CN108484768B

CN108484768B - Anti-resistin immune neutralizing antibody and application thereof in treatment of breast cancer

Info

Publication number: CN108484768B
Application number: CN201810227299.2A
Authority: CN
Inventors: 袁增强; 程金波; 高宇豪; 廖亚金
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS
Priority date: 2018-03-20
Filing date: 2018-03-20
Publication date: 2021-03-30
Anticipated expiration: 2038-03-20
Also published as: CN108484768A

Abstract

The invention discloses an anti-resistin immune neutralizing antibody and application thereof in treating breast cancer, belonging to the field of biological medicine. The anti-resistin immune neutralizing antibody is obtained by selecting antigen polypeptide, selecting an immune mode, purifying antibody serum, measuring antibody concentration and detecting antibody effect, and can be used for treating breast cancer under an obesity model.

Description

Anti-resistin immune neutralizing antibody and application thereof in treatment of breast cancer

Technical Field

The invention relates to an anti-resistin immune neutralizing antibody and application thereof in treating breast cancer, belonging to the field of biological medicine.

Background

Breast cancer is a major problem that afflicts female health worldwide, with about 140 million people diagnosed with breast cancer and about 50 million people dying from the disease every year, breast cancer being the first cause of death in women between the ages of 40-55. The annual average growth rate of breast cancer in China is 3% -4%, the breast cancer becomes the cancer with the fastest growth rate of death rate in cities, and particularly, the Shanghai, Beijing, Tianjin and coastal areas are high-incidence areas of breast cancer in China and already account for the first place of the incidence rate of female malignant tumors.

The occurrence and development of breast cancer are mainly attributed to the following three reasons: 1. genetic factors. For example, some patients have a significant familial predisposition to develop breast cancer inherited from their parents through specific genetic genes (BRCA1/BRCA 2). 2. Estrogen disorders. High fat diet is one of the main causes of abnormal increase of estrogen, since the breast is chronically stimulated by abnormal estrogen, resulting in the canceration of breast tissue. 3. Environmental factors. The unreasonable dietary structure and the stimulation of ionizing radiation toxin can cause the canceration of normal mammary epithelial cells.

At present, the unreasonable dietary structure and the obesity caused by genetic factors are closely related to the onset of breast cancer. Clinically, the incidence rate and clinical grade of breast cancer are positively correlated with obesity, and excessive adipose tissues provide energy for proliferation and migration of tumor cells, and meanwhile, the adipose tissues also provide shelter for metastasis of the tumor cells and chemical drug treatment.

Adipose tissue is reported to be closely related to the occurrence of breast cancer, not only as a storage place for lipid droplets, but also as an important secretory organ of the body. On the one hand, excessive adipose tissue causes body glucose and insulin resistance, and excessive levels of glucose and insulin provide sufficient energy and signal stimulation for tumor cell proliferation to metastasize. On the other hand, obesity causes adipose tissues to be in a hypoxic state, induces the expression of hypoxia inducible factor (HIF1 α), transforming growth factor- β (TGF- β), Matrix Metalloproteinases (MMPs), etc., further remodels the tumor microenvironment and induces tumor angiogenesis and metastasis. In addition, obesity is often accompanied by chronic inflammation of adipose tissue, and inflammatory factors secreted from adipose tissue, such as interleukin family, tumor necrosis factor α (TNF α), etc., participate in shaping tumor microenvironment and promoting tumor occurrence and development.

At present, the treatment of breast cancer is mainly a means of assisting radiotherapy and chemotherapy by operation. Clinically, breast cancer is mainly classified into the following four types according to the expression of Estrogen Receptor (ER), Progesterone Receptor (PR), and HER 2: luminal epithelial type A (Luminal subtype A, ER +/PR +, HER2-), Luminal epithelial type B (Luminal subtype B, ER +/PR +, HER2+), HER2 overexpression (HER2over-expression subtype, ER-, PR-, HER2+), triple negative breast cancer (ER-, PR-, HER 2-). Clinically adjunctive to different chemotherapeutic treatments, depending on the molecular subtype, such as female receptor/progestin receptor targeted drug therapy (tamoxifen) for ER/PR positive patients while blocking estrogen synthesis with aromatase inhibitors; the targeted drug hessidine against HER 2; the common chemotherapeutic drug cisplatin and the like are used for the triple negative patients. However, clinical treatment of breast cancer is often accompanied by tumor recurrence and chemotherapy resistance, especially in obese patients, and it is difficult to achieve and maintain and achieve the effect of treating tumors by reducing lipopexia and resisting obesity. Therefore, the search for a new safe and effective method for inhibiting breast cancer has become a hot research.

Resistin protein (Resistin) is produced and secreted by adipocytes in mice and mainly by adipocytes and mononuclear macrophages in humans. Resistin is reported to be closely related to obesity and type II diabetes, clinically, the Resistin level in serum is positively related to the malignancy degree of a breast cancer patient, the Resistin can promote the migration of breast cancer cells cultured in vitro, and meanwhile, in vitro experiments prove that the Resistin can promote the proliferation of breast cancer stem cells and promote the chemotherapy resistance of breast cancer.

Currently, an immune neutralizing antibody against Resistin is not used in vivo experiments, and is difficult to prepare and high in cost. We hope to search Resistin specific peptide fragment for the preparation of immune neutralizing antibody by bioinformatics, prepare polyclonal antibody in large scale, design mouse obesity model and verify the treatment of breast cancer.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an effective preparation method of an anti-murine resistin immune neutralizing antibody.

The preparation method comprises the steps of selecting antigen polypeptide, selecting an immunization mode and purifying antibody serum. And further determining the antibody concentration and antibody effect.

The antigen polypeptide is a Resistin protein specific polypeptide segment which comprises a polypeptide sequence EAIDKKIKQDF (the 30 th-40 th amino acid from the N end of a murine Resistin full-length sequence).

The immunization mode is that antigen polypeptide liquid is obtained by coupling BSA (bovine serum albumin) through cysteine at the C end of a peculiar polypeptide segment of Resistin protein, and the antigen polypeptide liquid is evenly mixed with Freund's complete adjuvant, and then 8 positions of subcutaneous tissues of the left and right back sides, subcutaneous tissues of the abdomen, muscles of the left and right back legs and popliteal lymph nodes of the left and right back legs of a male New Zealand rabbit for 8 weeks are injected with the antigen-adjuvant mixed liquid; then in 2, 4, 5, 6 and 7 weeks, the rabbit 8-week rapid adjuvant and the antigen polypeptide liquid are mixed uniformly in advance, and the antigen-rapid adjuvant mixed liquid is injected at the 8 positions.

The purified antibody serum is obtained by utilizing the specific affinity purification of antigen antibody.

In another aspect, the invention provides the use of an anti-murine resistin neutralizing immunoantibody in the treatment of a breast tumor that is obesity induced and that acts by in situ injection.

The invention takes protein polypeptide with the sequence comprising EAIDKKIKQDF as an antigen to immunize a New Zealand rabbit, obtains an anti-Resistin immune neutralizing antibody by purifying serum through antigen-antibody affinity purification, and the obtained anti-Resistin immune neutralizing antibody can obviously reduce the weight and the volume of the mammary tumor of an obese mouse, thereby achieving effective control and treatment effects.

Drawings

The symbols appearing in the figures of the present invention represent P <0.05, P <0.01, P < 0.001.

FIG. 1 shows the monomer structure (a), trimer (b), hexamer structure (c) of Resistin protein and full-length sequence information (d) of Resistin. Where the arrow indicates and the red color is labeled (1#) for the peptide fragment selected for the preparation of the immune neutralizing antibody.

FIG. 2 shows a scheme of immunization scheme for preparing antibodies by antigen injection in New Zealand rabbits. To the subcutaneous, thigh muscle and popliteal lymph nodes of the back of New Zealand rabbits at week 0 were injected Freund's complete adjuvant (Sigma-Aldrich Co.) in admixture with antigen (1:1, 500. mu.g). Then, the same part was injected with a mixture of rabbit 8-week rapid adjuvant (Beijing Boolong immuno-technology Co., Ltd.) and antigen (1:1, 250. mu.g) at weeks 2, 4, 5, 6, and 7. Antibody identification was performed by ear-marginal vein blood collection at week 8, and new zealand rabbit whole blood was collected via common carotid artery at week 9.

FIG. 3 shows that Western Blot verifies the ability of New Zealand rabbit serum to recognize Resistin in differentiated mature 3T3-L1 adipocytes.

FIG. 4 shows affinity purification of anti-Resistin antibodies in New Zealand serum using BSA coupled # 1 polypeptide, antibody concentration in the eluate as determined by Coomassie blue staining after protein electrophoresis after elution of the eluate.

FIG. 5ELISA experiment shows the recognition of Resistin in the supernatant of 3T3-L1 adipocytes by purified antibody, with a positive control of commercial anti-Resistin antibody (PEPROTECH) and a negative control of rabbit IgG (FIG. a). Panel b shows gradient dilutions of purified antibodies followed by ELISA assays to detect binding of Resistin protein and polypeptide # 1 in supernatants from 3T3-L1 adipocytes.

FIG. 6 shows a flowchart of administration of Resitin antibody for treatment of breast cancer in mice in an obese model. After induction of female C57BL/6 mice obesity with a 12-week high fat diet, mice were injected with a murine breast cancer cell line E0771(5 x 10) into the second mammary fat pad of normal/obese mice⁵) Then, anti-Resistin immune neutralizing antibody (0.7mg/kg body weight/time) or equivalent rabbit IgG was injected in situ into mammary gland fat pad on

days

1, 4, 8, and 12 while detecting relevant indexes, and the mice were sacrificed and counted on day 13.

FIG. 7 weight histogram (a) and visceral fat weight histogram (b) of fat pads of the contralateral mammary glands of the chest of normal/obese mice in the absence or presence of anti-Resistin antibody administration.

FIG. 8 daily measurement of tumor volume in normal/obese mice given anti-Resistin antibody or rabbit IgG.

FIG. 9 is a graph of the weight of breast tumors in mice (b) measured for the presence or absence of anti-Resistin antibodies given under normal and obese conditions, and a visual graph of the size of each group of tumors (a) after the mice were sacrificed on day 13.

Detailed Description

In the following examples, the new zealand rabbits used for antigen injection were male, 8 weeks old, and C57BL/6 female mice were fed with a high-fat diet for 12 weeks from the postnatal fourth week, with a fat ratio of 60%, unless otherwise specified.

Unless otherwise indicated, the reagents and materials used in the following examples are analytical grade reagents and are commercially available or purchased from a regular channel, wherein the purity of # 1 polypeptide required for immunizing New Zealand rabbits is > -95%.

According to the invention, the control group and the anti-Resistin immune neutralizing antibody group are subjected to statistical analysis, and P <0.05 indicates that the difference between the groups has statistical significance.

Example 1 Large Scale preparation of Resistin Immunoisoning antibodies

In mice, Resistin is secreted mainly by mature adipocytes and exists mainly as trimers and hexamers in vivo. Firstly, analyzing a region with stronger immunogenicity of a Resistin protein sequence by utilizing B cell epitope prediction analysis software, then analyzing and predicting the region with stronger antigenicity by utilizing protein three-dimensional structure analysis software, selecting a sequence which is exposed on the surface of a protein 3D structure and has stronger antigenicity according to the positions of a protein monomer and a polymer 3D structure to synthesize a short peptide, and carrying out subsequent experimental evaluation.

The 1# polypeptide (N-terminal alpha helix, sequence EAIDKKIKQDF) is used as an antigen, and the C-terminal of the polypeptide is coupled with BSA through cysteine, so that antigen presentation is facilitated, and immunity is enhanced.

According to the protocol of FIG. two, 500. mu.l Freund's complete adjuvant (Sigma-Aldrich Co.) was mixed with 500. mu.l of antigen polypeptide solution (1. mu.g/. mu.l in physiological saline) in advance at week 0, and put on ice and mixed well with a vacuum mixer. The antigen-adjuvant mixture was injected into 8 sites of 8 weeks old male New Zealand rabbits, including subcutaneous sites on the left and right sides of the back, subcutaneous sites on the abdomen, muscles of the left and right hind legs, and popliteal lymph nodes of the left and right hind legs, at about 120. mu.L per spot. Then, in 2, 4, 5, 6 and 7 weeks, 250 μ L of rabbit 8-week rapid adjuvant (Beijing Boolong immunization technology Co., Ltd.) and 250 μ L of antigen polypeptide solution (1 μ g/μ L, dissolved in physiological saline) were rapidly mixed uniformly, and then the antigen-adjuvant mixture was injected into the above 8 sites, and each site was about 60 μ L.

And (3) collecting about 1ml of blood of the auricular marginal vein at the 8 th week, placing the blood in an anticoagulation tube, standing the blood for 20 minutes at room temperature, centrifuging the blood for 15 minutes at 3500 rpm of a centrifuge at 4 ℃, sucking supernatant (serum), subpackaging and storing the blood at the temperature of 20 ℃ below zero. Meanwhile, the serum of the New Zealand rabbit is diluted by primary anti-diluent in a ratio of 1:500, and the recognition of the antibody generated in the serum to the Resistin protein in 3T3-L1 fat cells is verified by a protein immunoblotting experiment (3T 3-L1 precursor cell sample is used as a negative control (no Resistin is expressed), and Resistin antibody produced by PEPROTECH company is used as a positive control). The experimental results show that Resistin antibody is generated in the serum of the new Zealand rabbit after immunization, and the Resistin with full length and a secretory form can be specifically recognized (figure 3).

About 100mL of new zealand rabbit whole blood was collected via common carotid artery at week 9, left to stand at room temperature for 2 hours, centrifuged at 3500 rpm in a 4 ℃ centrifuge for 30 minutes, about 50mL of supernatant (serum) was aspirated, frozen at-80 ℃ with liquid nitrogen, and used for subsequent purification experiments.

Example 2 purification of Resistin Immunoisoning antibodies and Activity detection

In order to obtain a large amount of pure anti-Resistin neutralizing immunoantibodies, we purified the antibodies in the serum using antigen-conjugated sepharose,

1. 1mg of # 1 polypeptide antigen-BSA fusion protein was dissolved in coupling buffer overnight at 4 deg.C (coupling buffer formulation: 100mM NaHCO₃500mM NaCl, pH adjusted to 8.3).

2. 1ml of cyanogen bromide (CNBr) -activated Sepharose4B beads were activated in 20ml of cold 1mM HCl solution at 4 ℃ for 15 minutes.

3. The beads were washed twice with 20ml 1mM HCl.

4. Activated agarose beads were incubated overnight with dialyzed 1# polypeptide antigen-BSA fusion protein in a shaker at 4 ℃.

6. The protein-coupled sepharose beads were washed 2 times with 15ml of coupling buffer, centrifuged and the supernatant discarded.

7. 5ml of 0.1M Tris-HCl buffer (pH8.0) was added to block the uncoupling site on the beads and left overnight at 4 ℃.

8. Next, antisera were purified by antigen protein coupling to Sepharose beads:

1) sepharose beads were first washed 5 times with acid-base buffer. (acid-base buffer formulation: 0.1M acetic acid/sodium acetate, pH 4.0; 0.1M Tris-HCl, 0.5M NaCl, pH 8.0).

2) The beads were incubated overnight with serum diluted 2-fold in PBS buffer on a shaker at 4 ℃ (PBS buffer formulation: 10mM Na₂HPO₄，1.8mM KH₂PO₄137mM NaCl, 2.7mM KCl, pH adjusted to 7.4).

3) The bead and serum mixture was passed through a purification column to collect the flow-through and the flow-through was stored at-20 degrees celsius for ELISA testing.

4) The beads were washed 3 times with 10ml PBS buffer.

5) The column was washed with 10ml of 150mM NaCl-HCl (pH 5) solution.

6) The antibody was eluted with 7ml of elution buffer and the solution was neutralized with 500. mu.l of saturated phosphate buffer. (eluent formulation: 150mM NaCl, pH adjusted to 2.5 with hydrochloric acid; saturated phosphate buffer formulation: adding Na to PBS buffer₂HPO₄Solid until saturated).

7) The antibody solution was dispensed into 1ml tubes and stored at-80 ℃.

The antibody solution was subjected to protein electrophoresis and quantified by staining with Coomassie Brilliant blue. The concentration of antibody in the eluate was determined to be about 150. mu.g/ml (FIG. 4).

Next, ELISA assay was used to determine whether the purified neutralizing antibody could recognize the 1# antigen polypeptide and the natural Resistin protein.

10ml of mature 3T3-L1 adipocyte culture supernatant (the supernatant was collected after mature adipocytes were cultured in FBS-free DMEM medium for 12 hours) was centrifuged at 3000 rpm for 10min, filtered through a 0.22 μm filter and dried to powder in a freeze dryer. Dissolving the above powder with antigen coating solution to 2mg/ml (antigen coating solution formula: Na)₂CO₃0.159g/100ml，NaHCO₃0.293g/100ml) and coated in 96-well ELISA plates for ELISA detection. As shown in FIG. 5a, the purified anti-Resistin antibody recognized Resistin protein well in nature at a dilution ratio of 1:500, and the negative control was rabbit IgG (and purified antibody)Consistent at a 1:500 dilution concentration), the positive control was a commercial anti-Resistin antibody (PEROTECH, Inc., dilution ratio 1: 2000). In addition, mature 3T3-L1 adipocyte culture supernatant or 1# polypeptide antigen coated ELISA plate, under the condition of gradient dilution, the titer of the purified anti-Resistin antibody is detected, the experimental result is shown in figure 5b, under the condition of gradient dilution, the recognition of the antibody to two antigens is weakened in a gradient manner, and the specificity of the purified antibody is shown.

Example 3 Resistin Immunity-neutralizing antibody is effective in treating obesity-induced breast tumor hyperproliferation

First, a mouse obesity model was constructed. Female C57BL/6 mice of uniform size and weight were selected and fed on a high fat diet (60% lipid) from postnatal week 4 for 12 weeks to mice obese.

As shown in the scheme of FIG. 6, the second mammary fat pad on the right side of the mice was injected with C57BL/6 mouse-derived E0771 breast cancer cells 5 x 10 on week 13⁵One of the cells (cells resuspended in 100. mu.l PBS buffer) was injected with 0.7mg/kg body weight/time of purified anti-Resistin antibody into the adipose tissue surrounding the tumor tissue of the second mammary fat pad on the right side of normal/obese mice on

days

1, 4, 8 and 12, respectively, and the control group was dissolved in the eluate after neutralization with saturated phosphate buffer of example 2 using an equal volume of rabbit IgG.

On the sixth day after injection of breast cancer cells E0771, a touch-visible breast tumor appeared in the mammary fat pad, and the major diameter (cm) and the minor diameter (cm) of the tumor generated in each mouse were measured using a vernier caliper, and the tumor volume was calculated using the formula. Tumor volume (cm)³) 0.5 long diameter (cm) short diameter (cm)². From the sixth day, the major and minor diameters of the tumor were measured at the same time every day for 8 consecutive days, and the size of the tumor was calculated and compared as a graph. As shown in fig. 8, the breast tumor volume of the mice in the obese (high fat diet) group was significantly larger than that of the mice in the normal (normal diet) group, and in addition, the breast tumor volume of the mice was significantly reduced after the injection of the anti-resistance immune neutralizing antibody in the obese group.

Further, the mice were sacrificed on day 13, and tumor tissue, visceral adipose tissue, and left mammary fat pad (tumor-contralateral adipose tissue) were taken out of the mice, weighed, and photographed as in fig. 9. The experimental result shows that the weight of the breast tumor of the obese group of mice is obviously higher than that of the normal weight group of mice; after treatment with anti-Resistin immunoneutralizing antibody, the mouse mammary tumor volume was significantly reduced to normal group levels (FIG. 9), whereas the anti-tumor effect of the anti-Resistin immunoneutralizing antibody was exerted independent of the reduction in adipose tissue weight (FIG. 7), suggesting that the in vivo effect of the anti-Resistin immunoneutralizing antibody was exerted by direct binding to Resistin protein and repressing the tumor growth promoting effect of Resistin protein.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> military medical research institute of military science institute of people's liberation force of China

<120> an anti-resistin immune neutralizing antibody and application thereof in treating breast cancer

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 11

<212> PRT

<213> mouse

<400> 1

Glu Ala Ile Asp Lys Lys Ile Lys Gln Asp Phe

1 5 10

Claims

1. An anti-Resistin immune neutralizing antibody is characterized in that an antigen is a Resistin protein specific polypeptide segment, and the amino acid sequence of the Resistin protein specific polypeptide segment is shown as SEQ ID NO: 1 is shown.

2. The anti-resistin neutralizing immunoantibody of claim 1, being a polyclonal antibody of rabbit origin, said rabbit comprising a New Zealand rabbit.

3. The method for preparing the anti-resistin neutralizing antibody of claim 1 or 2, comprising selecting an antigenic polypeptide with an amino acid sequence as shown in SEQ ID No.1, selecting an immunization mode, purifying antibody serum.

4. The method of claim 3, further comprising determining antibody concentration and antibody effect.

5. The method according to claim 3, wherein the immunization mode is that bovine serum albumin is coupled at the C end of the Resistin protein specific polypeptide segment to obtain antigen polypeptide liquid, and the antigen polypeptide liquid and Freund's complete adjuvant are uniformly mixed to obtain antigen-adjuvant mixed liquid; injecting the antigen-adjuvant mixed solution into 8 positions of the old male New Zealand rabbit, including subcutaneous tissues of the left and right sides of the back, subcutaneous tissues of the abdomen, muscles of the left and right back legs and popliteal lymph nodes of the left and right back legs in 8 weeks;

and then, uniformly mixing the fast adjuvant and the antigen polypeptide liquid of the rabbit for 8 weeks in advance in 2, 4, 5, 6 and 7 weeks to obtain an antigen-fast adjuvant mixed liquid, and injecting the antigen-fast adjuvant mixed liquid at the 8 positions.

6. The method of claim 3, wherein the purified antibody serum is obtained by specific affinity purification of antigen-antibody.

7. Use of an anti-resistin immunoneutralizing antibody according to claim 1 or 2 in the manufacture of a medicament for the treatment of obesity induced breast tumours.