CN115991773B

CN115991773B - Pharmaceutical composition containing human serum albumin

Info

Publication number: CN115991773B
Application number: CN202210985801.2A
Authority: CN
Inventors: 曹立烜; 熊建勋
Original assignee: Yingling Wuhan Biotechnology Co ltd
Current assignee: Yingling Wuhan Biotechnology Co ltd
Priority date: 2022-08-15
Filing date: 2022-08-15
Publication date: 2023-11-17
Anticipated expiration: 2042-08-15
Also published as: CN115991773A

Abstract

The application relates to a pharmaceutical composition containing human serum albumin. The application prepares a specific monoclonal antibody aiming at endoglin, which can effectively inhibit proliferation of colorectal cancer cells and can directly regulate expression of TGF-beta 1 at the downstream of endoglin by inhibiting activity of endoglin. The paclitaxel/albumin nanoparticle and the endoglin specific monoclonal antibody are combined, so that the tumor inhibition rate can be effectively improved, and a better treatment effect is achieved.

Description

Pharmaceutical composition containing human serum albumin

Technical Field

The application relates to the field of biology, in particular to a pharmaceutical composition containing human serum albumin.

Background

Colorectal cancer is a common malignancy, including colon cancer and rectal cancer. The incidence of colorectal cancer is, in order from high to low, rectum, sigmoid colon, cecum, ascending colon, descending colon, and transverse colon, and in recent years, there is a trend toward the proximal end (right half colon). The onset of the disease is closely related to life style, heredity, large intestine adenoma and the like. The onset age is aged, and the ratio of men to women is 1.65:1. at present, about 312 thousands of new cancer cases are generated annually in China, and on average, 8550 people are diagnosed every day, 6 people are diagnosed as cancer every minute, and 5 people die from the cancer. The number of cancer death cases is 270 ten thousand per year.

In the tumors with the highest incidence of five diseases in China, the number of digestive tract tumors is four, so that the incidence of gastric cancer, esophageal cancer and liver cancer is stable in practice. However, there is a clear upward trend in the incidence of colorectal cancer. The incidence rate of colorectal cancer rises rapidly, and white-collar people are particularly obvious and have a tendency to younger. Colon and rectum cancers are collectively referred to as colorectal or colorectal cancers. Experts show that the incidence rate of colorectal cancer rises rapidly, the incidence rate of colorectal cancer is particularly obvious in white-collar people in large cities such as northern Guangdong, and the trend of younger occurrence is predicted by scientists, so that the incidence rate of colorectal cancer in the near future probably exceeds lung cancer and gastric cancer, and the colorectal cancer becomes the first of the cancer. The rejuvenation of colorectal cancer, in addition to genetic factors, exacerbation of urbanization and changes in the dietary structure of the population are also important reasons, and urban white-collar under high intensity working pressure is of particular concern.

Research on pathogenesis of colorectal cancer generally suggests that the pathogenesis of colorectal cancer is complex, and genetic material abnormality, genetic abnormality, unhealthy eating habits and the like are main causes of colorectal cancer. From a genetic perspective, the onset of colorectal cancer is mainly manifested as familial adenomatous polyposis and hereditary non-polyposis colorectal cancer. Adenomatous polyposis gene has the function of inhibiting oncogene, and its loss of function results in the elevation of p-calnexin level, which directly induces tumor occurrence. The occurrence of hereditary non-polyposis colorectal cancer is related to the defect of the mismatching repair system. Hereditary non-polyp colorectal cancer is a susceptible pestilence group of tumors, and from the current research, the change of the mismatching repair related genes has great influence on the formation of tumors. Changes in Hmshl, hmsh2, hpsml, hpsm2 and GTBP genes are the main causes of tumor induction. In addition, high-fat low-cellulose diet and long-term smoking are causative factors of colorectal cancer. At present, common colorectal cancer chemotherapeutic drugs in the market include 5-FU, capecitabine, oxaliplatin, irinotecan, cetuximab, bevacizumab, panitumumab and the like. The main monoclonal antibody medicine is cetuximab, bevacizumab and panitumumab. But the variety of drugs is not yet abundant.

The growth and metastasis of malignant solid tumors depend on the formation of new blood vessels in tumor tissues, which acquire nutrients through tumor blood vessels, excrete metabolites and import tumor cells into the host, which are closely related to tumor growth, invasion and metastasis, while CD ₁₀₅ Also participates in angiogenesis, is a new vessel mark. From this, it is expected that CD ₁₀₅ Plays an important role in diagnosis, prognosis and treatment of human malignant tumor. CD (compact disc) ₁₀₅ Also known as endoglin, which is a surface of endothelial cell membranesThe glycoprotein is one of the components of the transforming growth factor beta receptor complex, but can be present independently on the cell surface. CD (compact disc) _105/ endoglin regulates endothelial and interstitial signaling by participating in TGF- β receptor signaling, and plays an important role in tumor angiogenesis. Tumor vascular growth is an important condition for tumorigenesis, growth, and invasive metastasis. CD (compact disc) _105/ endoglin provides a new therapeutic target for anti-tumor vascular treatment. The targeted therapy for inhibiting tumor angiogenesis or selectively damaging tumor blood vessels is a new ideal tumor treatment method after traditional surgery, chemotherapy and radiotherapy. In the mouse experiments, the CD is shown _105/ endoglin as acting target and CD resisting _105/ The endoglin antibody is taken as a carrier, and a compound combined with the endoglin antibody, such as a chemotherapeutic drug, biotoxin, radionuclide, and the like, can specifically guide to a tumor part, directly kill tumor cells, has obvious tumor inhibiting effect, and has no obvious adverse effect. In the recent CD ₁₀₅ In the research of antitumor blood vessels serving as targets, the oral vaccine induces CD8+ cell mediated immune response, so that lung metastasis of breast cancer of a cancer cell line is effectively inhibited, and the effect of antitumor blood vessel targeted therapy in future breast cancer treatment is predicted. Because the anti-angiogenesis treatment has the advantages of high efficiency, specificity, difficult generation of drug resistance, small toxic and side effects and the like, the CD is applied _105/ The endoglin monoclonal antibody has broad prospect in combination with chemotherapeutics, biotoxins and radionuclides for anti-tumor treatment.

However, at present, research on endoglin monoclonal antibodies is not enough, and research is not enough, so that corresponding medicaments are still to be further developed.

Disclosure of Invention

The application overcomes the defects of the prior art and provides a novel method for treating colorectal cancer.

In one aspect, the application provides a monoclonal antibody specific for endoglin.

In one aspect, the application provides a monoclonal antibody a11 directed against endoglin, the amino acid sequence of the light chain variable region:

amino acid sequence of heavy chain variable region:

in another aspect of the application, there is provided the use of a monoclonal antibody directed against endoglin for the preparation of a pharmaceutical composition for the treatment of colorectal cancer.

Further, the colorectal cancer is caused by a human colorectal cancer cell line HCT 116.

Human Serum Albumin (HSA) is a natural carrier of human endogenous substances and hydrophobic molecules, and HSA has a special transport mechanism, so that the concentration of a drug combined with the HSA in tumor cells can be greatly improved. Thus in a further aspect, the present application also provides albumin paclitaxel nanoparticles.

The nanometer example is that paclitaxel is dissolved in acetone, a certain amount of human albumin is taken to be dissolved in water, the medicine carrying ratio (medicine/albumin mol ratio) is set to be 10:1, the paclitaxel acetone solution is dropwise added into the albumin water solution under the stirring condition, the stirring is continued for 6min, then acetone is removed by rotary evaporation, water is added for volume fixing, and high-pressure homogenization is carried out, thus obtaining the albumin paclitaxel nanoparticle solution.

In another aspect, the application also provides the use of a monoclonal antibody directed against endoglin and albumin paclitaxel nanoparticles for the preparation of a pharmaceutical composition for the treatment of colorectal cancer.

Furthermore, the albumin paclitaxel nanoparticle can also be prepared by other conventional preparation methods in the field.

In some embodiments, the pharmaceutical composition, wherein the antibody comprises the amino acid sequence: a variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homology to the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO. 2.

The pharmaceutical compositions of the application are useful for administration to a subject in need thereof. In particular, the pharmaceutical compositions of the application are useful for treating cancer, and in certain embodiments, for treating cancer in which endoglin is expressed, e.g., a therapeutically effective amount or a prophylactically effective amount. In some embodiments, the cancer is selected from: adrenal gland cancer, anal cancer, AIDS-related cancer, alveolar soft tissue sarcoma, bile duct cancer, one bile duct cancer, bladder cancer, bone cancer, brain and spinal cord cancer, breast cancer, metastatic breast cancer, carotid aneurysm, cervical cancer, HPV-related cervical cancer, chondrosarcoma, chordoma, borborrelia renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, connective tissue proliferative microcirculatory cell tumor, ependymoma, endometrial cancer, ewing & #8217 sarcoma, extraosseous mucoid chondrosarcoma, fibroblastic bone, fibrodysplasia of bone, gall bladder or bile duct cancer, gastric cancer, gastroesophageal junction (GEJ) cancer, gestational trophoblastoma, germ cell tumor, head and neck cancer, islet cell tumor, kaposi sarcoma, kidney cancer, leukemia, liposarcoma/malignant lipoma, liver cancer, hepatocellular carcinoma liver cancer (HCC), lymphoma, non-hodgkin's lymphoma (NHL), lung cancer, small Cell Lung Cancer (SCLC), non-small cell lung cancer (NSCLC), myeloblastoma, melanoma, meningioma, merkel cell carcinoma, multiple endocrine tumor, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, renal metastasis carcinoma, coryne, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma head and neck cancer (SCCHN), gastric cancer, synovial sarcoma, testicular cancer, thymus cancer, thymoma, thyroid cancer and uterine cancer.

The pharmaceutical compositions of the present disclosure may be manufactured using methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, freeze-drying, and the like.

The solid oral compositions may be prepared by conventional mixing, filling or tabletting methods. For example, it can be obtained by the following method: the active compound is mixed with solid auxiliary materials, the resulting mixture is optionally milled, if desired with other suitable auxiliary materials, and the mixture is then processed to granules, giving a tablet or dragee core. Suitable excipients include, but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, and the like.

The pharmaceutical composition comprising the present application is physically introduced into a subject. Routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. In certain embodiments, administration is by a non-parenteral route, in certain embodiments, oral administration. Other non-parenteral routes include topical, epidermal or mucosal routes of administration, e.g., intranasally, vaginally, rectally, sublingually or topically. Administration may also be performed, for example, one, multiple times, and/or over one or more extended periods of time.

Advantageous effects

The application prepares a specific monoclonal antibody aiming at endoglin, which can effectively inhibit proliferation of colorectal cancer cells and can directly regulate expression of TGF-beta 1 at the downstream of endoglin by inhibiting activity of endoglin. The paclitaxel/albumin nanoparticle and the endoglin specific monoclonal antibody are combined, so that the tumor inhibition rate can be effectively improved, and a better treatment effect is achieved.

Drawings

FIG. 1 shows a diagram of the results of monoclonal antibody specificity identification

FIG. 2 proliferation effect of monoclonal antibodies on cancer cells

FIG. 3 influence of monoclonal antibodies on TGF- β1 Gene expression in cancer cells

FIG. 4 is a graph showing tumor-inhibiting effect of albumin-paclitaxel nanoparticles combined with monoclonal antibodies

Detailed Description

The present disclosure is further described below in connection with specific embodiments, however, these embodiments are merely illustrative of the present disclosure and do not limit the scope of the present disclosure. Also, the present disclosure is not limited to any particular preferred embodiment described herein. It should be understood by those skilled in the art that equivalent substitutions and corresponding modifications of the technical features of the present disclosure still fall within the protection scope of the present disclosure. The reagents used in the examples below are commercially available products, and the solutions may be formulated using techniques conventional in the art, unless otherwise specified.

EXAMPLE 1 preparation of endoglin monoclonal antibodies

Recombinant endoglin was used as an immunogen (Prospec, cat# CYT-525) and was prepared as a 1mg/ml solution.

Three SPF-class 6-week-old female Balb/c mice, labeled 1, 2, and 3 mice, were selected and first bred for two weeks to adapt to the environment. Three immunizations and one booster immunization were performed, respectively. Wherein the first immunization is that antigen 0.1 mg+0.15 ml of sterile physiological saline+0.3 ml of Freund's complete adjuvant, the subcutaneous multipoint injection is carried out after three weeks, the second immunization is carried out, the subcutaneous multipoint injection of antigen 0.1 mg+0.15 ml of sterile physiological saline+0.3 ml of Freund's incomplete adjuvant, the third immunization is carried out after three weeks, the subcutaneous multipoint injection of antigen 0.1 mg+0.15 ml of sterile physiological saline+0.3 ml of Freund's incomplete adjuvant is carried out, and after three weeks, the intraperitoneal injection of mice is carried out after the dilution of antigen 0.1mg with physiological saline 0.15 ml. Serum titers were determined after 5 days, and showed that the serum titers of mouse number 2 reached up to 1:25000, significantly higher than other mice. Therefore, spleen of mouse No.2 with highest serum titer was selected for cell fusionAnd (5) combining. The eyes were first dug out and serum was collected. Preparation of spleen cell suspension of immunized mice, counting living cells so that the final concentration of cell suspension is 5X 10 ⁷ /ml。

SP2/0 myeloma cells (1X 10) were mixed in a 5:1 ratio in a 50ml centrifuge tube ⁷ Individual/ml) and immunized murine spleen cells (5X 10) ⁷ 25ml of 1640 medium was added thereto, centrifuged at 1000rpm/min for 10min, and the supernatant was discarded. Lightly beating the bottom of the tube with fingers to disperse the precipitated cells, and placing the dispersed cells in a constant-temperature water bath box at 37 ℃ for heat preservation; 1ml of 50% PEG4000 was added in 1min while gently shaking; slowly add serum-free 1640 to dilute, slowly add serum-free 1640 (15 ml), terminate PEG action; centrifuging and precipitating at 1000rpm/min for 10min, and sucking supernatant; 1ml is added dropwise every 1min, and a centrifuge tube is slowly rotated while adding for 5min; the bottom of the tube was tapped, 1ml of RPMI-1640 medium containing 20% fetal bovine serum HAT was added, the mixture was mixed and counted, and the mixture was added to a 96-well cell culture plate containing feeder cells after 24 hours of culture. Setting culture plates with different cell numbers for ensuring the growth rate of the fused cells; placing 5% CO ₂ Culturing in a 37℃incubator, sucking 50. Mu.L of the culture supernatant from each well on days 4, 6, 8 and 10, adding 50. Mu.L of HAT culture solution, culturing with HT culture solution for one week on day 15, and then using normal culture solution. When the surviving cell clone occupies more than 1/10 area of the visual field, the liquid is changed, 100 mu l of culture liquid is sucked out, and positive cells are detected by indirect ELISA. The result shows that the total positive rate reaches 52.3% when the total positive rate is 71 holes. Cloning positive hybridoma cells, subcloning four times by adopting a limiting dilution method continuously until all cell holes with cell clone growth are positive, and considering subcloning to be successful. After four subcloning, two cells with strongest positivity were obtained, a11 and F15, respectively.

Preparation of monoclonal antibody: a6-8 week normal male Balb/c mouse was given by intraperitoneal injection of 0.5ml of liquid paraffin. After 7d, the A11 and F15 hybridoma cells in the logarithmic growth phase were collected, centrifuged at 1000rpm X5 min, and the supernatant was discarded, and the cell concentration was adjusted to 1X 10 with physiological saline sterilized by autoclaving in advance ⁶ Each mouse was intraperitoneally injected with 0.5ml of the cell suspension, about 5X 10, per ml ⁵ Individual impuritiesAnd (3) an hybridoma cell. The abdominal state of the mice is observed, a large amount of ascites can be generated after 10 days of inoculation, and the obvious increase of the abdomen of the mice can be seen. When the abdomen of the mice is large enough and the movement is inconvenient (about 14 days), the mice are sacrificed after the eyeballs are picked for blood collection. The epidermis was cut off with sterile scissors in an ultra clean bench, the peritoneum was exposed, the peritoneum was lifted with sterilized forceps and cut off at the uppermost end, and ascites was collected. After the ascites is collected, the residual ascites is sucked out by using normal saline, and the residual ascites is packaged separately and marked as ascites in washing liquid. After accumulated extraction and collection, centrifugation is carried out, after the floating fat on the uppermost layer is removed, the yellow clarified liquid is transferred into an EP pipe, the impurities on the bottom layer are discarded, and the mixture is purified by a column and utilizedThe Ultra-15 centrifugal filter removes substances smaller than 50KD so as to achieve the aim of improving the concentration of the antibody, and the titers of the concentrated A11 monoclonal antibody and F15 monoclonal antibody are respectively 1:160000 and 1:320000, so that the effect is better. SDS-PAGA electrophoresis experiments determine the purified sample bands, and the results show that the two target proteins have protein bands at 25KD and 55KD and have fewer hetero proteins.

EXAMPLE 2 Endoglin monoclonal antibody A11 specificity identification

Monoclonal antibody specificity assay: ELISA plates coated with the immunogen endoglin recombinant protein, BSA, AFP, CEA, and 1:4000 dilution of antibody were added. Negative and blank wells were set simultaneously. The enzyme-labeled instrument measures A450nm. P/N (a certain dilution of purified antibody A450 nm-blank A450 nm)/(negative control A450 nm-blank A450 nm) ]. Gtoreq.2.1 was positive. The results are shown in FIG. 1.

As can be seen from the specific identification result of the ELISA system shown in FIG. 1, the P/N value of the A11 monoclonal antibody against endoglin protein is obviously larger than 2.1, the monoclonal antibody is positive, and the detection result against other substances is negative, which indicates that the system can specifically detect target antigen protein, and also indicates that the A11 monoclonal antibody has better specificity.

Example 3 affinity and sequence identification of endoglin monoclonal antibody A11

Antibody binding kinetics rate constants were determined using biological membrane interferometry (BLI, forteBio oct RED 96). BLI test lineA biosensor (ForteBio) with AHC (Anti-hIgG Fc Capture) was used to Capture Anti-antibody endoglin monoclonal antibody (750 ng/mL), a 0.5nM shift (shift) was obtained, and then the biosensor was immersed in different concentrations (i.e., 0, 0.625, 1.25, 2.5, 5, 10, 20, and 40 nM) of recombinant endoglin protein in an electrophoresis buffer containing 0.1% Bovine Serum Albumin (BSA), 0.1% Tween-20, 250mM NaCl, 2.7mM KCl, 10mM Na ₂ HPO ₄ 1.8mM KH ₂ PO ₄ ). The rate constant was calculated using a curve fitting analysis of the binding reaction for 5 minutes binding and 15 minutes separation interaction time (curve fitting analysis;1:1 Langmuir model), resulting in an equilibrium dissociation constant (KD=Kd/Ka) value for the antibody as shown in FIG. 1.

TABLE 1 Endoglin monoclonal antibody A11 dissociation constant

Referring to Table 1, it can be seen that the A11 antibody has a lower KD value, representing that the antibody of the present application has a high affinity for endoglin.

Reverse transcription of total RNA of hybridoma cells into cDNA; amplifying antibody light chain variable region IgVL (κ) and heavy chain variable region VH sequences using degenerate primers and a Phusion kit; purifying the PCR amplification product by using a gel recovery kit; and connecting the amplified PCR product to a T vector according to the specification of a T vector cloning kit, converting the E.coli competent cells, amplifying the strain, extracting plasmids, and then carrying out DNA sequencing to obtain a monoclonal antibody variable region sequence. Sequencing results show that the amino acid sequence of the light chain variable region of the monoclonal antibody A11 is shown as SEQ ID NO.1, and the sequence of the heavy chain variable region is shown as SEQ ID NO. 2.

Example 4 Effect of endoglin monoclonal antibody A11 on proliferation and Gene expression of cancer cells

Human colorectal cancer cell line HCT116 was maintained supplemented with 10% foetal cattleSerum, 100U/ml penicillin and 100U/ml streptomycin in DMEM medium and at 37℃with 5% CO ₂ Is incubated in a saturated humid environment.

The above treated HCT116 was placed in 96-well plates with a density of 1X 10 per well ⁴ Cells, the culture plate is placed in the DMEM medium, and the culture plate contains 5% CO at 37 DEG C ₂ Is cultured for 24 hours until the cells adhere to the wall. A blank control group (without any treatment), a positive control LY364947 (10. Mu.g/ml, 10. Mu.l), a low-dose group of A11 (1. Mu.g/ml, 10. Mu.l), a medium-dose group of A11 (10. Mu.g/ml, 10. Mu.l), a high-dose group of A11 (100. Mu.g/ml, 10. Mu.l) were added to each of the above-mentioned adherent cells in this order, and culturing was continued for 48 hours according to the protocol. After the preset time is reached, the original culture medium is discarded, 20 mu l of CCK-8 reagent is added into each hole to avoid generating bubbles, cells are incubated with the culture medium containing the CCK-8 reagent for 2 hours, the color of the culture solution is developed, and the absorbance value (A value) of each hole at 450nm is measured by an enzyme-labeled instrument. Cell viability (%) (cell activity) was calculated according to the formula: (experimental group A value-blank group A value)/(control group A value-blank group A value). Times.100%, the proliferation of each group of cells is expressed as cell activity.

As shown in fig. 2, HCT116 cell activity after mab a 11-mediated drying was significantly reduced (P < 0.05) compared to the control group without any intervention, with no significant difference (P > 0.05) compared to the positive control group LY 364947; within the same intervention time, there was a statistical difference in HCT116 cell activity between each mab dose group, suggesting that there was a dose dependence of the mab on the cytotoxic effect of HCT116, with only (36.17 ±1.86)% of cell activity under a11 high dose group treatment conditions.

The cells from each group were collected and the lower pellet was washed 3 times with cold PBS and lysed in immunoprecipitation assay lysis buffer, and the cells were collected and sonicated. And (3) carrying out protein extraction on a small amount of supernatant after ice cracking, regulating the content of each group of proteins after measurement, mixing with a protein loading buffer solution, and carrying out Western blot experiment, wherein the whole sample lysate is separated through sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane at low temperature. Membranes were blocked with 5% bovine serum albumin for 1h at room temperature and incubated overnight at 4℃with shaking in GAPDH, TGF- β1 primary antibody. TBST is washed three times and then is placed in horseradish peroxidase-coupled secondary antibody at 3 ℃ for incubation for 1h, filter membrane color development is carried out by using enhanced chemiluminescent reagent with the secondary antibody, an analytical imager is used for photographing images, and related software QuantityOne-4.6.5 is used for analyzing data.

As shown in fig. 3, overall, the protein expression of TGF- β1 was significantly reduced (P < 0.05) in HCT116 cells following mab a 11-dry prognosis compared to the control group without any intervention; specifically, the TGF-beta 1 expression of HCT116 cells among each monoclonal antibody dose group has dose dependency, and under the treatment condition of the A11 high-dose group, the TGF-beta 1 expression amount is only (23 61+/-1.96)% relative to the blank control group. This also demonstrates that the a11 mab of the present application is capable of directly modulating the expression of TGF- β1 downstream thereof by inhibiting the activity of endoglin. The positive control had less effect on the expression level of TGF-. Beta.1.

EXAMPLE 5 paclitaxel albumin nanoparticle preparation

10mg of paclitaxel is dissolved in 2ml of acetone, a certain amount of human serum albumin is taken and dissolved in 15ml of water, the medicine carrying ratio (medicine/albumin molar ratio) is set to be 8:1 and 10:1, the paclitaxel acetone solution is dropwise added into the albumin aqueous solution under the stirring condition, the stirring is continued for 6min, the acetone is removed by rotary evaporation, the water is added to fix the volume to 20ml, the paclitaxel albumin nanoparticle (or called albumin paclitaxel nanoparticle) solution is obtained after high-pressure homogenization, the particle size is measured by adopting a particle size meter, and the result is shown in table 2.

TABLE 2 particle size of paclitaxel albumin nanoparticles with different drug loading ratios

Medicine-carrying ratio	Particle size nm
		8:1	156.4±1.6
10:1	164.8±2.0

From the results of the measured particle sizes, the particle sizes of the paclitaxel/albumin nanoparticles with different drug loading ratios are different.

Taking paclitaxel albumin nanoparticle with the molar ratio of 10:1, placing the paclitaxel albumin nanoparticle in a refrigerator at the temperature of 4 ℃ for a week in a dark place, measuring the particle size by a Markov particle analyzer to determine the stability, freeze-drying 2mL of nanoparticle solution, re-dissolving the nanoparticle solution in 1mL of organic solvent (methanol), swirling for 1min to denature and precipitate the protein, centrifuging at 10000rpm for 10min to discard the protein precipitate, properly diluting the supernatant with methanol, measuring the ultraviolet absorbance at the wavelength of 227nm to obtain the drug concentration, and calculating the drug loading DL (%) = (W1/W2) 100% according to the following formula, wherein W1 is the drug mass in the freeze-dried nanoparticle, and W2 is the total mass of the freeze-dried nanoparticle. The results show that the particle size of the particles which are placed for one week is hardly changed, the particles are uniformly distributed, and the stability is good. The drug loading of paclitaxel in the prepared nano-particles is 10.59%.

EXAMPLE 6 Albumin paclitaxel in combination with monoclonal antibody treatment experiments

Inoculating HCT116 cell strain of colorectal cancer cell line into sterile culture flask, adding into 100×10 strain containing 10% calf serum ³ U/L penicillin, 100X 10 ³ In RPMI1640 medium of U/L streptomycin, the temperature is constant at 37 ℃ and CO is 5% ₂ And culturing in a saturated humidity incubator, wherein the cells grow in a single-layer adherence manner, and are passaged for 1 time every 3-4 d, and are digested for 3min by 0.02% EDTA (ethylene diamine tetraacetic acid), and are blown into a cell suspension by a culture solution, and inoculated according to the required concentration. Building a tumor-bearing animal model: HCT116 cells were inoculated subcutaneously in the right proximal axilla of BALB/C nude mice at an inoculum size of 0.2ml cell suspension with a total cell count of 2X 10 ⁷ And each. Experimental grouping and processing: mice were randomly divided into 2 groups of 10 animals each, male and female halves.

(1) A11 antibody group: while inoculating HCT116 cells, injecting endoglin monoclonal antibody A11 μg,0.2ml by intraperitoneal injection, and boosting 1 time per week;

(2) Physiological saline group: while inoculating HCT116 cells, injecting physiological saline into the abdominal cavity, wherein the physiological saline is 0.2ml, and the physiological saline is strengthened 1 time per week;

(3) Albumin paclitaxel group: while inoculating HCT116 cells, 100 μg,0.2ml of paclitaxel albumin nanoparticle prepared in example 5 was injected intraperitoneally, 1 time a week;

(4) Albumin paclitaxel in combination with a11 mab treatment group: while inoculating HCT116 cells, 100 μg,0.1ml of paclitaxel albumin nanoparticle prepared in example 5 was injected intraperitoneally; antibody A11 μg,0.2ml was injected 1 week after 2h intervals;

(5) Positive control group: while the HCT116 cells were seeded, LY364947 100 μg,0.2ml, 1-week booster was injected intraperitoneally;

after 3 weeks of treatment, the size of the subcutaneous tumor was measured using vernier calipers. The tumor volume calculation method comprises the steps of measuring the maximum front and rear diameter (a), the left and right diameter (b) and the upper and lower diameter (c 1 times, three diameters are perpendicular to each other and record volume change by using a vernier caliper, wherein the tumor volume calculation method comprises the step of calculating the tumor volume (mln) of the mice by a multiplied by c multiplied by pi/6, and the tumor inhibition rate is equal to the average tumor volume of a physiological saline control group, the average tumor volume of a treatment group, and the average tumor volume of the physiological saline control group multiplied by 100%.

From the experimental results of fig. 4, it is shown that the a11 antibody group, the albumin-paclitaxel group, the positive control group, and the albumin-paclitaxel combined mab group significantly inhibit the growth of tumors compared with the physiological saline group, and the difference is significant (P < 0.05). From the results of fig. 4, it can also be seen that the albumin paclitaxel combined with the a11 monoclonal antibody has a synergistic better promoting therapeutic effect, and the tumor inhibition rate reaches (93.6±2.03%).

While the application has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains and as may be applied to the essential features hereinbefore set forth. Such modifications and variations are intended to fall within the scope of this disclosure and/or the appended claims. It is to be understood that the present disclosure is not limited to particular methods or compositions, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting of the application.

Claims

1. A monoclonal antibody a11 directed against endoglin, characterized by the amino acid sequence of the light chain variable region being:

DLVMTQTAPSVPVTPGESVSISCRSTDTRYTSMKKDCLYWFLQRPGQSPQLLIYSCVNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCIKILPYMCQFGSGTKLEIK

the amino acid sequence of the heavy chain variable region is:

VKPGGSLKLSCAASGERMQRNFMSWVRQTPDKRLEWVAIPACKQCMCYYPDSVKGRFTISRDQDKQTLYLQMSSLKSEDTAMYYCVGTTGNGWFQRWGQGTTVTVS。

2. use of the monoclonal antibody a11 of endoglin according to claim 1 for the preparation of a pharmaceutical composition for the treatment of colorectal cancer.

3. The use according to claim 2, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

4. Use of the monoclonal antibody a11 of endoglin and human serum albumin loaded with paclitaxel according to claim 1 for the preparation of a pharmaceutical composition for the treatment of colorectal cancer.

5. The use according to claim 4, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

6. The method according to claim 4, wherein the paclitaxel-loaded human serum albumin is prepared by dissolving paclitaxel in acetone, adding paclitaxel acetone solution dropwise into albumin aqueous solution under stirring according to a drug loading ratio of 10:1, stirring for 6min, removing acetone by rotary evaporation, and homogenizing under high pressure.