CN113583124B

CN113583124B - Anti-progastrin release peptide monoclonal antibody and preparation method thereof

Info

Publication number: CN113583124B
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: 2023-12-15
Anticipated expiration: 2041-06-16
Also published as: CN113583124A

Abstract

The invention belongs to the technical field of antibody medicines, and relates to an anti-progastrin release peptide monoclonal antibody and a preparation method thereof. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the monoclonal antibody are shown in SEQ ID NO.5-10 respectively. By utilizing the anti-progastrin release peptide monoclonal antibody and the preparation method thereof, the anti-progastrin release peptide monoclonal antibody can be better prepared, and the prepared anti-progastrin release peptide monoclonal antibody 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 medicines, 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 extremely high morbidity and mortality, wherein small cell Lung Cancer (Small Cell Lung Cancer, SCLC) is a Lung Cancer with higher malignancy and lower cure rate, accounting for about 15% -20% of Lung Cancer. SCLC is characterized by short tumor multiplication time, poor prognosis, lowest differentiation degree, easy metastasis and survival rate of only 1-7% in 5 years. However, SCLC is sensitive to radiotherapy and chemotherapy, for example, comprehensive treatment means such as systemic chemotherapy and local radiotherapy are adopted in early detection, and the survival rate of 3 years can reach more than 30%, so that early diagnosis and treatment of SCLC are important.

Progastrin release peptide (Pro-Gastrin 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, and the positive rate is as high as 76%, so that the method has been used for SCLC early clinical diagnosis. PGRP consists of a signal peptide, gastrin releasing peptide (Gastrin Releasing Peptide, GRP), cleavage site, constant region and 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 release peptide antibody is one of SCLC targeting diagnosis and treatment directions, and can lay a certain technology and material foundation for diagnosis and treatment of SCLC patients.

Disclosure of Invention

The primary object of the invention is to provide an anti-progastrin release peptide monoclonal antibody which can be better used for SCLC targeting diagnosis and treatment.

To achieve this object, in a basic embodiment, the present invention provides an anti-progastrin releasing peptide monoclonal antibody, wherein 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), LCDR3 (light chain complementarity determining region 3) are shown in SEQ ID NOS.5-10, respectively.

In a preferred embodiment, the present invention provides an anti-progastrin-releasing peptide monoclonal antibody, wherein the heavy chain variable region V of said monoclonal antibody _H The amino acid sequence of (a) is shown as SEQ ID NO.3, and the light chain variable region V _L The amino acid sequence of (C) is shown as SEQ ID NO. 4.

In a preferred embodiment, the invention provides an anti-progastrin-releasing peptide monoclonal antibody, wherein the monoclonal antibody is a mouse monoclonal antibody.

The second object of the present invention is to provide a method for preparing the anti-progastrin-releasing peptide monoclonal antibody, so that the anti-progastrin-releasing peptide monoclonal antibody can be better prepared, and the prepared anti-progastrin-releasing peptide monoclonal antibody can be better used for SCLC targeted diagnosis and treatment.

To achieve the object, in a basic embodiment, the present invention provides a method for preparing the above-mentioned anti-progastrin release peptide monoclonal antibody, the method comprising the steps of:

(1) Immunizing a mouse with a mouse progastrin release peptide or a fragment thereof, and taking spleen cells of the mouse;

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

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

(4) Injecting the monoclonal cell strain obtained in the step (3) into the abdominal cavity of a mouse, and extracting ascites for monoclonal antibody purification and identification.

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

the murine progastrin release peptide or the fragment thereof is a constant region amino acid peptide fragment of the murine progastrin release peptide,

the mice are Balb/c mice.

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

In a preferred embodiment, the present invention provides a method for preparing the above-mentioned anti-progastrin release peptide monoclonal antibody, wherein in step (4), the monoclonal antibody is purified by using a protein a-sepharose affinity chromatography column.

In a preferred embodiment, the present invention provides a method for preparing the anti-progastrin release peptide monoclonal antibody, wherein the preparation method further comprises the following steps:

(5) Extracting total RNA from the monoclonal cell strain obtained in the step (3), reversely transcribing the total RNA into cDNA, amplifying variable regions of heavy chains and light chains of antibodies, respectively connecting the variable regions to a vector, converting the variable regions into competent cell postextract plasmids, and sequencing.

In a more preferred embodiment, the present invention provides a method for preparing the anti-progastrin release peptide monoclonal antibody, wherein in the step (5), the vector used for ligation is pMD 19-T, and the competent cell used is DH5 alpha.

A third object of the present invention is to provide the use of the above-mentioned anti-progastrin-releasing peptide monoclonal antibody for the preparation of 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-mentioned anti-progastrin release peptide monoclonal antibody for the preparation of an agent or medicament for diagnosing or treating lung cancer.

The anti-progastrin release peptide monoclonal antibody and the preparation method thereof have the advantages that the anti-progastrin release peptide monoclonal antibody can be better prepared, and the prepared anti-progastrin release peptide monoclonal antibody 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 immune mice, cell fusion, cloning culture and a series of cell screening experiments _L And heavy chain variable region V _H Sequence. Experiments prove that the monoclonal antibody has specificity and provides a novel method for targeted treatment of tumors.

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

Drawings

FIG. 1 is a graph showing the ultraviolet absorption detection of the washing and elution products of protein A-agarose affinity chromatography in the example, wherein I is a hybrid protein washing peak and II is a monoclonal antibody elution peak.

FIG. 2 is a SDS-PAGE identification before and after ascites purification of protein A-Sepharose anti-progastrin release peptide monoclonal antibody in the examples, wherein 1 is peritoneal effusion, 2-4 is wash protein (2 is peak I top branch, 3 and 4 are peak I bottom branch), 5-8 is monoclonal antibody (5 is peak II top branch, 6 and 7 are peak II top branch, 8 is peak II bottom branch, two protein bands are antibody heavy chain and light chain, respectively), and M is protein molecular weight standard.

FIG. 3 is a chart showing purity identification of purified monoclonal antibodies in example by double dilution SDS-PAGE, wherein 1:1×;2:10×;3:20×;4:40×;5:80×;6:160×;7:320×.

Detailed Description

The following describes the embodiments of the present invention further with reference to examples and drawings.

Example 1: preparation of anti-progastrin release peptide monoclonal antibodies

1. Immunogen (antigen)

The constant region amino acid peptide fragment of murine progastrin release peptide (rPGRP) was prepared according to the method disclosed in the description of the embodiment section of chinese patent application CN 200510065889.2.

2. Immunization of mice

Balb/c mice were immunized with rPGRP at a dose of 100 ug/dose. Uniformly mixing and emulsifying the antigen and the equivalent Freund's complete adjuvant, and injecting the mixture into an immunized mouse intraperitoneally; after two weeks, the same dose of antigen is taken and uniformly mixed with the equal amount of Freund's incomplete adjuvant for emulsification, and the mice are immunized by subcutaneous multipoint and intraperitoneal injection at the back; dissolving the same dose of antigen in normal saline after two weeks, and performing tail vein injection to strengthen immunity; the same dose was given for intravenous immunization three days prior to fusion.

3. Hybridoma cell preparation

(1) Preparation of feeder cells

Balb/c mice were cervical removed, sacrificed, immersed in 75% alcohol solution for 5 minutes, placed in an ultra clean bench, and fixed with the abdomen facing up on an anatomic platen. Cutting off the abdominal skin to fully expose the peritoneum; sucking basic culture solution-8 ml by using a syringe, injecting into the abdominal cavity of a mouse, fixing the syringe to leave a needle in the abdominal cavity, and lightly massaging the abdominal cavity by holding an alcohol cotton ball for 1-2 minutes; pumping back the liquid in the abdominal cavity by using the original injector, injecting the liquid into a centrifuge tube, centrifuging at 1000rpm for 10 minutes, and discarding the supernatant; suspending and mixing the precipitated cells with 5ml culture solution, counting, and adjusting cell concentration to 2×10 according to counting result ⁵ cells/ml. The cell suspension was added to 96-well plates at 0.1ml per well in 5% CO ₂ Is cultured in an incubator at 37 ℃. Prepared the day before use.

(2) Cell fusion

Spleen cells and myeloma cell suspensions were prepared from basal medium and counted by white blood cell count. Respectively taking 1×10 components ⁸ Spleen cells and 2-3×10 ⁷ Mixing the suspension of myeloma cells into the same centrifugal tube, centrifuging, flicking the bottom of the centrifugal tube to paste the cells, adding 0.7ml 50% PEG solution to melt the cellsAfter 90s, 25ml of incomplete broth was added for dilution and the PEG was removed by centrifugation. Adding cells into HAT culture solution 10ml, inoculating into 96-well culture plate, and placing 0.1ml per well in the presence of 5% CO ₂ Is cultured in an incubator at 37 ℃. After successful fusion, hybridoma cells are subjected to cloning culture to screen cell strains secreting specific monoclonal antibodies. Hybridoma cells were cultured for two weeks with HAT selective medium at 1/2-2/3 of the amount of the medium at intervals of 3 days starting on day 4 after fusion, with HT medium after two weeks. Then transferring into common culture solution containing 5% CO ₂ Is continuously cultured in an incubator at 37 ℃. Hybridoma cells expressing the anti-progastrin release peptide antibody were screened by ELISA and subjected to cloning culture.

4. Cloning culture and stability culture of monoclonal cell strain

Positive well cells were counted accurately, diluted and the cell concentration was adjusted to 10cells/ml, and 0.1ml was added to each well of a 96-well plate to average 1 cell per well. And (5) timely replacing and detecting, and taking the positive hole of the single clone for cloning culture again. When all the cell clone producing holes are positive, a cell strain secreting monoclonal antibodies can be considered to be selected. And (3) continuously culturing the screened cell strains capable of secreting monoclonal antibodies for 3 months, changing the liquid once in 2-3 days according to the growth condition of the cells, detecting the OD value of the monoclonal antibodies in the supernatant once per week, and discarding the decrease of the OD value to finally obtain the cell strains F-B6, E-B5, F-B3, D-D3 and E-C6 capable of stably expressing the monoclonal antibodies of the progastrin release peptide.

5. Ascites preparation and purification

(1) Ascites preparation

One week before hybridoma cell inoculation, mice were intraperitoneally injected with 0.5ml of primordial liquid paraffin. Centrifuging at 1000rpm for 10min, collecting cultured hybridoma cells, discarding supernatant, mixing with serum-free culture solution, and regulating cell number to 1-2×10 ⁵ cells/ml, 0.5ml per mouse was intraperitoneally injected. Collecting ascites, inoculating for 1-2 weeks to make it clear that the abdomen of mice is obviously enlarged, sterilizing the skin of lower abdomen, extracting ascites with sterilizing syringe, and extracting 3-5ml of ascites once per mouse for 1-2 daysAfter water regeneration and accumulation, pumping again by the same method, centrifuging the pumped ascites for 15 minutes at 3000rpm, collecting supernatant, measuring titer, and sub-packaging for freezing storage.

(2) Ascites purification

Centrifuging about 1.5ml of prepared mouse ascites at 10000g for 10min, removing jelly precipitate, and diluting the sample to 2ml with 0.02mol/L PBS for use; protein a-agarose affinity chromatography column was immobilized, washed with 25ml of double distilled water to remove alcohol preservative, then passed through the column with 10ml of 0.1mol/L sodium citrate buffer (ph=4.6), then washed with 20ml of 0.02mol/L phosphate buffer (ph=7.0), equilibrated with 3mol/L NaCl, 0.02mol/L PBS, and the column equilibrated with 25 ml; slowly pouring 2ml of prepared ascites sample into the column; washing with 25ml of 0.5mol/L NaCl, 0.02mol/L PBS and 25ml of 0.02mol/L phosphate buffer (pH=7.0) to remove unbound proteins, and collecting the respective effluents for detection; eluting the bound monoclonal antibodies with 30ml glycine-hydrochloric acid buffer at ph=2.4, collecting the effluent, 2ml per tube, and adjusting the elution eluate per tube to neutral (ph=7.0) as soon as possible with Tris-HCl buffer at ph=9.0 for detection (flow rates during the above purification are all 2 ml/min); the washing and elution of the affinity chromatography samples were monitored at 280nm using UV absorption and recorded.

As a result, as shown in FIG. 1, the ascites fluid sample after purification was separated into 2 ultraviolet light absorption peaks, namely, 2 separable protein fractions.

Fractions were collected stepwise, assayed for immune activity by indirect ELISA, and subjected to SDS-PAGE detection. The results are shown in Table 1, the specific activity of the component II is obviously higher than that of the component I and ascites, which shows that the component II contains a large amount of antibodies, and the component II is an isolated and purified monoclonal antibody; the component I has extremely low antibody content and is other hetero protein, and the specific activity of the purified antibody is obviously higher than that before purification. SDS-PAGE showed that bands 2-4 (peak I) contained a large amount of hetero-protein as shown in FIG. 2; lanes 5-8 (peak II) have two distinct protein bands, which, in combination with ELISA results, indicate that peak II is a purified monoclonal antibody and that the two protein bands are the heavy and light chains of the antibody, respectively.

TABLE 1 ELISA detection results (OD values) of different components before and after purification

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

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

Example 2: identification of monoclonal antibodies prepared in example 1

(1) The monoclonal antibody titer was determined by indirect enzyme-linked immunosorbent assay (ELISA) and the specific activity was calculated as the concentration/titer of purified antibody, and the results are shown in Table 2, indicating that the specific activity of the purified antibody was higher than before purification.

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

(3) SDS-PAGE detection is carried out after the purified monoclonal antibody is subjected to double dilution to identify the purity of the purified antibody, and the result is shown in figure 3, which shows that the purified antibody protein is successfully purified, and the light chain and the heavy chain of the antibody after melting can be clearly observed.

(4) IgG against HBs with myeloma cell culture supernatant as negative control ₁ The subclasses of MAbs were positive controls, and the subclasses of monoclonal antibodies were identified according to the mouse monoclonal antibody detection kit of Sigma Co. USA, and the results are shown in Table 4, indicating that the MAb cell lines F-B6, E-A6, F-B3 and E-C6 were IgG _2b D-D3 is IgG ₁ 。

(5) The cross reaction of the monoclonal antibody and hepatitis B surface antigen (HBSAg) is measured by an indirect ELISA method, the negative and positive control of the HBSAg are respectively a negative and positive sample of the hepatitis B surface antibody, the rPGRP negative is a primary cell culture solution, the positive is a positive culture supernatant of a known antibody, and the PGRP _(42～53) 、PGRP _(68～82) The positive control is the serum of immunized mice, and the result is shown in Table 5, which shows that 5 monoclonal antibodies have no cross with hepatitis B surface antigenCross-reacting E-C6 antibodies with PGRP _(68～82) Synthetic peptides react, D-D3 reacts with PGRP _(42～53) Antigen reaction, the other 3 strains react with PGRP _(42～53) And PGRP _(68～82) No reaction occurs.

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

TABLE 3 recovery rate after ascites purification and monoclonal antibody content

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 anti-progastrin releasing peptide monoclonal antibody

1. Acquisition of anti-progastrin Release peptide monoclonal antibody Gene sequence

(1) RNA extraction and cDNA conversion

Culturing fresh E-B5 hybridoma cells, and centrifuging to collect 10 ⁷ The above cells; extracting total RNA of the cells by using Trizol reagent; mu.L of total RNA, 2.5. Mu.L of oligo (dT) 12-18 primer (10 mM) and 5. Mu.L of dNTPs were mixed, incubated at 70℃for 5 minutes and then allowed to stand on ice for 5 minutes, or reverse transcription was used as appropriateEnzyme (BPI) is subjected to denaturation; 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 reacted at 42℃for 1 hour; the reaction was terminated by incubating at 70℃for 15 minutes, and the obtained cDNA was stored at-20 ℃.

(2) Gene acquisition

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

2. Anti-progastrin release peptide monoclonal antibody gene sequencing

(1) Connection

Mu.l of Vector (pMD 19-T Vector, taKaRa), 3. Mu.l of recovered product, 4. Mu.l of ligase mixture (TaKaRa), and the mixture was mixed and reacted at 16℃for 2 hours.

(2) Transformation

Taking out competent cells (DH 5 alpha) preserved at-80deg.C, and thawing slowly on ice; adding the connection product into competent cells, mixing, and standing on ice for 30min; heat shock at 42 ℃ for 90s; after 2min of ice bath, 800. Mu.l of non-resistant LB medium was added; culturing at 37deg.C for 45min; centrifuging at 8000rpm for 1min, discarding most supernatant, leaving about 50-100 μl, re-suspending thallus, selecting LB plate with corresponding resistance, and plating; air-dried, and cultured upside down in an incubator at 37℃overnight.

(3) Authentication

The monoclonal shaking bacteria were picked up from the plate, 1. Mu.l of the bacterial liquid was used as a template, M13F/R was used as a primer, and PCR was verified (program: 95℃3min,95℃30s, annealing temperature 55℃60s, 72℃40s,25 cycles, 72℃3 min).

(4) Sequencing

Sequencing of the recombinant plasmid was completed by Beijing-ing large protein research and development center Co., ltd, and the sequencing results were as follows:

(1) Expression of monoclonal antibody heavy chain variable region V _H And light chain variable region V _L The nucleotide sequences of (2) are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.

(2) Heavy chain variable region V of monoclonal antibody _H The amino acid sequence of (a) is shown as SEQ ID NO.3, and the light chain variable regionV _L The amino acid sequence of (C) is shown as 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 modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof. The foregoing examples or embodiments are merely illustrative of the invention, which may be embodied 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 are intended to be encompassed within the scope of the invention.

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Claims

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

the heavy chain variable region V of the monoclonal antibody _H The amino acid sequence of (a) is shown as SEQ ID NO.3, and the light chain variable region V _L The amino acid sequence of (2) is shown as SEQ ID NO. 4;

the monoclonal antibody is a mouse monoclonal antibody.

2. Use of the monoclonal antibody according to claim 1 for the preparation of a reagent or medicament for diagnosing or treating lung cancer.