Antihuman PCSK9 monoclonal antibody and application thereof

Abstract

The invention discloses a brand-new PCSK9 antibody. The PCSK9 antibody is sieved through a phage display technology, then a full-length gene sequence is obtained through a gene engineering technology, and applied to preparation of the antibody, and the prepared antibody is high in yield and short in period and is a humanized antibody. The invention further discloses an anti-tumor effect of the antihuman PCSK9 antibody, which expands the way of thinking for research on occurrence and development of tumors in the future, and lays foundations for preparing the antihuman PCSK9 antibody into monoclonal antibody medicines used for treating tumors in the later period.

Claims

1. An antihuman PCSK9 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain having an amino acid sequence shown in SEQ ID NO: 7 and the light chain having an amino acid sequence shown in SEQ ID NO: 8.

2. The antihuman PCSK9 monoclonal antibody according to claim 1, wherein the antihuman PCSK9 monoclonal antibody is a fully humanized antibody.

3. The antihuman PCSK9 monoclonal antibody according to claim 1, wherein the antihuman PCSK9 monoclonal antibody contains an Fab fragment.

4. The antihuman PCSK9 monoclonal antibody according to claim 1, wherein the heavy chain contains a CDR1, a CDR2 and a CDR3 having an amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

5. The antihuman PCSK9 monoclonal antibody according to claim 1, wherein the light chain contains a CDR1, a CDR2 and a CDR3 having an amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

6. A pharmaceutical composition for treating a cancer comprising an effective amount of the antihuman PCSK9 monoclonal antibody of claim 1.

7. The pharmaceutical composition for treating the cancer according to claim 6, wherein the cancer is a colorectal cancer.

8. A method for treating a cancer comprising a step of administering to a subject in need of treatment by the antihuman PCSK9 monoclonal antibody of claim 1.

9. The method according to claim 8, wherein the cancer is a colorectal cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows SDS-PAGE identification results of a cell supernatant;

(2) FIG. 2 shows SDS-PAGE detection results of a PCSK9-purified antibody;

(3) FIG. 3 is a tumor inhibition curve of an antibody to nude mice; and

(4) FIG. 4 shows inhibition of the antibody on a tumor size of the nude mice.

DETAILED DESCRIPTION

(5) The application is described in detail hereinafter with reference to specific embodiments.

Embodiment 1

Screening of an Fab Phage Library to Obtain an Antihuman PCSK9 Positive Monoclonal Antibody

(6) 1. A PCSK9 protein (GenScript Biotech Corporation) was diluted to a concentration of 100 ug/ml, and 1.5 ml of the PCSK9 protein was added into an immune tube, and coated overnight at 4° C. A coating buffer in the immune tube was discarded next day, and washed with a PBS for three times. 2% skimmed milk powder was prepared and sealed in the immune tube for 3 hours to 4 hours. Meanwhile, a natural humanized Fab phage display library constructed according to the report of Hans J. W. de Haard et al. (Antibody PhageDisplay, 2002, p87-100) was also sealed for 1 hour, then a phage was added into the immune tube, and turned over repeatedly for 2 hours, and the phage bound to the immune tube was eluted with 0.1% Tween 20 and neutralized. The neutralized phage was infected with TG1 for next round of screening.

(7) 2. After 3 to 5 rounds of screening, the eluted phage was infected with Escherichia coli TG1 (Beyotime, D0389) and subjected to plate coating, then 100 clones were randomly selected for Elisa identification of the phage.

(8) 3. The clones were inoculated in a 96-well plate and cultured at 37° C. until OD.sub.600 reached about 0.5; a VSCM13 auxiliary phage was added, and cultured overnight; and the amplified phages were blocked with the 2% skimmed milk powder, respectively added into an ELISA plate coated with 1 μg/ml PCSK9 antigen and BSA (BSA is negative control), incubated at a room temperature for 1 hour, washed with PBST for 5 times, then added with a rabbit anti-M13 phage HRP conjugate (GE Healthcare Life Sciences), incubated at a room temperature for 1 hour, washed with PBST for 5 times, added with a TMB peroxy substrate, and read at 450 nm wavelength. Finally, a monoclonal antibody No. 6 with a high affinity was screened out as PA6.

(9) 4. DNA sequencing was performed on the PA6 to obtain sequences of a variable region of heavy chain (VH) and a variable region of light chain (VL) of the PA6, wherein the complete sequences of the VH and the VL were shown in SEQ ID NO:7 and SEQ ID NO:8, wherein a CDR1, a CDR2 and a CDR3 of the VH respectively had an amino acid sequence shown in SEQ ID NO:1-3, and a CDR1, a CDR2 and a CDR3 of the VL respectively had an amino acid sequence shown in SEQ ID NO:4-6.

Embodiment 2

Expression and Purification of an Fab Fragment of a Positive Monoclonal PA6 Antibody

(10) 1. A screened PA6 antibody with a high affinity was infected with TG1 and amplified, then a bacterial liquid was collected, a plasmid was extracted by a plasmid extraction kit (omega, D6950-01), and Gene III of the plasmid was removed.

(11) 2. The obtained Genelll-excised plasmid was transfected into Escherichia coli TG I, cultured in a 2YTA medium at 37° C. to a logarithmic phase, added with 1 mM IPTG, and subjected to induced expression at 37° C. overnight. 3. A supernatant was collected by centrifugation and purified with a Protein A (CWBIO, CW0894S) to obtain a PA6 Fab protein.

Embodiment 3

Expression and Purification of a Positive Clone in a Full-Length Antibody IgG1 Form

(12) 1. Construction of a heavy chain expression plasmid: a CH1-VH part in an Fab fragment of a PA6 antibody was amplified by PCR, and the CH1-VH part was bound to a Fc fragment (referring to a variable region gene of a fully humanized monoclonal antibody against PCSK9 and an application thereof for details, with the patent No. CN 104861071 A) stored in a laboratory by using a T4 ligase. Then, a Kozak sequence (SEQ ID NO: 9: AAG CTT GCCACC), a signal peptide and a restriction enzyme site were added to an N-terminal of a variable region of heavy chain VH. An amplified heavy chain fragment and an expression vector UCOE-Mu-P were digested by restriction enzymes NgoMIV and Nhel, and the fragment was bound to the vector after enzyme digestion.

(13) 2. Construction of a light chain expression plasmid: a complete light chain part of the Fab fragment of the PA6 antibody was amplified by PCR. A Kozak sequence (SEQ ID NO: 9: AAG CTT GCCACC), a signal peptide and a restriction enzyme site were added to an N-terminal of a variable region of light chain VL. An amplified light chain fragment and an expression vector UCOE-Mu-P were digested by restriction enzymes NgoMIV and Nhel, and the fragment was bound to the vector after enzyme digestion.

(14) 3. Induced expression: the heavy and light chain plasmids expressing the PCSK9 antibody were co-transfected intoCHO-S cells (BeNa Bio, 338010) with a transfection reagent; after a cell viability was stable, MTX (50 nm, 100 nm, 200 nm and 500nm) and puromycin (Solarbio, P8230) were added for pressure screening until a cell state was stable, and a cell supernatant was taken and ran in gel to see whether a target antibody thereof was expressed. After the expression was confirmed, the cells were spread into a 96-well plate with an average of 2 cells per well. After the cells grew to a monoclonal state, a supernatant was taken for ELISA detection, after 2 to 3 rounds of subclone screening, then the cells were subjected to extended culture to a 250 ml shake flask, and regularly added with a supplement to increase an antibody production, and the cells in the shake flask were sampled, counted and analyzed when the supplement was added each time. Specifically, as shown in Table 1-3, cell strains stably expressing the antihuman PCSK9 monoclonal antibody were subjected to extended culture, and used to prepare the antibody through shake flask culture, three independent experiments were respectively performed, the cells were extended to the shake flask, and two shake flasks were selected for parallel experiments. Specific results of sample counting of one and/or two shake flasks were randomly taken every 2 days to 3 days and the cells were treated.

(15) TABLE-US-00001 TABLE 1 Cell density Cell viability Time (10.sup.6) (%) Cell treatment Cell culture method May 21, 2018 0.8 99 Extended to 250 ml shake CD-FortiCHO + flask for 60 ml respectively 4 mM of Glutamax + May 23, 2018 2.18/6.20 98/98 Supplemented with 5% of 1% of ACA Feed C (with white precipitate on flask body) May 25, 2018 11.6/5.3  97/98 Supplemented with 10% of Feed C May 28, 2018 Basically died/2.7 .sup. 10/48.2 Collected 80 ml of supernatant respectively

(16) TABLE-US-00002 TABLE 2 Cell density Cell viability Time (10.sup.6) (%) Cell treatment Cell culture method Jun. 1, 2018 0.8 99 Extended to two 250 ml CD-FortiCHO + 4 mM shake flasks for 80 ml of Glutamax + 1% of respectively ACA Jun. 4, 2018 11.3/6.8 98/97 Supplemented with 10% of Feed C Jun. 6, 2018 14.15/8.0  98/98 Supplemented with 10% of Feed C Jun. 8, 2018 4.15/5.8 85.3/72.5 Collected 160 ml of supernatant respectively

(17) TABLE-US-00003 TABLE 3 Cell density Cell viability Time (10.sup.6) (%) Cell treatment Cell culture method Jun. 10, 2018 0.8 95 Extended to 250 ml CD-FortiCHO + 4 mM of shake flask for 60 ml Glutamax + 1% of respectively ACA + 1% of HT Jun. 11, 2018 1.0 98 Untreated Jun. 14, 2018 4.34 98 Supplemented with 1 ml of Glutamax Supplemented with 10% of Feed2 Jun. 16, 2018 7.56 99 Supplemented with 10% of Feed2 Jun. 18, 2018 10.75 99 Supplemented with 10% of Feed2 Jun. 21, 2018 6.5/5.15 97 Untreated Jun. 23, 2018 — — Collected 80 ml of supernatant respectively

(18) When the cell viability in the shake flask reached 70% or below, the cell supernatant was collected, and a sample was taken for SDS-PAGE identification. Results were shown in FIG. 1. It could be seen that there was really a target band in the supernatant to express the antihuman PCSK9 antibody, and the supernatant was purified in a later stage.

Embodiment 4

Purification of a PCSK9 Monoclonal Antibody

(19) 1. Preparation of a Buffer

(20) Water and a buffer were filtered with a 0.45 μm filter membrane before use.

(21) Binding/impurity washing buffer: 0.15 M NaCl, 20 mM Na2HPO4, pH 7.0.

(22) Elution buffer: 0.1 M glycine, pH 3.0.

(23) Neutralization buffer: 1 M Tris-HCl, pH 8.5.

(24) 2. Preparation of a Sample

(25) The supernatant at −80° C. above was placed at 4° C. for natural thawing, and the entire supernatant with a volume of about 150 ml was ultrafiltrated and concentrated to a total volume of about 10 ml by using an ultrafiltration tube (Thermo) of 15 ml 50 KD (molecular weight cut-off), diluted twice with the bound buffer, and finally filtered with the 0.45 μm filter membrane to reduce impurities, thus improving a protein purification efficiency and preventing a column from blockage.

(26) 3. Purification of the Sample

(27) 1) rProtein G Beads were loaded into a 1 ml chromatography column, and chromatography was balanced with a bound solution of 5 times the column volume, so that a filler was placed in the same buffer system as the target antibody to protect the antibody.

(28) 2) The sample was added into the well-balanced rProtein G Beads to ensure full contact between the target antibody and the rProtein G Beads, thus improving a recovery rate of the target antibody, and an outflow was collected.

(29) 3) The sample was washed with an impurity washing solution of 10 times the column volume to remove a non-specifically adsorbed foreign protein, and the impurity washing solution was collected.

(30) 4) The bound antibody was eluted with an eluent of 5 times the column volume, and the eluent was collected into a centrifuge tube added with the neutralized buffer in advance (5 ml of eluent was added with 0.5 ml of neutralized buffer), namely a target antibody component.

(31) 5) The collected eluate was desalted and concentrated by using an ultrafiltration tube of 15 ml 50 KD (molecular weight cut-off), and washed twice with a PBS during the period, and finally, about 1 ml of the concentrated solution was collected, and temporarily stored at 4° C. for concentration and purity detection.

(32) 4. Concentration and Purity Detection of a Purified Antibody

(33) A concentration of a purified antibody was detected by a Bradford Protein Assay Kit, and the concentration was finally determined to be 2.775 mg/ml. An outflow component, an impurity washing component and an elution component were detected by using SDS-PAGE to observe a purification effect, and as shown in FIG. 2, according to the arrow in the figure, obvious heavy and light chain belts appeared after sample reduction, and finally, the purity was indicated to be more than 95%.

Embodiment 5

Application of an Antihuman PCSK9 Antibody Obtained Above in Treating a Tumor

(34) After the antibody is purified, a concentration was marked, and the antibody was sub-packaged and stored at −80° C. A colon cancer cell (BeNa Bio, BNCC100275) was inoculated to a mouse, the PCSK9 antibody was used to treat the mouse, and a tumor size was observed. Specific operation steps were as follows:

(35) 1. 10 female nude mice in 6 weeks were purchased, SW480 cells were extended into 12 large dishes of 10 cm, and a cell inoculation amount of each mouse was ensured to be 5×10.sup.6.

(36) 2. The cells were digested by trypsin, and collected, and a cell inoculation amount of each mouse was ensured to be 5×10.sup.6. The 10 nude mice were divided into a control group and an antibody group, the cells were injected subcutaneously, and the mice were observed until a tumor grew to a certain size (about 200 mm.sup.3 in volume).

(37) 3. The PCSK9 antibody was prepared, and each nude mouse in the antibody group was injected with a dose of 10 mg/g in a tail intravenous mode according to a body weight; and the nude mice in the control group were injected with the same volume of normal saline, a tumor size of each nude mouse was measured every other week, and the antibody was continuously injected. Then, the tumor size was measured, and the experiment operation was repeated every week for one month.

(38) 4. Finally, tumors of the nude mice were taken out, and tumor sizes of the experimental group and the control group were compared and analyzed. Results were shown in FIG. 3 and FIG. 4. As shown in FIG. 3, after the tumor sizes of the nude mice were analyzed, tumor growth of the treatment group was found to be obviously inhibited compared with that of the control group. As shown in FIG. 4, the tumors of the mice were taken out, and the upper row was the control group and the lower row was the treatment group. The results showed that the tumor growth of the antibody group was significantly inhibited compared with that of the control group.