody_HThe amino acid sequence of (A) is shown in SEQ ID NO.3, and the variable region V of the light chain is shown in_LThe amino acid sequence of (A) is shown in SEQ ID NO. 4.

(3) The amino acid sequences of HCDR1 (heavy chain complementarity determining region 1), HCDR2 (heavy chain complementarity determining region 2), HCDR3 (heavy chain complementarity determining region 3), LCDR1 (light chain complementarity determining region 1), LCDR2 (light chain complementarity determining region 2), and LCDR3 (light chain complementarity determining region 3) of the monoclonal antibodies are shown in SEQ ID Nos. 5-10, respectively.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is intended to include such modifications and variations. The foregoing examples or embodiments are merely illustrative of the present invention, which may be embodied in other specific forms or in other specific forms without departing from the spirit or essential characteristics thereof. The described embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. The scope of the invention should be indicated by the appended claims, and any changes that are equivalent to the intent and scope of the claims should be construed to be included therein.

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Claims

1. An anti-progastrin-releasing peptide monoclonal antibody, characterized in that: the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the monoclonal antibody are respectively shown in SEQ ID NO. 5-10.

2. The monoclonal antibody of claim 1, characterized in that: the heavy chain variable region V of the monoclonal antibody_HThe amino acid sequence of (A) is shown in SEQ ID NO.3, and the variable region V of the light chain is shown in_LThe amino acid sequence of (A) is shown in SEQ ID NO. 4.

3. The monoclonal antibody according to claim 1 or 2, characterized in that: the monoclonal antibody is a mouse monoclonal antibody.

4. A method for preparing the monoclonal antibody of claim 3, comprising the steps of:

5. The method of claim 4, wherein: in the step (1), the step (c),

the mouse is a Balb/c mouse.

6. The method of claim 4, wherein: in the step (2), the myeloma cells are SP2/0 cells, and the culture medium used for selective culture is HAT selective culture medium.

7. The method of claim 4, wherein: in the step (4), the monoclonal antibody is purified by using a protein A-agarose affinity chromatography column.

8. The method of claim 4, further comprising the steps of:

9. The method of claim 8, wherein: in step (5), the vector used for ligation was pMD 19-T, and the competent cell used was DH5 α.

10. Use of a monoclonal antibody according to one of claims 1 to 3 for the preparation of a reagent or a medicament for the diagnosis or treatment of lung cancer.