CN114644710A

CN114644710A - Anti-progastrin release peptide single-chain antibody and preparation method and application thereof

Info

Publication number: CN114644710A
Application number: CN202210257952.6A
Authority: CN
Inventors: 周小林; 郭玉凤
Original assignee: China Institute for Radiation Protection
Current assignee: China Institute for Radiation Protection
Priority date: 2022-03-16
Filing date: 2022-03-16
Publication date: 2022-06-21

Abstract

The invention belongs to the technical field of antibody drugs, and relates to an anti-progastrin release peptide single-chain antibody, and a preparation method and application thereof. The single-chain antibody comprises a heavy chain variable region V_HAnd light chain variable region V_LAnd optionally comprises a variable region V for linking to the heavy chain_HAnd light chain variable region V_LThe connecting peptide of (1), theHeavy chain variable region V_HComprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of the three are respectively shown as SEQ ID NO.5-7, the light chain variable region V_LComprises LCDR1, LCDR2 and LCDR3, and the amino acid sequences of the three are respectively shown in SEQ ID NO. 8-10. The anti-progastrin release peptide single-chain antibody and the preparation method thereof can be used for better preparing the anti-progastrin release peptide single-chain antibody, and the prepared anti-progastrin release peptide single-chain antibody can be better used for SCLC targeted diagnosis and treatment.

Description

Anti-progastrin release peptide single-chain antibody and preparation method and application thereof

Technical Field

The invention belongs to the technical field of antibody drugs, and relates to an anti-progastrin release peptide single-chain antibody, and a preparation method and application 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, and 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%. However, the early symptoms of the SCLC patient are not obvious, the early diagnosis obtained through pathological and imaging examination is not easy, the disease condition is often developed to a wide stage when the diagnosis is confirmed, and the optimal treatment time is missed, so the early diagnosis and treatment of the 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 SCLC early clinical diagnosis. 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, therapeutic antibody drugs are gradually occupying the global market and used for diagnosis and treatment of diseases such as malignant tumors and autoimmune diseases. The development of the anti-PGRP monoclonal antibody and the genetic engineering 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.

The humanized genetic engineering antibody comprises a full-length antibody, a small antibody and the like. Wherein, the full-length antibody comprises a chimeric antibody and a CDR grafted antibody; small antibodies include single chain antibodiesAntibodies, Fab and F (ab)' 2 fragment antibodies, bispecific antibodies, domain antibodies, and the like. Among them, single chain antibodies (ScFv) are the smallest functional fragments of antibody molecules that retain the antigen binding site, and their full length is about 1/6 of the whole antibody molecule. The key technology of ScFv is to use a linker peptide consisting of about 15-25 amino acid residues to link the Heavy chain variable region (V) of antibody_H) And the Light-chain variable region (V)_L) And performing connection recombination. Compared with the complete antibody, the ScFv can reduce the immunogenicity, has smaller molecular weight and is easier to enter tumor tissues. People can also prepare novel antibodies to carry therapeutic drugs according to the treatment requirements, and can adopt various modes such as pronucleus, eukaryotic cells and the like to express in large quantities, thereby reducing the production cost.

Disclosure of Invention

The primary object of the present invention is to provide an anti-progastrin releasing peptide single chain 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 single chain antibody comprising a heavy chain variable region V_HAnd light chain variable region V_LAnd optionally comprises a variable region V for linking to the heavy chain_HAnd light chain variable region V_LThe linker peptide of (1) or (2),

the heavy chain variable region V_HComprises HCDR1 (heavy chain complementarity determining region 1), HCDR2 (heavy chain complementarity determining region 2) and HCDR3 (heavy chain complementarity determining region 3), the amino acid sequences of which are respectively shown in SEQ ID NO.5-7,

the light chain variable region V_LComprises LCDR1 (light chain complementarity determining region 1), LCDR2 (light chain complementarity determining region 2) and LCDR3 (light chain complementarity determining region 3), and the amino acid sequences of the three are respectively shown in SEQ ID NO. 8-10.

In a preferred embodiment, the present invention provides an anti-progastrin-releasing peptide single chain antibody, wherein the heavy chain variable region V is_HThe amino acid sequence of (A) is shown as SEQ ID NO.3, and the light chain variable region V_LThe amino acid sequence of (A) is shown in SEQ ID NO. 4.

In a preferred embodiment, the present invention provides an anti-progastrin-releasing peptide single chain antibody, wherein the linker peptide is (Gly)₄Ser)₃。

In a preferred embodiment, the present invention provides an anti-progastrin-releasing peptide single chain antibody, wherein the amino acid sequence of said single chain antibody is as shown in SEQ ID No. 1.

The second objective of the present invention is to provide a method for preparing the anti-progastrin releasing peptide single-chain antibody, so as to be able to better prepare the anti-progastrin releasing peptide single-chain antibody, and the prepared anti-progastrin releasing peptide single-chain antibody 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 anti-progastrin-releasing peptide single-chain antibody, the method comprising the steps of:

(1) recombining the nucleotide sequence expressing the anti-progastrin release peptide single-chain antibody into a prokaryotic expression vector, constructing a recombinant expression vector expressing the anti-progastrin release peptide single-chain antibody, and introducing the recombinant expression vector into genetic engineering bacteria to construct recombinant engineering bacteria;

(2) and (2) performing fermentation culture on the recombinant engineering bacteria obtained in the step (1), and separating and purifying the thalli obtained by centrifugation to obtain the purified anti-progastrin release peptide single-chain antibody.

In a preferred embodiment, the present invention provides a method for producing the above-mentioned anti-progastrin-releasing peptide single-chain antibody, wherein in step (1), the nucleotide sequence is represented by SEQ ID No. 2.

In a preferred embodiment, the present invention provides a method for producing the above-mentioned anti-progastrin-releasing peptide single-chain antibody, wherein in the step (1), the prokaryotic expression vector is selected from the group consisting of pET-28a (+), pET-32a (+), pGEX-4T-1 and pE-SUMO.

In a preferred embodiment, the present invention provides a method for producing the above-mentioned anti-progastrin-releasing peptide single-chain antibody, wherein in step (1), the genetically engineered bacterium is selected from escherichia coli BL21(DE3), BL21, BL21(AI), BL21(DE3) PLySs or Rosetta (DE 3).

In a preferred embodiment, the present invention provides a method for producing the above-mentioned anti-progastrin-releasing peptide single-chain antibody, wherein in step (2),

IPTG is added in the process of fermentation culture for overnight induction culture,

the separation and purification is to collect the centrifugal thallus, and the protein is purified by using a nickel metal chelating chromatographic column after the ultrasonic crushing.

The third purpose of the present invention is to provide the use of the anti-progastrin releasing peptide single chain antibody 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 single-chain antibody for the preparation of an agent or medicament for the diagnosis or treatment of lung cancer.

The invention has the advantages that the anti-progastrin releasing peptide single-chain antibody and the preparation method thereof can be used for better preparing the anti-progastrin releasing peptide single-chain antibody, and the prepared anti-progastrin releasing peptide single-chain antibody can be better used for SCLC targeted diagnosis and treatment.

The present invention utilizes a linker peptide (Gly)₄Ser)₃Connecting the variable regions of the heavy chain and the light chain to obtain a PRGP-ScFv single-chain antibody gene, and successfully constructing a prokaryotic expression system. Obtaining a target single-chain antibody with a 6 × His tag through affinity purification; the specificity and the affinity of the single-chain antibody are detected by Western-blot and ELISA, which shows that the PGRP-ScFv single-chain antibody can be specifically combined with PGRP, and the PGRP-ScFv has targeting property. Therefore, the PRGP-ScFv single-chain antibody has potential medical and pharmaceutical values, and provides a new method for the targeted therapy of tumors.

Drawings

FIG. 1 is a diagram showing the identification of pET-PGRP-ScFv recombinant plasmid constructed in example 1, wherein lane 1: v_LPCR amplification products; lane 2: v_HPCR amplification products; lane 3: PCR amplification products of PGRP-ScFv; swimming deviceLane 4: linker PCR amplification product.

FIG. 2 is an SDS-PAGE pattern of the anti-PGRP single-chain antibody purified in example 2.

FIG. 3 is a Western-blotting identification chart of an anti-PGRP single-chain antibody in example 3.

FIG. 4 is a graph showing the concentrations of the anti-PGRP single-chain antibody of example 3 at an initial concentration of 0.5mg/ml after standing at different temperatures for 3 hours.

FIG. 5 is a graph showing SDS-PAGE detection results of the anti-PGRP single-chain antibody of example 3 at an initial concentration of 0.5mg/ml after standing at 4 ℃ for various periods of time.

Detailed Description

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

Example 1: construction of PGRP-ScFv prokaryotic expression System

1. PGRP-ScFv single-chain antibody expression Gene acquisition

(1) PGRP-ScFv single-chain antibody expression gene amplification

Amplifying V by PCR method by using cell strain cDNA secreting monoclonal antibody against progastrin releasing peptide as template_L、V_HAnd connecting peptide gene fragment, and purifying by a Gel recovery Kit (TaKaRaAgarose Gel DNA Purification Kit) to obtain 3-segment gene fragment. PCR amplification is carried out by taking 3 gene fragments as templates to obtain the expression gene of the PGRP-ScFv single-chain antibody. The PCR reaction system is as follows: 10 × buffer: 5 μ L, dNTP mix: 5 μ L, F, R1.5 μ L each, template (V)_L、V_HAnd linker gene fragment) 2 μ L each, Taq DNA polymerase: 0.5. mu.L, ddH₂O make up to 50. mu.L. PCR amplification reaction conditions: 30 cycles of 94 ℃ for 30s, 54 ℃ for 30s and 72 ℃ for 1 min; 10min at 72 ℃. The PCR reaction products were analyzed by 1.5% agarose gel electrophoresis.

The complete PGRP-ScFv expression gene was ligated to pGEM-T Easy Vector, overnight ligation at 4 ℃: 2 × quick connect buffer: 5 mu l of the solution; pGEM-T Easy Vector: 0.5 mul; PCR products: 3.5 mul; t is₄DNA ligase: 1 μ l.

(2) Conversion of ligation products

Taking out Escherichia coli DH5 alpha competent cells 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 mul of LB culture medium without resistance is added; culturing at 37 deg.C for 45 min; centrifuging at 8000rpm for 1min, discarding most of supernatant, reserving about 50-100 μ l, resuspending thallus, selecting Amp resistant LB plate, and coating; air-dried and cultured in an incubator at 37 ℃ for overnight in an inverted state.

(3) Screening and identification of recombinant plasmids

And selecting the well-grown monoclonal colonies on the plate, and screening the strains by using a positive clone screening method. Positive clones were transferred to 5mL of a liquid medium having ampicillin resistance, cultured overnight at 37 ℃ and 180rpm, and plasmids were extracted and identified using a plasmid miniprep.

2. Prokaryotic expression vector construction

(1) Carrying out double enzyme digestion on plasmid pGEM-T-PGRP-ScFv containing the single-chain antibody expression gene fragment and a prokaryotic expression plasmid vector pET-28a (+) by using restriction enzymes EcoRI and Not I respectively, and carrying out water bath at 37 ℃ for 4 h; separating the target fragment from the vector by 1.5% agarose gel electrophoresis, recovering the target fragment and the vector, and₄the PGRP-ScFv-expressing gene was recombined into pET-28a (+) by ligation with DNA Ligase at 4 ℃ overnight.

TABLE 1 double digestion System of pGEM-T-PGRP-ScFv and pET-28a (+)

TABLE 2 ligation reaction System of pET-PGRP-ScFv

(2) The pET-PGRP-ScFv recombinant plasmid is transformed into Escherichia coli DH5 alpha competent cells, and after Kan resistance screening, plasmids are extracted and identified, and the result is shown in figure 1. The result shows that the pET-PGRP-ScFv recombinant plasmid is successfully constructed.

Example 2: anti-PGRP single-chain antibody expression purification

1. Prokaryotic expression of anti-PGRP single-chain antibody

(1) The constructed recombinant plasmid pET-PGRP-ScFv was transformed into E.coli BL21(DE3) and cultured on LB solid medium plate overnight in an incubator at 37 ℃.

(2) BL21/pET-PGRP-ScFv single colonies were picked from the plate in an ultraclean bench in 5mL LB liquid medium containing 100. mu.g/mL Kan, cultured overnight at 37 ℃ with shaking at 180 rpm.

(3) Transferring the overnight cultured bacteria liquid to 100mL LB liquid medium at a ratio of 1:100, shake culturing at 37 deg.C for 2-3 hr to OD₆₀₀About 0.6 to 0.8; adding IPTG (isopropyl thiogalactoside) with different concentrations for induction, selecting different induction temperatures and induction times, and performing shake culture at 180 rpm; collecting thallus, centrifuging at 4 deg.C and 8500rpm for 15min, and removing supernatant; resuspending the thalli in precooled PBS buffer solution, washing, centrifuging at 8000rpm for 10min at 4 ℃, and discarding supernatant; the bacterial cells were suspended in PBS buffer, 1mM protease inhibitor PMSF was added, and the bacterial solution was placed on ice and disrupted by ultrasonication until the cells were completely disrupted.

(4) Adding 100 μ L of whole bacteria solution into 5 xSDS loading buffer solution, standing at 100 deg.C for 5min for SDS-PAGE electrophoresis detection; centrifuging the lysed whole bacteria solution at 4 ℃ and 13000rpm for 30 minutes, collecting the supernatant, taking 100 mu L of the supernatant, adding 5 xSDS loading buffer solution, and standing for 5 minutes at 100 ℃ for SDS-PAGE electrophoresis detection; the pellet was resuspended in PBS buffer, 100. mu.L of the supernatant was added to 5 XSDS loading buffer and allowed to stand at 100 ℃ for 5min for SDS-PAGE detection. Protein samples were analyzed by 12% SDS-PAGE.

2. Denaturation, purification and renaturation of anti-PGRP single-chain antibody inclusion body protein

The expressed bacterial pellet was resuspended in precooled 50mmol/L Tris-HCl, 100mmol/L NaCl, 1mmol/L EDTA (pH7.0) to dissolve, the bacteria were disrupted by sonication, centrifuged at 30000g for 30min at 4 ℃. The pellet was washed with 3mol/L urea and 50mmol/L Tris-HCl (pH7.0), 30000g, and centrifuged at 4 ℃ for 30min to collect inclusion bodies. 6mol/L guanidine hydrochloride and 0.1mol/L Tris-HCl (pH7.0), shaking overnight at 4 ℃ to dissolve the inclusion bodies. The precipitate was removed by centrifugation at 30000g for 30min at 4 ℃ and filtered through a 0.45 μm filter and the supernatant was used for purification. The sample was loaded on a nickel metal chelate affinity column, the column was washed with 6mol/L urea, 50mmol/L Tris-HCl and 50mmol/L imidazole (pH7.0), and ScFv bound to the nickel column was eluted with a solution containing 250mmol/L imidazole, 6mol/L urea and 50mmol/L Tris-HCl. The eluate was added to a dialysis bag and sufficiently renatured with TEA buffer for 24h, and then dialysis was continued with PBS for 24 h. Ultrafiltering and concentrating the sample to a proper volume, determining the protein content by using a BCA method, and subpackaging and storing at-20 ℃.

The results of SDS-PAGE analysis are shown in FIG. 2, and show that the anti-PGRP single-chain antibody with the 6 XHis tag was successfully purified.

Example 3: identification of anti-PGRP Single chain antibody

1. Western-blotting detection of anti-PGRP single-chain antibody

Taking 5 mu L of the purified product, and carrying out 12% SDS-PAGE electrophoresis; transferring the gel to a cellulose acetate membrane after electrophoresis, and rotating the membrane for 1h30min at 100V; TBST washing for 5min, 5% skimmed milk powder overnight sealing at 4 ℃, based on 6 His-tag short peptides at the tail end of an expression product, incubating for 2h at room temperature by using a mouse anti-His-tag antibody, fully washing for 3 times by using PBS, adding diluted horseradish peroxidase-labeled goat anti-mouse IgG polyclonal antibody, incubating for 1h at room temperature, washing for 3 times by using PBS-Tween, 10min each time, and then developing by using Diaminobenzidine (DBA).

The Western-blotting detection result is shown in FIG. 3, and the result shows that the fusion protein 6 XHis-PGRP-ScFv is successfully purified.

2. anti-PGRP single-chain antibody affinity and stability detection

The ScFv antigen-binding activity of the anti-PGRP single-chain antibody, namely the affinity determination of the anti-PGRP single-chain antibody, was carried out by a competitive ELISA method. The specific detection method comprises the following steps:

antigen PGRP_(31-98)(see CN200510065889.2 for preparation method) and BSA protein were diluted to 3. mu.g/mL with PBS, coated to ELISA plate wells at 100. mu.L/well, and incubated overnight at 4 ℃; discarding the solution in the wells, washing with PBST for 3 times, adding 200 μ L of blocking solution into each well, and blocking at 37 deg.C for 2 h;discarding the solution in the wells, adding 100 μ L of anti-PGRP-ScFv and monoclonal antibody with different concentration ratios, adding 100 μ L of PBS to the negative control group, incubating for 1h at 4 ℃, washing the plate for 3 times with PBS (containing 0.25% Tween20), and adding 100 μ L (2000 x) of goat anti-mouse Ig labeled with HRP; 4c incubation for 1h, wash plate 3 times with PBS (containing 0.25% Tween 20); adding a color development solution (DAB) to react for about 15min at room temperature, stopping the reaction by 2M concentrated sulfuric acid, reading the absorbance value (OD value) at the wavelength of 450nm by using an enzyme-labeling instrument, and recording the reading.

Detection of 6 XHis-PGRP-ScFv antibody and PGRP by ELISA method_(31-98)The strength of affinity. Specifically, based on the above ELISA experiment, 6 XHis-PGRP-ScFv antibody was diluted and the affinity thereof was measured at different concentration gradients.

The stability of the 6 XHis-PGRP-ScFv antibody was determined by SDS-PAGE. Specifically, the single-chain antibody is placed at different temperatures for 3 hours, and sampling is carried out to detect the concentration change of the single-chain antibody; meanwhile, the single-chain antibody is placed at 4 ℃ for different time, and the degradation degree of the single-chain antibody is detected through gray level analysis after SDS-PAGE electrophoresis, so that the stability of the single-chain antibody is identified.

The results of the affinity assay are shown in Table 3, and show that the single-chain antibody can specifically recognize and bind to PGRP_(31-98)And at lower concentrations still maintains a better affinity to progastrin releasing peptide (PGRP).

The stability test results are shown in fig. 4 and 5, and the results show that: after the single-chain antibody with the initial concentration of 0.5mg/ml is respectively placed at the temperature of 25 ℃, 35 ℃, 45 ℃, 55 ℃, 65 ℃ and 75 ℃ for 3 hours, the single-chain antibody concentrations are respectively 0.420mg/ml, 0.289mg/ml, 0.092mg/ml, 0.032mg/ml, 0.020mg/ml and 0.031 mg/ml; after the single-chain antibody is placed at 4 ℃ for 1d, 2d, 3d, 5d and 7d, the antibody is degraded by 16.74%, 39.87%, 40.97%, 70.94% and 79.02% respectively.

TABLE 3 affinity assay for anti-PGRP Single chain antibodies

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 single chain antibody, characterized in that:

the single-chain antibodyComprising a heavy chain variable region V_HAnd light chain variable region V_LAnd optionally comprises a variable region V for linking to the heavy chain_HAnd light chain variable region V_LThe linker peptide of (1) or (2),

the heavy chain variable region V_HComprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of the three are respectively shown in SEQ ID NO.5-7,

the light chain variable region V_LComprises LCDR1, LCDR2 and LCDR3, and the amino acid sequences of the three are respectively shown in SEQ ID NO. 8-10.

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

3. Single chain antibody according to claim 1 or 2, characterized in that: the connecting peptide is (Gly)₄Ser)₃。

4. The single chain antibody of claim 1, characterized in that: the amino acid sequence of the single-chain antibody is shown in SEQ ID NO. 1.

5. A method for preparing a single chain antibody according to any one of claims 1 to 4, comprising the steps of:

6. The method of claim 5, wherein: in the step (1), the nucleotide sequence is shown as SEQ ID NO. 2.

7. The method of claim 5, wherein: in the step (1), the prokaryotic expression vector is selected from pET-28a (+), pET-32a (+), pGEX-4T-1 or pE-SUMO.

8. The production method according to claim 5, characterized in that: in the step (1), the genetically engineered bacteria are selected from escherichia coli BL21(DE3), BL21, BL21(AI), BL21(DE3) PLySs or Rosetta (DE 3).

9. The method of claim 5, wherein: in the step (2),

10. Use of a single chain antibody according to any one of claims 1 to 4 for the preparation of a reagent or medicament for the diagnosis or treatment of lung cancer.