Mutants of recombinant immunoregulatory protein of Ganoderma lucidum and applications thereof
20190352345 ยท 2019-11-21
Inventors
Cpc classification
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
Mutants of recombinant immunoregulatory protein of Ganoderma lucidum (rLZ-8) and applications thereof are provided. It is found by the present invention that: an anti-EGFR (epidermal growth factor receptor) domain exists in a structure of the rLZ-8; particularly, the domain through positive potential characteristics thereof induces a killing effect to an abnormal EGFR-expressed tumor. Based on the above scientific discovery, with utilizing computational simulation technology, the mutants of the rLZ-8, having better antitumor effects, are obtained.
Claims
1-20. (canceled)
21: A protein mutant able to treat cancer, wherein: the mutant is obtained through artificial mutation of recombinant immunoregulatory protein of Ganoderma lucidum (rLZ-8); and related mutation locations comprise 6.sup.th-9.sup.th amino acids, 16.sup.th-20.sup.th amino acids, 41.sup.st-46.sup.th amino acids and 68.sup.th-74.sup.th amino acids.
22: The mutant, as recited in claim 21, wherein the mutant is obtained through single-point mutation at one of the mutation locations.
23: The mutant, as recited in claim 21, wherein the mutant is obtained through multi-point mutation at the mutation locations.
24: The mutant, as recited in claim 21, wherein: through binding with epidermal growth factor receptor (EGFR), the mutant is able to treat abnormal EGFR-expressed tumors and related diseases.
25: The mutant, as recited in claim 24, wherein the abnormal EGFR-expressed tumors and the related diseases comprise a liver cancer and tumors related to liver metastasis.
26: The mutant, as recited in claim 24, wherein the abnormal EGFR-expressed tumors comprise a lung cancer.
27: The mutant, as recited in claim 24, wherein the abnormal EGFR-expressed tumors comprise a breast cancer.
28: The mutant, as recited in claim 24, wherein the abnormal EGFR-expressed tumors comprise a rectocolonic cancer.
29: The mutant, as recited in claim 21, wherein: the mutant is able to be obtained through a recombinant expression system of gene engineering technology, the recombinant expression system comprises a prokaryotic expression system, a fungus expression system and a mammalian cell expression system.
30: The mutant, as recited in claim 21, wherein the mutant is able to be used independently or combined with other drugs.
31: The mutant, as recited in claim 30, wherein an effective dose of the mutant is adopted to serve as a treatment method for killing abnormal EGFR-expressed tumors.
32: The mutant, as recited in claim 21, wherein the mutant is able to serve as a main active material for preparing various formulations of drugs and for administration in various in vitro manners.
33: The mutant, as recited in claim 32, wherein the various drug formations comprise freeze-dried powders, injection solution, tablets and inhalants, which enable the main active material to reach a tumor location.
34: The mutant, as recited in claim 32, wherein the drug formations are able to be administrated in various manners, comprising oral administration, intravenous injection, intravenous drip and subcutaneous injection, which enable the mutant to reach a tumor location.
35: The mutant, as recited in claim 21, wherein the mutant is able to be used in any dose, and combined with any dose of other drugs.
36: A method for treating abnormal EGFR-expressed tumors in a subject, comprising steps of: administrating a composition which comprises a therapeutically effective amount of mutant to the subject, wherein the mutant is obtained through artificial mutation of recombinant immunoregulatory protein of Ganoderma lucidum (rLZ-8); and related mutation locations comprise 6.sup.th-9.sup.th amino acids, 16.sup.th-20.sup.th amino acids, 41.sup.st-46.sup.th amino acids and 68.sup.th-74.sup.th amino acids.
37: The method, as recited in claim 36, wherein the mutant is able to be applied in treating a liver cancer and tumors related to liver metastasis.
38: The method, as recited in claim 36, wherein the mutant is able to be applied in treating a lung cancer.
39: The method, as recited in claim 36, wherein the mutant is able to be applied in treating a breast cancer.
40: The method, as recited in claim 36, wherein the mutant is able to be applied in treating a rectocolonic cancer.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0060] The preferred embodiments of the present invention are merely for explaining the present invention, not for limiting the present invention in any way.
First Preferred Embodiment: Prokaryotic and Eukaryotic Expressions of Recombinant Immunoregulatory Protein of Ganoderma lucidum (rLZ-8) and Mutants Thereof
[0061] According to the first preferred embodiment, target protein represents rLZ-8 and mutants thereof, and target gene represents genes of rLZ-8 and mutants thereof.
[0062] Construction of Prokaryotic Expression Strain for rLZ-8 and Mutants Thereof and Expressions of rLZ-8 and Mutants Thereof Therein
[0063] According to the first preferred embodiment, Escherichia coli BL21(DE3) strain which is representative in a prokaryotic expression system is adopted for expression of the target protein. Codons of the target gene are optimized; then according to a direction of a T7 promoter of a pET-28a vector, DNA sequences of Xba I restriction site and ribosome binding site are added at a 5 terminal of the target gene, termination codon and Xho I restriction site are added at a 3 terminal of the target gene, for gene synthesis. Through ligating to the pET-28a prokaryotic expression vector by the two restriction sites of Xba I and Xho I, a recombinant expression plasmid is constructed. After being validated by sequencing, the BL21(DE3) strain is transformed through heat shock, positive clones are processed with kanamycin resistance screening, and a genetic engineering strain containing the recombinant expression vector is obtained. The obtained genetic engineering strain is placed at 37 C. and cultured until OD6000.6; the expression of the target protein is induced at 30 C. with isopropyl--D-thiogalactoside (IPTG); after collected thallus is disrupted, supernatant is collected through centrifugation for Western blot detection. Results shown in
[0064] Construction of Constitutive Pichia pastoris Strain for rLZ-8 and Mutants Thereof and Expressions of rLZ-8 and Mutants Thereof Therein
[0065] According to codon usage bias of Pichia pastoris, codons of the target gene are optimized; then according to a direction of a GAP promoter in a constitutive expression vector of pGAPZ A plasmid, DNA sequences of Xho I restriction site and Kex2 and Stel 3 hydrolytic enzyme sites are added at the 5 terminal of the target gene, the termination codon and the Xba I restriction site are added at the 3 terminal of the target gene, for gene synthesis. Through the restriction enzyme ligation method, a recombinant expression vector is constructed and is sequenced for validation. According to a specification of the pGAPZ A plasmid of Invitrogen Corporation, X33 Pichia pastoris is electrically transformed and processed with Zeocin resistance screening, and a constitutive recombinant X33 Pichia pastoris expression strain for the target protein is obtained. The obtained genetic engineering strain is placed at 30 C. and cultured until OD6006, and then is further cultured for 48 hours to enable the target protein to secrete expression; supernatant is collected through centrifugation for Western blot detection. Results shown in
[0066] Expressions of rLZ-8 and Mutants Thereof in Mammalian Cell
[0067] The expression of the target protein is made with FreeStyle MAX CHIO Expression System of Invitrogen Corporation. Codons of the target gene are optimized; and, Asc I and Xho I restriction sites are added for gene synthesis.
[0068] Through the restriction enzyme ligation method, the target gene is transferred into a pSecTag2 A expression vector, and a recombinant target gene expression vector is obtained. Through FreeStyle MAX Reagent, the recombinant target protein expression vector is transfected into a CHO-S cell and then processed with hygromycin B resistance screening, so as to obtain a recombinant target protein cell strain. The obtained cell strain secretes and expresses the target protein at a condition of 37 C., 8% CO.sub.2 and 135 rpm; supernatant is collected through centrifugation for Western blot detection, thereby observing the expression of the target protein. Results shown in
Second Preferred Embodiment: Preparation of rLZ-8 and Mutants Thereof Through Fermentation Engineering Technology
[0069] Construction and expression screening of the rLZ-8 and the mutants thereof are made with adopting the same expression vector and strain. According to the second preferred embodiment, Pichia pastoris is adopted for genome recombination and construction; the obtained strains after early shake-flask culture is screened, for screening out an engineering strain can be applied in fermentation tank scale preparation. In the following description, target protein represents rLZ-8 and mutants thereof.
[0070] Detailed Process of Fermentation Technology
[0071] Working seed recovery: taking out constitutive working seeds from a refrigerator of 80 C.; slowly unfreezing at a room temperature; adding 10 L working seeds into a 100 mL shake flask containing 10 mL YPD (yeast peptone dextrose) liquid medium; and, shake-culturing for 24 hours at a condition of 28.5 C. and 225 rpm.
[0072] Constitutive seed solution culture: keeping an OD (optical density) value of a bacteria solution of the recovered working seeds at 2-6; taking 1 mL bacteria solution, and adding into a 2 L conical flask containing 400 mL YPD medium; and, shake-culturing at a condition of 28.5 C. and 225 rpm, until the OD value of the bacteria solution is approximately equal to 6, wherein the obtained seed solution can be applied in fermentation tank culture.
[0073] Culture in Fermentation Tank:
[0074] {circle around (1)} calibrating equipment, further comprising steps of: calibrating a pH electrode of the fermentation tank (processing pH6.86 and pH4.00 calibrations with pH calibration liquid before sterilizing the fermentation tank); calibrating a dissolved oxygen electrode (after sterilizing and cooling the medium in the fermentation tank, calibrating through stirring for more than 30 minutes with a maximum ventilation rate, an optimum temperature, an optimum pH value and a highest rotation speed of the culture condition); and calibrating a flow of a peristaltic pump;
[0075] {circle around (2)} preparing 3.5 L BSM (basic salt medium), and adding into a 7.5 L fermentation tank; then adding 4 mL antifoaming agent (can be sterilized) into the fermentation tank; sterilizing the medium, the fermentation tank and pipelines at a temperature of 121 C. and a high pressure for 30 minutes;
[0076] {circle around (3)} after sterilizing and cooling the medium in the fermentation tank, setting following parameters, wherein: temperature is set to be 29 C., rotation speed is set to be 800 rpm, ventilation rate is set to be 8 L/min and the pH value of the medium is set to be 6.0; and for sterile operation, further adding 0.22 m filtering membrane for degerming, 8 mL Biotin and 17 mL PTM1 microelements;
[0077] {circle around (4)} through the sterile operation, connecting an inoculation port of the fermentation tank with a seed solution addition port; adding the above 400 mL YPD bacteria solution in the 2 L conical flask into the fermentation tank through the peristaltic pump, wherein a total volume in the fermentation tank is 3.9 L; fermenting and culturing; wherein: for the fermentation tank, a stirring speed is set to be 800 rpm, a temperature is set to be 29 C., and a DO (dissolve oxygen) value is kept at about 20%; when necessary, pure oxygen can be introduced into the fermentation tank;
[0078] {circle around (5)} taking a sample once every 6 hours; measuring OD600, a cell wet weight and a cell dry weight, and analyzing a growth situation of yeast; observing the bacterium solution by naked eyes and under a microscope; removing bacterial contaminants; and collecting supernatant;
[0079] {circle around (6)} adding PTM1 microelements into cooled 500/glycerinum after high-pressure sterilization, and uniformly mixing, wherein 12 mL microelements are added for every liter of glycerinum; and then adding the glycerinum into the medium through the peristaltic pump, wherein: during an addition process of glycerinum, the DO value cannot be lower than 20%; and an oxygen supply can be ensured through increasing the stirring speed and introducing the pure oxygen;
[0080] {circle around (7)} sampling strains of every time point, and processing with SDS-PAGE electrophoresis, so as to measure an expression quantity thereof; analyzing the constitutive rLZ-8, and fermenting the supernatant; and
[0081] {circle around (8)} when fermenting for 126 hours, adding 20 mL phosphoric acid into the fermentation tank, so that a certain amount of ammonium hydroxide enters the culture solution and supports fermentation as a nitrogen source; when fermenting for 150 hours, stopping culturing.
[0082] Results and Analysis
[0083] It can be seen from
Third Preferred Embodiment: Separation and Purification Process of rLZ-8 and Mutants Thereof
[0084] 1. Experimental Methods
[0085] Through many experiments, fermentation solution is successively processed with microfiltration, ultrafiltration, cation exchange chromatography and hydrophobic interaction chromatography, for separation and purification of the rLZ-8 and the mutants thereof. Because physicochemical property differences between the rLZ-8 and the rLZ-8 mutants are relatively small, a purification process of the rLZ-8 is similar as that of the rLZ-8 mutants, and the rLZ-8 mutants can be successively prepared through the purification process of the rLZ-8. However, the purification process of the rLZ-8 is not the best separation and purification process of every mutant. The third preferred embodiment does not optimize the purification process of any mutant, but merely describes the purification process of the rLZ-8 in detail as follows.
[0086] {circle around (1)} Microfiltration Process of Fermentation Solution
[0087] Washing a well-preserved 0.7 m.sup.2 hollow fiber microfiltration column with 3 L injection water, so as to remove 50 ppm sodium hypochlorite protective solution in the microfiltration column; washing the microfiltration column with 5 L 0.5M sodium hydroxide solution containing 2000 ppm sodium hypochlorite; and, measuring a pH value and an endotoxin content.
[0088] Microfiltration: measuring a conductance and a pH value of the supernatant after centrifugation, and processing with the microfiltration column, wherein: the peristaltic pump has a rotation speed of 40 rpm/min and is at first gear; and, upper and lower pressure gauges are both controlled to be <0.1 Mpa. After microfiltration, washing the microfiltration column with injection water; collecting column washing liquid, and mixing with the sample, wherein: an addition amount of the total volume is at least 0.25 times the volume of the original fermentation solution.
[0089] Preparing 5 L 0.5M sodium hydroxide solution containing 2000 ppm sodium hypochlorite; washing the microfiltration column with the sodium hydroxide solution; washing the microfiltration column with 10 L injection water until the pH value thereof is neutral; and preserving the microfiltration column with a certain amount of 50 ppm sodium hypochlorite.
[0090] Fermenting and centrifuging the supernatant; after microfiltration, respectively sampling for SDS electrophoresis detection; fermenting and centrifuging the supernatant, and detecting the bacterial endotoxin.
[0091] {circle around (2)} Ultrafiltration Process of Fermentation Solution
[0092] Washing a well-preserved 0.6 m.sup.2 filtering membrane with 2 L injection water, so as to remove 0.1M sodium hydroxide protective solution in the filtering membrane; washing the filtering membrane with 3 L 1M sodium hydroxide; then washing with acid liquid until the pH is neutral; measuring the pH value and a water flux.
[0093] Ultrafiltration: concentrating and desalting the sample after microfiltration by a tangential flow filtration system, wherein: a CONC-DIFI-CONC program is set and selected for concentrating; 20 L injection water is added for desalting; and according to a protein content, the sample is determined to be concentrated 5 times.
[0094] After completing ultrafiltration, a final pH value and a final conductance of the sample are measured.
[0095] Washing and preserving the filtering membrane: after processing the sample by the filtering membrane, washing the filtering membrane with 5 L 1M sodium hydroxide until reaching an original water flux and becoming neutral; and preserving the filtering membrane with 0.1M sodium hydroxide alkali liquid.
[0096] The sample after ultrafiltration is processed with SDS electrophoresis detection.
[0097] {circle around (3)} Cation Chromatographic Purification Process
[0098] Adding 5 L collected target protein after ultrafiltration into a mobile phase A mother solution (namely 0.02M anhydrous sodium acetate solution with pH of 3.6) of cation chromatography, and adjusting a pH value with glacial acetic acid until the pH becomes 3.6; filtering through a plate filter with a 0.22 m filtering membrane; and preparing for sample loading.
[0099] Equilibrating chromatographic column: filling BPG (140/500) column with 1.5 L filler of Capto S, washing the column with 6 L injection water, until 20% ethyl alcohol and 0.2M sodium acetate protective solution in the column are completely removed; measuring endotoxin, wherein the chromatographic column can be used only when the endotoxin measurement result is smaller than 0.25 EU/ml; equilibrating six column volumes with the mobile phase A, wherein a flow speed is 30 L/h and an instrument protection pressure is set to be 2 bar; measuring a conductance, until the pH value and the conductance reach corresponding values of the mobile phase A.
[0100] Sample loading: loading the sample after filtration through an A3 pump, wherein a flow speed is 20 L/h; after completing loading, collecting a flow-through peak, and measuring a pH value, a conductance range and an absorption value range of 280 nm ultraviolet; eluting a non-combined peak with 8 L phase A buffer solution at a flow speed of 30 L/h, until the non-combined peak reaches an original ultraviolet absorption value; collecting the non-combined peak, and measuring a conductance range.
[0101] Eluting: according to the protection pressure, setting the flow speed, and conducting the stage eluting; washing with 12 L 10% mobile phase B (namely a solution containing 0.02M anhydrous sodium acetate and 1.5M sodium chloride with a pH value of 3.6), and then washing with 6 L 30% mobile phase B, respectively until an eluting peak reaches the original ultraviolet absorption value; collecting the eluting peaks of every stage, and measuring the conductance range and the absorption value range of 280 nm ultraviolet; taking out a certain volume of the collected eluting peak for SDS electrophoresis and high-performance liquid chromatography (HPLC) measurement; wherein: for every stage, if a purity of the eluting peak containing the target protein is lower than 50%, the eluting peak is abandoned.
[0102] {circle around (4)} Hydrophobic Chromatographic Purification Process
[0103] Sample processing: adding 2 L collected target protein after cation ultrafiltration into an HIC (hydrophobic interaction chromatography) phase B mother solution by volume, and adjusting a pH value with acetic acid, until the pH value becomes 5.0; then filtering through the plate filter with the 0.22 m filtering membrane; and preparing for sample loading.
[0104] Equilibrating chromatographic column: filling the BPG (140/500) column with 1.0 L filler of Capto Phenyl; washing the column with 5 L injection water, until 20% ethyl alcohol protective solution in the column is completely removed; washing with 6 L 1M sodium hydroxide alkali liquid, and then washing with the injection water, until the pH value is neutral; processing with endotoxin detection, wherein the column can be used only when passing the detection; equilibrating eight column volumes with the mobile phase B, wherein a flow speed is 30 L/h and an instrument protection pressure is set to be 2 bar.
[0105] Sample loading: loading the sample after filtration through a sample loading pump, wherein a flow speed is 20 L/h, after completing loading, collecting a flow-through peak, and measuring a pH value and a conductance range; eluting a non-combined peak with 6 L mobile phase B buffer solution at a flow speed of 20 L/h, until the non-combined peak reaches the original ultraviolet absorption value; collecting the non-combined peak, measuring a conductance range and electrophoresis.
[0106] Eluting: setting the flow speed to be 30 L/h, and conducting the stage eluting; washing with 25 L 60% phase B, and then washing with 6 L 30% phase B, respectively until an eluting peak reaches the original ultraviolet absorption value; collecting the eluting peaks of every stage, and measuring the conductance range and the absorption value range of 280 nm ultraviolet; taking out a certain volume of the collected eluting peak for SDS electrophoresis and HPLC measurement; wherein: for every stage, if a purity of the eluting peak containing the target protein is lower than 90%, the eluting peak is abandoned.
[0107] 2. Results and Analysis
[0108] The rLZ-8 purification results are verified as follows. The electrophoretic results shown in
[0109] The same samples are further processed with liquid phase detection. For the 30% mobile phase B eluting sample (shown in
Fourth Preferred Embodiment: Endocytosis of rLZ-8 in Way of Macropinocytosis
[0110] 1. Experimental Methods
[0111] With an ultrahigh resolution imaging system, the endocytosis way of the rLZ-8 is screened through an inhibitor experiment and a co-localization experiment.
[0112] {circle around (1)} Endocytosis Morphology Observation of rLZ-8
[0113] The rLZ-8 is labeled by an Alexa Fluor 568 fluorescent probe; then 10 g/mL fluorescent labeled rLZ-8 acts on Hep G2 cells for 3 hours; whether the rLZ-8 enters the cell is observed with the ultrahigh resolution imaging system; and obtained images are reconstructed, fitted and further analyzed through an image analysis software of Imaris.
[0114] {circle around (2)} Inhibitor Screening
[0115] Inhibitors of different internalization ways are selected, respectively EIPA (macropinocytosis inhibitor), Wortmannin/LY294002 (P13K inhibitor), Nystatin+Progesterone (caveolae inhibitor) and Chlorpromazine (clathrin inhibitor); the inhibitors respectively act on the Hep G2 cells, and inhibitory effects of the inhibitors on rLZ-8 endocytosis are observed.
[0116] {circle around (3)} Influence of rLZ-8 Endocytosis on Microfilaments During the macropinocytosis process, the microfilaments play an important role that can be disrupted and recombined. Thus, through observing the influence of rLZ-8 endocytosis on the microfilaments, whether the endocytosis is in the way of macropinocytosis is judged.
[0117] {circle around (4)} Co-Localization Research of rLZ-8 Respectively with Macropinocytosis Markers of Dextran and BSA (Bovine Serum Albumin)
[0118] Dextran and BSA are classic markers of macropinocytosis. In order to observe the endocytosis way of the rLZ-8, the rLZ-8 and the Dextran/BSA together act on the Hep G2 cells, so as to observe whether a co-localization phenomenon occurs.
[0119] 2. Experimental Results
[0120] The endocytosis way of the rLZ-8 is researched according to the above methods, and results thereof are showed in
[0121] It can be known from
Fifth Preferred Embodiment: Endocytosis of rLZ-8 after Binding with EGFR (Epidermal Growth Factor Receptor)
[0122] 1. Experimental Methods
[0123] Considering that the high internalization degree of the rLZ-8 may be caused by the internalization after binding with a receptor on the cytomembrane, the rLZ-8 and the receptor on the cytomembrane are screened. Relationships between the different receptors on the cytomembrane and the tLZ-8 are measured through an immunofluorescence method. Fluorescent labels are made respectively on antibodies of EGFR, c-Met, PDGFR, N-cadherin and LDLR, and co-localization situations thereof with the rLZ-8 are respectively observed.
[0124] 2. Experimental Results
[0125] The immunofluorescence results are showed in
Sixth Preferred Embodiment: No Fusion Between rLZ-8 after Internalization and Lysosome
[0126] 1. Experimental Methods
[0127] During the normal macropinocytosis internalization process, the absorbed material through the early endosome and the late endosome will fuse with the lysosome and then be degraded. Thus, whether the rLZ-8 experiences the above process after internalization endocytosis is observed. Through the immunofluorescence method, Rab5 (early endosome marker), Rab7 (late endosome marker) and Lamp1 (lysosome marker) are made with fluorescent labels, and the co-localization phenomena between the markers and the rLZ-8 are observed.
[0128] 2. Experimental Results
[0129] The co-localization phenomena respectively between the rLZ-8 and the Rab5, between the rLZ-8 and the Rab7, and between the rLZ-8 and the Lamp1 are showed in
[0130] It can be known from
Seventh Preferred Embodiment: Tumor Cell Death Caused by Occupation of Large Amount of Membrane after Internalization of rLZ-8
[0131] 1. Experimental Methods
[0132] With the ultrahigh resolution imaging system, the whole change process of the cell after the internalization of the rLZ-8 is observed in real time; and, combined with the above researches, the reason why the rLZ-8 causes the tumor cell death is judged.
[0133] 2. Experimental Results
[0134] Real-time dynamic observation results are showed in
[0135] If the above mechanism is correct, when the rLZ-8 stops entering the cell, the cell will not continue shrinking and dying.
[0136] As shown in
Eighth Preferred Embodiment: Influences of rLZ-8 Mutants on Model Mice with Orthotopic Transplantation Tumor of Human Liver Cancer Cells
[0137] 1. Experimental Methods
[0138] {circle around (1)} Experimental Materials and Reagents
[0139] NOG Mice of 6-8 weeks old are selected. The mice have a weight of 18-22 g and are bought from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice are fed under an SPF environment at Northeast Normal University; during the experiment, the temperature is controlled at (202) C., the humidity is controlled at 48%, and the mice are illuminated every 12 hours alternately. The experimental reagents comprise: human liver cancer Hep G2 cell strain, DMEM (Dulbecco's modified eagle medium), fetal bovine serum, PBS (phosphate buffer saline), pancreatin-EDTA, DMSO (dimethyl sulfoxide), 0.05% trypsin, rLZ-8, rLZ-8 mutants, and sorafenib as the positive control drug.
[0140] {circle around (2)} Experimental Equipment and Apparatus
[0141] The experimental equipment and apparatus comprise: carbon dioxide constant-temperature incubator, full-automatic cell counting analyzer, table centrifuge, electronic scales, micropipette, culture flask, pipette, fixator, injector, scissor and tweezers.
[0142] {circle around (3)} Experimental Procedure
[0143] The DMEM containing 10% fetal bovine serum is selected for the human liver cancer Hep G2 cells, and then is placed in 5% CO.sub.2 constant-temperature incubator at 37 C. for culturing. The Hep G2 cells at the logarithmic phase are digested by pancreatin and thereafter washed with serum-free DMEM; the living cell concentration is adjusted to 1108 cells per milliliter; 20 L cell suspension (with a ratio of DMEM to Matrigel being 1:1) is taken and inoculated at the liver of the NOG mice, so as to construct the orthotopic transplantation tumor model of Hep G2 liver cancer cells in the NOG mice. Two weeks later, the model mice are divided into groups randomly, and each group has 10 mice. The administration method is caudal vein injection, once a day, for consecutive 28 days (qd28, i.v.). The negative control group is injected with normal saline; the groups of the rLZ-8 and the mutants thereof are administrated with a dose of 0.5 mg/kg; the sorafenib control group is administrated with a dose of 50 mg/kg. During the experiment, if the mouse dies, the dead mouse is weighted on the same day, and the tumor tissue is taken out to be weighted. For the living mouse after administration for 28 days, the mouse is killed, and then the tumor tissue is taken out to be weighted. The death time and death number of the mice in each group are recorded in detail; the survival conditions of the mice of the experimental groups and the control groups are analyzed, and the survival rate is calculated, wherein the survival rate is obtained through subtracting the death number of the mice from the total number of the mice, and then dividing the difference by the total number of the mice.
[0144] 2. Experimental Results
[0145] {circle around (1)} Analysis of Influences of rLZ-8 Mutants on Mice Weight
[0146] After consecutive administration with caudal vein injection for 28 days, weights of the mice of the negative control group and the sorafenib control group are obviously decreased; the weights of the mice of the rLZ-8 control group have no obvious change, for the rLZ-8 mutants, compared with the weights before experiment, the weights of the mice of the groups of rLZ-8 (K16A/S18A/K41A/D45A), rLZ-8 (K16A/K41A), rLZ-8 (D45A) and rLZ-8 (S18A) are obviously decreased, while the weights of the mice of the groups of rLZ-8 (D70K) and rLZ-8 (L17K/D70K) are slightly increased. The above results indicate that: the rLZ-8 and the mutants thereof are able to adjust the physical condition of the mice; and the combinations of the sorafenib respectively with the rLZ-8 (D70K) and the rLZ-8 (L17K/D70K) also show the relatively good effect.
TABLE-US-00001 TABLE 1 Influences of rLZ-8 mutants on weight of model mice with transplantation tumor of Hep G2 liver cancer cells Mice weight before experiment Mice weight after Experimental group (g) experiment (g) Negative control group 20.4 1.3 17.4 1.8 Sorafenib control group 19.8 1.0 19.6 1.6 rLZ-8 control group 19.9 0.9 19.8 1.4 rLZ-8 (K16A/S18A/K41A/D45A) 20.2 1.1 17.3 1.5 rLZ-8 (K16A/K41A) 19.8 0.8 17.2 1.3 rLZ-8 (D45A) 70.4 1.2 17.4 1.6 rLZ-8 (S18A) 20.3 1.3 17.6 1.4 rLZ-8 (R9A) 20.1 0.8 19.3 1.3 rLZ-8 (D70K) 20.2 1.2 20.7 1.7 rLZ-8 (L17K/D70K) 20.7 1.4 21.0 1.8 rLZ-8 (D20H/D68K) 20.0 1.3 20.1 1.5 rLZ-8 (D20H) 19.5 1.2 19.7 1.7 rLZ-8 (L17K) 20.2 1.3 20.1 1.4 rLZ-8 (K41D/K46E/K74E) 20.1 1.1 20.0 1.2 rLZ-8 (K46E/K74E) 20.8 1.2 20.1 1.7 rLZ-8 (K46E) 19.7 1.0 19.4 1.8 rLZ-8 (D70K) + sorafenib 20.2 1.2 17.4 1.2 rLZ-8 (L17K/D70K) + sorafenib 20.4 0.9 17.0 0.9
[0147] {circle around (2)} Influences of rLZ-8 Mutants on Tumor Weight of Mice
[0148] For the tumor weight, after weighting the stripped tumor, the mean value of the tumor weights of each group is calculated. It can be seen that: after administration for 28 days, compared with the negative control group, the groups of rLZ-8 (K16A/S18A/K41A/D45A), rLZ-8 (K16A/K41A), rLZ-8 (D45A) and rLZ-8 (S18A) have no obvious tumor weight difference and have nearly no tumor inhibitory effect; compared with the rLZ-8 control group, the groups of rLZ-8 (D70K) and the rLZ-8 (L17K/D70K) have the obviously increased tumor inhibitory effects; the other mutants have the tumor inhibitory effect nearly equal to or slightly better that that of the rLZ-8 control group. The above results indicate that: K16A/S18A/K41A/D45 is the key amino acids that influence the rLZ-8 antitumor activity; and on the rLZ-8 structure, the mutants near the above antitumor activity domain with the increased positive potential or the decreased surrounding negative potential show the better antitumor effect. The combinations of the sorafenib respectively with the rLZ-8 (D70K) and the rLZ-8 (L17K/D70K) also show the relatively good effect.
TABLE-US-00002 TABLE 2 Influences of rLZ-8 mutants on tumor weight of mice with Hep G2 liver cancer transplantation tumor Mice tumor weight after Experimental group experiment (g) Negative control group 1.43 0.87 Sorafenib control group 0.82 0.37 rLZ-8 control group 0.62 0.53 rLZ-8 (K16A/S18A/K41A/D45A) 1.62 0.72 rLZ-8 (K16A/K41A) 1.46 0.93 rLZ-8 (D45A) 1.44 0.80 rLZ-8 (S18A) 1.50 0.71 rLZ-8 (M1A/R9A/D100A) 0.78 0.67 rLZ-8 (R9A) 0.65 0.43 rLZ-8 (D70K) 0.35 0.21 rLZ-8 (L17K/D70K) 0.42 0.23 rLZ-8 (D20H/D68K) 0.51 0.27 rLZ-8 (D20H) 0.60 0.31 rLZ-8 (L17K) 0.58 0.33 rLZ-8 (K41D/K46E/K74E) 1.25 0.53 rLZ-8 (K46E/K74E) 0.69 0.42 rLZ-8 (K46E) 0.75 0.44 rLZ-8 (D70K) + sorafenib 0.59 0.23 rLZ-8 (L17K + D70K) + sorafenib 0.52 0.18
[0149] {circle around (3)} Influences of rLZ-8 Mutants on Mice Survival Rate
[0150] The experimental results indicate that: the mutants with the increased positive potential near the rLZ-8 antitumor activity domain, such as the rLZ-8 (D70K) and the rLZ-8 (L17K/D70K), have a certain effect on lengthening the survival time of the mice.
TABLE-US-00003 TABLE 3 Influences of rLZ-8 mutants on survival rate of mice with Hep G2 liver cancer transplantation tumor Survival rate of mice after Experimental group experiment (%) Negative control group 30 Sorafenib control group 50 rLZ-8 control group 60 rLZ-8 (K16A/S18A/K41A/D45A) 30 rLZ-8 (K16A/K41A) 20 rLZ-8 (D45A) 50 rLZ-8 (S18A) 30 rLZ-8 (M1A/R9A/D100A) 60 rLZ-8 (R9A) 50 rLZ-8 (D70K) 80 rLZ-8 (L17K/D70K) 90 rLZ-8 (D20H/D68K) 70 rLZ-8 (D20H) 60 rLZ-8 (L17K) 70 rLZ-8 (K41D/K46E/K74E) 40 rLZ-8 (K46E/K74E) 60 rLZ-8 (K46E) 60 rLZ-8 (D70K) + sorafenib 60 rLZ-8 (L17K + D70K) + sorafenib 50
[0151] In conclusion, compared with the rLZ-8, the mutants with the increased positive potential near the rLZ-8 antitumor activity domain, such as the rLZ-8 (D70K) and the rLZ-8 (L17K/D70K), show the better antitumor activity on the model mice with the orthotopic transplantation tumor of the human liver cancer cells, and have the certain effect on lengthening the survival time of the tumor-bearing mice. The combinations of the mutants with the sorafenib also show the relatively good effect.
Ninth Preferred Embodiment: Influences of rLZ-8 Mutants on Model Mice with Orthotopic Transplantation Tumor of Human Lung Cancer Cells
[0152] 1. Experimental Methods
[0153] {circle around (1)} Experimental Materials and Reagents
[0154] The human lung cancer A549 cell strain is selected. The NOG Mice of 6-8 weeks old are selected. The mice have a weight of 18-22 g and are bought from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice are fed under the SPF environment at Northeast Normal University; during the experiment, the temperature is controlled at (202) C., the humidity is controlled at 48%, and the mice are illuminated every 12 hours alternately. The experimental reagents comprise: DMEM, fetal bovine serum, PBS, pancreatin-EDTA, DMSO, 0.05% trypsin, rLZ-8, and rLZ-8 mutants.
[0155] {circle around (2)} Experimental Equipment and Apparatus
[0156] The experimental equipment and apparatus comprise: carbon dioxide constant-temperature incubator, full-automatic cell counting analyzer, table centrifuge, electronic scales, micropipette, culture flask, pipette, fixator, injector, scissor and tweezers.
[0157] {circle around (3)} Grouping and Administration
[0158] The DMEM containing 10% fetal bovine serum is selected for the human lung cancer A549 cells, and then is placed in the CO.sub.2 constant-temperature incubator at 37 C. for culturing. The A549 cells at the logarithmic phase are digested by pancreatin and thereafter washed with serum-free DMEM; the living cell concentration is adjusted to 1108 cells per milliliter; 20 L cell suspension is taken and inoculated at the lung of the NOG mice; two weeks later, the orthotopic transplantation tumor model of A549 lung cancer cells is constructed in the NOG mice.
[0159] 2. Experimental Results
[0160] {circle around (1)} Analysis of Influences of rLZ-8 Mutants on Mice Weight
[0161] The rLZ-8 and the mutants thereof are able to adjust the physical condition of the model mice with the A549 lung cancer transplantation tumor, wherein: the mutants with the increased positive potential near the rLZ-8 antitumor activity domain have the certain positive adjustment effect on the increase of the mice weight.
TABLE-US-00004 TABLE 4 Influences of rLZ-8 mutants on weight of model mice with A549 lung cancer transplantation tumor Mice weight before experiment Mice weight after Experimental group (g) experiment (g) Negative control group 21.4 1.4 17.5 1.8 rLZ-8 control group 21.0 1.1 20.8 1.5 rLZ-8 (K16A/S18A/K41A/D45A) 20.2 1.2 17.6 1.4 rLZ-8 (K16A/K41A) 19.9 0.8 17.4 1.5 rLZ-8 (D45A) 20.2 1.1 17.5 1.5 rLZ-8 (S18A) 20.5 1.3 17.8 1.6 rLZ-8 (R9A) 20.2 0.7 20.0 1.4 rLZ-8 (D70K) 20.0 1.2 21.1 1.2 rLZ-8 (L17K/D70K) 20.1 1.4 21.2 1.5 rLZ-8 (D20H/D68K) 20.0 1.5 20.6 1.7 rLZ-8 (D20H) 19.7 1.3 19.9 1.7 rLZ-8 (L17K) 20.1 1.2 20.1 1.4 rLZ-8 (K41D/K46E/K74E) 20.7 1.3 19.1 1.3 rLZ-8 (K46E/K74E) 20.3 1.0 20.0 1.5 rLZ-8 (K46E) 20.7 1.3 20.4 1.6
[0162] {circle around (2)} Influences of rLZ-8 Mutants on Mice Tumor Weight
[0163] It can be seen that: after administration for 28 days, compared with the negative control group, the groups of rLZ-8 (K16A/S18A/K41A/D45A), rLZ-8 (K16A/K41A), rLZ-8 (D45A) and rLZ-8 (S18A) have no obvious tumor weight difference and have nearly no tumor inhibitory effect; compared with the rLZ-8 control group, the groups of rLZ-8 (D70K) and rLZ-8 (L17K/D70K) have the obviously increased tumor inhibitory effect; the other mutants have the tumor inhibitory effect nearly equal to or slightly better than that of the rLZ-8 control group.
TABLE-US-00005 TABLE 5 Influences of rLZ-8 mutants on tumor weight of model mice with A549 lung cancer transplantation tumor Mice tumor weight after Experimental group experiment (g) Negative control group 1.23 0.74 rLZ-8 control group 0.60 0.45 rLZ-8 (K16A/S18A/K41A/D45A) 1.22 0.83 rLZ-8 (K16A/K41A) 1.26 0.77 rLZ-8 (D45A) 1.28 0.68 rLZ-8 (S18A) 1.20 0.70 rLZ-8 (R9A) 0.69 0.53 rLZ-8 (D70K) 0.36 0.28 rLZ-8 (L17K/D70K) 0.40 0.26 rLZ-8 (D20H/D68K) 0.49 0.28 rLZ-8 (D20H) 0.51 0.32 rLZ-8 (L17K) 0.53 0.30 rLZ-8 (K41D/K46E/K74E) 1.20 0.62 rLZ-8 (K46E/K74E) 0.63 0.41 rLZ-8 (K46E) 0.65 0.31
[0164] {circle around (3)} Influences of rLZ-8 Mutants on Mice Survival Rate
[0165] Compared with the rLZ-8, the mutants with the increased positive potential near the rLZ-8 antitumor activity domain, such as the rLZ-8 (D70K) and the rLZ-8 (L17K/D70K), can obviously lengthen the survival time of the model mice with the A549 lung cancer transplantation tumor.
TABLE-US-00006 TABLE 6 Influences of rLZ-8 mutants on survival rate of model mice with A549 lung cancer transplantation tumor Mice survival rate after Experimental group experiment (%) Negative control group 20 rLZ-8 control group 50 rLZ-8 (K16A/S18A/K41A/D45A) 10 rLZ-8 (K16A/K41A) 20 rLZ-8 (D45A) 30 rLZ-8 (S18A) 30 rLZ-8 (R9A) 50 rLZ-8 (D70K) 80 rLZ-8 (L17K/D70K) 80 rLZ-8 (D20H/D68K) 70 rLZ-8 (D20H) 70 rLZ-8 (L17K) 70 rLZ-8 (K41D/K46E/K74E) 40 rLZ-8 (K46E/K74E) 50 rLZ-8 (K46E) 40
Tenth Preferred Embodiment: Influences of rLZ-8 Mutants on Model Mice with Orthotopic Transplantation Tumor of Human Breast Cancer Cells
[0166] 1. Experimental Methods
[0167] {circle around (1)} Experimental Materials and Reagents
[0168] The experimental materials and reagents comprise: human breast cancer MCF7 cell strain, 17 estradiol sustained release tablet, BALB/c female nude mice, DMEM, fetal bovine serum, PBS, pancreatin-EDTA, DMSO, 0.05% trypsin, rLZ-8, and rLZ-8 mutants.
[0169] {circle around (2)} Experimental Equipment and Apparatus
[0170] The experimental equipment and apparatus comprise: carbon dioxide constant-temperature incubator, full-automatic cell counting analyzer, table centrifuge, electronic scales, micropipette, culture flask, pipette, fixator, injector, scissor and tweezers.
[0171] {circle around (3)} Grouping and Administration
[0172] The DMEM containing 10% fetal bovine serum is selected for the human breast cancer MCF7 cells, and then is placed in the CO.sub.2 constant-temperature incubator at 37 C. for culturing. One day before the experiment, 0.5 mg of 17 estradiol sustained release tablet is embedded below the neck skin of the BALB/c nude mice. The MCF7 cells at the logarithmic phase are digested by pancreatin and thereafter washed with serum-free DMEM; 5106 MCF7 cells are inoculated in the left second breast of the BALB/c nude mice, so as to construct the orthotopic transplantation tumor model of MCF7 breast cancer cells in the BALB/c nude mice.
[0173] 2. Experimental Results
[0174] After administration for 28 days, compared with the negative control group, the groups of rLZ-8 (K16A/S18A/K41A/D45A), rLZ-8 (K16A/K41A), rLZ-8 (D45A) and rLZ-8 (S18A) have no obvious tumor weight difference and have nearly no tumor inhibitory effect; compared with the rLZ-8 control group, the groups of rLZ-8 (D70K) and rLZ-8 (L17K/D70K) have the obviously increased tumor inhibitory effect.
TABLE-US-00007 TABLE 7 Influences of rLZ-8 mutants on tumor weight of model mice with MCF7 breast cancer transplantation tumor Mice tumor weight after Experimental group experiment (g) Negative control group 1.03 0.24 rLZ-8 control group 0.74 0.35 rLZ-8 (K16A/S18A/K41A/D45A) 1.02 0.33 rLZ-8 (K16A/K41A) 1.12 0.37 rLZ-8 (D45A) 1.08 0.28 rLZ-8 (S18A) 1.04 0.30 rLZ-8 (M1A/R9A/D100A) 0.77 0.32 rLZ-8 (R9A) 0.79 0.31 rLZ-8 (D70K) 0.52 0.28 rLZ-8 (L17K/D70K) 0.49 0.26 rLZ-8 (D20H/D68K) 0.60 0.27 rLZ-8 (D20H) 0.61 0.22 rLZ-8 (L17K) 0.63 0.31 rLZ-8 (K41D/K46E/K74E) 1.10 0.42 rLZ-8 (K46E/K74E) 0.73 0.24 rLZ-8 (K46E) 0.75 0.32
Eleventh Preferred Embodiment: Influences of rLZ-8 Mutants on Model Mice with Orthotopic Transplantation Tumor of Human Colon Cancer Cells
[0175] 1. Experimental Methods
[0176] {circle around (1)} Experimental Materials and Reagents
[0177] The experimental materials and reagents comprise: human colon cancer SW480 cell strain, BALB/c female nude mice, RPMI1630 medium, DMEM, fetal bovine serum, PBS, pancreatin-EDTA, DMSO, 0.05% trypsin, rLZ-8, and rLZ-8 mutants.
[0178] {circle around (2)} Experimental Equipment and Apparatus
[0179] The experimental equipment and apparatus comprise: carbon dioxide constant-temperature incubator, full-automatic cell counting analyzer, table centrifuge, electronic scales, micropipette, culture flask, pipette, fixator, injector, scissor and tweezers.
[0180] {circle around (3)} Grouping and Administration
[0181] The RPMII640 medium containing 10% fetal bovine serum is selected for the human colon cancer SW480 cells, and then is placed in the CO.sub.2 constant-temperature incubator at 37 C. for culturing. The SW480 cells at the logarithmic phase are digested by pancreatin and thereafter washed with serum-free RPMI1640 medium; 2106 cells are inoculated below the back skin of the BALB/c nude mice; after the tumor blocks for observation are formed and have the diameter larger than 0.5 cm, the mice are divided into groups randomly; and the transplantation tumor model of human colon cancer SW480 cells are constructed in the BALB/c nude mice.
[0182] 2. Experimental Results
[0183] After administration for 28 days, compared with the negative control group, the groups of rLZ-8 (K16A/S18A/K41A/D45A), rLZ-8 (K16A/K41A), rLZ-8 (D45A) and rLZ-8 (S18A) have no obvious tumor weight difference and have nearly no tumor inhibitory effect; compared with the rLZ-8 control group, the groups of rLZ-8 (D70K) and rLZ-8 (L17K/D70K) have the obviously increased tumor inhibitory effect.
TABLE-US-00008 TABLE 8 Influences of rLZ-8 mutants on tumor weight of model mice with SW480 colon cancer transplantation tumor Mice tumor weight after Experimental group experiment (g) Negative control group 3.13 0.64 rLZ-8 control group 1.94 0.43 rLZ-8 (K16A/S18A/K41A/D45A) 3.12 0.54 rLZ-8 (K16A/K41A) 3.12 0.47 rLZ-8 (D45A) 3.26 0.56 rLZ-8 (S18A) 3.01 0.39 rLZ-8 (M1A/R9A/D100A) 1.97 0.35 rLZ-8 (R9A) 1.89 0.37 rLZ-8 (D70K) 1.22 0.29 rLZ-8 (L17K/D70K) 1.09 0.33 rLZ-8 (D20H/D68K) 1.60 0.29 rLZ-8 (D20H) 1.54 0.45 rLZ-8 (L17K) 1.62 0.41 rLZ-8 (K41D/K46E/K74E) 3.10 0.48 rLZ-8 (K46E/K74E) 2.23 0.54 rLZ-8 (K46E) 2.15 0.62
Twelfth Preferred Embodiment: Affinity Test Experiment
[0184] 1. Experimental Methods
[0185] The Biacore T200 equipment (GE Company) is selected. Through the surface plasma resonance technology, the affinity of the rLZ-8 and the mutants thereof to the EGFR extracellular domain is tested. The CM5 chip is adopted to serve as the affinity test chip.
[0186] {circle around (1)} pH Screening for Coupling
[0187] The EGFR extracellular domain (with an amino acid sequence of 25-645) serves as the ligand for coupling; 1 mg EGFR freeze-dried powders are diluted with HEPES buffer solution to a concentration of 400 g/mL and set aside. Three 1.5 mL EP tubes are prepared; 10 L ligand are respectively added into the three tubes, then 90 L 10 mM sodium acetate with different pH values of 4.5, 5.0 and 5.5 are respectively added into the tubes and are mixed completely and uniformly; and a final concentration of the ligand is 40 g/mL. Moreover, 200 L 50 mM sodium acetate is added into another 1.5 mL EP tube. Results show that the condition of pH 5.0 is selected for ligand coupling.
[0188] {circle around (2)} Ligand Coupling
[0189] The ligand has a molecular weight of 70 kDa, the analyte (rLZ-8) has a molecular weight of 12.4 kDa. In theory, one rLZ-8 antibody can bind with one EGFR, and thus the stoichiometric ratio Sm is equal to 1. Through the following equations, the coupling amount RL of the ligand is calculated.
[0190] Through the experimental experience, the optimal reagent coupling amount is set to be 3*RL, and the coupling amount is set to be 1700 RU. The EGFR extracellular domain is successively coupled on the CM5 chip.
[0191] {circle around (3)} Preparation of Sample to be Tested
[0192] In the affinity test, in order to eliminate the influence of different concentrations on the affinity, every of the rLZ-8 and the mutants thereof are tested with eight concentrations, wherein: seven concentrations are gradient concentrations, and the last one is the test reference concentration. Detailed concentrations are showed in Table 9.
TABLE-US-00009 TABLE 9 Gradient concentrations of sample to be tested Sample number Sample concentration C1 256 nM C2 128 nM C3 64 nM C4 32 nM C5 16 nM C6 8 nM C7 4 nM C8 (reference) 128 nM
[0193] 2. Experimental Results
[0194] After fitting and calculating the result curves, the affinity results of the tested rLZ-8 and the mutants thereof are showed in Table 10.
TABLE-US-00010 TABLE 10 Affinity of rLZ-8 and mutants thereof Name K.sub.a (M .Math. s.sup.1) K.sub.d (s.sup.1) K.sub.D (M) rLZ-8 6.34 10.sup.4 5.83 10.sup.4 9.20 10.sup.9 rLZ-8 (K16A/S18A/K41A/ 1.39 10.sup.2 4.23 10.sup.3 3.06 10.sup.5 D45A) rLZ-8 (K16A/K41A) 2.20 10.sup.2 2.73 10.sup.4 1.24 10.sup.6 rLZ-8 (D45A) 1.20 10.sup.3 7.43 10.sup.4 6.20 10.sup.7 rLZ-8 (S18A) 3.72 10.sup.3 1.82 10.sup.3 4.89 10.sup.7 rLZ-8 (R9A) 6.12 10.sup.4 5.28 10.sup.4 8.63 10.sup.9 rLZ-8 (D70K) 2.03 10.sup.5 4.57 10.sup.4 2.25 10.sup.9 rLZ-8 (L17K/D70K) 2.15 10.sup.5 3.29 10.sup.4 1.53 10.sup.9 rLZ-8 (D20H/E68K) 1.29 10.sup.4 8.29 10.sup.5 6.43 10.sup.9 rLZ-8 (D20H) 5.87 10.sup.4 4.32 10.sup.4 7.36 10.sup.9 rLZ-8 (L17K) 1.01 10.sup.4 7.56 10.sup.5 7.49 10.sup.9 rLZ-8 (K41D/K46E/K74E) 3.53 10.sup.4 4.31 10.sup.4 1.22 10.sup.8 rLZ-8 (K46E/K74E) 6.57 10.sup.4 6.14 10.sup.4 9.35 10.sup.9
[0195] Affinities of the rLZ-8 and twelve mutants thereof are tested. Results show that: for the alanine recessive mutants at the key antitumor domain, respectively the rLZ-8 (K16A/S18A/K41A/D45A), rLZ-8 (K16A/K41A), rLZ-8 (D45A) and rLZ-8 (S18A), the affinity is obviously decreased by 2-3 orders of magnitudes.
[0196] Through calculating the potential at the protein surface, some representative mutation locations (such as the 70.sup.th amino acid and the 20.sup.th amino acid) are selected, and the amino acids which are highly likely to increase the surface positive potential are processed with mutation. Results show that: the affinities of the mutants of rLZ-8 (D70K), the rLZ-8 (L17K/D70K) and the rLZ-8 (D20H/E68K) are respectively increased by 2.3 times, 6 times and 1.4 times.
Thirteenth Preferred Embodiment: Formulations of Freeze-Dried Powders of rLZ-8 and Mutants Thereof
[0197] 1. Experimental Methods
[0198] American VirTis Wizard 2.0 freeze dryer is selected for exploring and determining the freeze-drying prescription and process of the rLZ-8 and the mutants thereof.
[0199] {circle around (1)} Freeze-Drying Prescription of rLZ-8 and Mutants Thereof
[0200] Through the single factor experiment, the stabilizer and the surfactant which affect the formulation of the freeze-drying prescription are observed, and the influences of adding different ingredients on the content, purity, insoluble particles and activity of the protein are determined. A weight ratio of the main active material and the stabilizer is 1:5, and a weight ratio of the main active material and the surfactant is 20:1.
[0201] Through screening of the combined prescription, the optimal freeze-drying prescription is determined, and the basic prescription designs are showed as follows.
TABLE-US-00011 TABLE 1 Prescription designs of rLZ-8 and mutants thereof Prescrip- tion Mycose Mannitol Lactose Twain 80 Poloxamer rLZ-8 number (mg) (mg) (mg) (mg) 188 (mg) (mg) 1 50 50 10 2 50 50 10 3 50 50 10 4 50 50 0.5 10 5 50 50 0.5 10 6 50 50 0.5 10 7 50 50 0.5 10 8 50 50 0.5 10 9 50 50 0.5 10
[0202] {circle around (2)} Freeze-Drying Process of rLZ-8 and Mutants Thereof
[0203] During the freeze-drying process, six sublimation drying temperatures of 23 C., 25 C., 27 C., 29 C., 31 C. and 33 C. are selected; six vacuum degrees of 100 mTorr, 150 mTorr, 200 mTorr, 250 mTorr, 300 mTorr and 350 mTorr are selected; six freeze-drying times of 50 hours, 60 hours, 70 hours, 80 hours, 90 hours and 100 hours are selected; and the single factor experiments are respectively conducted with the character as the evaluation index. Six vacuum drying temperatures of 25 C., 30 C., 35 C., 40 C., 45 C. and 50 C. are selected; six drying times of 15 hours, 20 hours, 25 hours, 30 hours, 35 hours and 40 hours are selected; six vacuum degrees of 100 mTorr, 150 mTorr, 200 mTorr, 250 mTorr, 300 mTorr and 350 mTorr are selected; and the single factor experiments are respectively conducted with the moisture as the evaluation index.
[0204] 2. Experimental Results
[0205] {circle around (1)} Freeze-Drying Prescription of rLZ-8 and Mutants Thereof
[0206] According to results shown in Table 12, at a high temperature condition, each stabilizer has little effect on the rLZ-8 that each index after 10 days is slightly decreased; and thus the stabilizers can all be applied in the subsequent prescription screening.
TABLE-US-00012 TABLE 12 Influences of stabilizers on rLZ-8 0 day 5 days 10 days Sample + Content Purity Activity Content Purity Activity Content Purity Activity stabilizer (mg) (%) (IC50) (mg) (%) (IC50) (mg) (%) (IC50) rLZ-8 + 10.11 99.75% 1.197 10.05 99.71% 1.188 10.01 99.69% 1.191 mannitol rLZ-8 + 10.09 99.77% 1.186 10.04 99.72% 1.179 10.02 99.65% 1.175 lactose rLZ-8 + 10.13 99.82% 1.188 10.07 99.80% 1.184 10.04 99.77% 1.185 mycose
[0207] According to results shown in Table 13, at a high temperature condition, each surfactant has little effect on the rLZ-8 that each index after 10 days is slightly decreased; and thus the surfactants can all be applied in the subsequent prescription screening.
TABLE-US-00013 TABLE 13 Influences of surfactants on rLZ-8 0 day 5 days 10 days Sample + Content Purity Activity Content Purity Activity Content Purity Activity surfactant (mg) (%) (IC50) (mg) (%) (IC50) (mg) (%) (IC50) 1 10.07 99.65% 1.177 10.04 99.62% 1.168 10.02 99.61% 1.166 2 10.08 99.57% 1.184 10.04 99.55% 1.179 10.03 99.53% 1.172
[0208] Experimental results of prescription combination screening are showed in Table 14. The data indicate that: during screening process, indexes of the 3.sup.rd, 6.sup.th and 9.sup.th prescriptions have the relatively large change; compared with the 4.sup.th, 5.sup.th, 7.sup.th and 8.sup.th prescriptions, insoluble particle amounts and change amounts of the 1.sup.st and 2.sup.nd prescriptions have no obvious change; the 4.sup.th, 5.sup.th, 7.sup.th and 8.sup.th prescriptions contain surfactant, which may affect the subsequent toxicity test. The 1.sup.st and 2.sup.nd prescriptions can be used as the standby prescriptions, but the injectable lactose in the 2.sup.nd prescription is difficult to obtain. Thus, the 1.sup.st prescription is selected as the freeze-drying prescription.
TABLE-US-00014 TABLE 14 Screening results of freeze-drying prescriptions of rLZ-8 0 day 5 days 10 days Insoluble Insoluble Insoluble Prescription Activity particle Purity Activity particle Purity Activity particle Purity number (IC50) (10 m\25 m) (%) (IC50) (10 m\25 m) (%) (IC50) (10 m\25 m) (%) 1 1.177 47.2\19.3 99.65% 1.186 48.4\19.6 99.47% 1.179 47.9\21.6 99.45% 2 1.184 50.2\19.6 99.57% 1.187 51.3\18.7 99.43% 1.192 52.2\20.6 99.42% 3 1.167 49.3\18.3 99.32% 1.341 120.5\40.2 95.42% 1.573 300.6\0.5 92.31% 4 1.188 48.5\19.3 99.41% 1.184 47.5\19.4 99.36% 1.124 44.3\18.9 99.54% 5 1.171 57.4\22.4 99.28% 1.177 52.1\23.2 99.18% 1.167 58.5\24.5 99.21% 6 1.164 52.1\23.6 99.34% 1.376 53.3\25.6 95.42% 1.752 52.8\28.8 92.31% 7 1.182 51.3\24.8 99.53% 1.187 55.5\28.8 99.63% 1.178 53.4\29.6 99.55% 8 1.183 52.4\28.9 99.76% 1.184 56.8\21.6 99.46% 1.189 54.2\26.5 99.66% 9 1.194 55.2\27.5 99.45% 1.401 58.4\28.8 95.42% 1.886 56.3\29.3 92.31%
[0209] {circle around (2)} Freeze-Drying Process of rLZ-8 and Mutants Thereof
[0210] Through exploring the freeze-drying condition, the optimal freeze-drying process is determined that: for the sublimation drying, the drying temperature is 30 C., the vacuum degree is 300 mTorr, and the drying time is 90 hours; for the vacuum drying, the drying temperature is 40 C., the vacuum degree is 250 mTorr, the drying time is 30 hours.
[0211] With the same freeze-drying prescription and the same freeze-drying process, the rLZ-8 mutants can also obtain the freeze-dried powders with the character and the moisture reaching the standard.