An Interleukin 21 Protein (IL21) Mutant and Use Thereof
20220025006 · 2022-01-27
Assignee
Inventors
Cpc classification
C07K2319/60
CHEMISTRY; METALLURGY
C07K16/24
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The invention relates to an interleukin-21 protein (IL21) mutant and application thereof. The mutant is with ILE at position 16 and SER at position 70 of the amino acid sequence of the wild-type IL21 both mutating into CYS, and a disulfide bond forming between the two mutated CYSs. The amino acid sequence of the wild-type IL21 is shown in SEQ ID NO.1. The invention also relates to a fusion protein containing the IL21 mutant protein and a use of the IL21 mutant protein or the fusion protein in the preparation of a medicine and, preferably, the medicine regulates or activates immunity or are an anti-tumor medicine. The invention also relates to the use of the IL21 mutant, the IL21/4 mutant or the fusion protein in the preparation of a formulation for promoting the differentiation and proliferation of B cell, the differentiation and proliferation of T cell, the differentiation and proliferation of NK cell.
Claims
1.-7. (canceled)
8. A mutant of interleukin-21 (IL21) comprising: (1) isoleucine (ILE) at position 16 and serine (SER) at position 70 of the amino acid sequence of wild-type IL21 both mutated into cysteine (CYS) amino acids, and a disulfide bond between the two mutated CYS amino acids, wherein the amino acid sequence of the wild-type IL21 is shown in SEQ ID NO. 1; or (2) isoleucine (ILE) at position 16, valine (VAL) at position 17, serine (SER) at position 70 and lysine (LYS) at position 112 of the amino acid sequence of wild type IL21 all mutated into cysteine (CYS) amino acids, and two groups of disulfide bonds formed at amino acid positions 16-70 and 17-112, wherein the amino acid sequence of the wild type IL21 is shown in SEQ ID NO. 1.
9. A mutant of interleukin-21 and interleukin-4 chimeric protein (IL21/4) comprising: (1) ILE at position 16 and SER at position 70 of the amino acid sequence of the interleukin-21 and interleukin-4 chimera (IL21/4) mutated into CYS amino acids, and a disulfide bond between the two mutated CYS amino acids; or (2) ILE at position 16, VAL at position 17, SER at position 70 and LYS at position 106 of the amino acid sequence of the interleukin-21 and interleukin-4 chimera (IL21/4) mutated into CYS amino acids, and two groups of disulfide bonds formed at amino acid positions 16-70 and 17-106; wherein the amino acid sequence of the interleukin-21 and interleukin-4 chimeric protein (IL21/4) is shown in SEQ ID NO. 2.
10. A nucleotide sequence encoding the IL21 mutant of claim 8.
11. A nucleotide sequence encoding the IL21/4 mutant of claim 9.
12. A fusion protein comprising the IL21 mutant of claim 8, wherein the fusion protein comprises: (1) a functional fragment 1 comprising the IL21 mutant; (2) a functional fragment 2 having monoclonal antibody function, and a connecting domain that connects functional fragment 1 and functional fragment 2.
13. A fusion protein comprising the IL21/4 mutant of claim 9, wherein the fusion protein comprises: (1) a first functional fragment comprising the IL21/4 mutant; (2) a second functional fragment having monoclonal antibody function, and a connecting domain that connects functional fragment 1 and functional fragment 2.
14. A medicine or pharmaceutical composition comprising a therapeutically effective amount of the IL21 mutant of claim 8 as an active ingredient, and a pharmaceutical excipient.
15. A medicine or pharmaceutical composition comprising a therapeutically effective amount of the IL21/4 mutant of claim 9 as an active ingredient, and a pharmaceutical excipient.
16. A medicine or pharmaceutical composition comprising a therapeutically effective amount of the fusion protein of claim 5 as an active ingredient, and a pharmaceutical excipient.
17. A medicine or pharmaceutical composition comprising a therapeutically effective amount of the fusion protein of claim 13 as an active ingredient, and a pharmaceutical excipient.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE EMBODIMENTS
[0053] Extraction, Purification and Preparation of Product of PCR and DNA Digestion, and Plasmid.
[0054] The product of PCR and DNA digestion was extracted by AxyPrep DNA gel extraction kit (AXYGEN). A small amount of plasmid was extracted and purified by Transgen Plasmid Mini Kit (Transgen, China). A medium amount of plasmid was extracted and purified by QIAGEN Plasmid Midi Kit. A large amount of plasmid was extracted and purified by Tiangen EndoFree Maxi Plasmid Kit. The particular procedure was carried out according to instructions.
[0055] Cell Culture.
[0056] CHO/dhFr− (Dihydrofolate reductase-deficient Chinese hamster ovary cells): The cells were cultured with IMDM medium containing 10% calf serum (Hyclone), 100 U/mL double antibody, 0.1 mM hypoxanthine and 0.016 mM thymine at 37° C. in a 5% CO.sub.2 incubator.
[0057] 293F cells: Human embryonic kidney cells, which were cultured with FreeStyle™ 293 Expression Medium in shake flasks at 125 rpm and 37° C. in a 5% CO.sub.2 incubator.
[0058] KOB cells: Adult T lymphoma cells with high expression of IL21 receptor, which were donated by WANG Shengdian group of the Institute of Biophysics of the Chinese Academy of Sciences. The cells were cultured with RPMI1640 medium containing 10% calf serum (Hyclone) and 100 U/mL double antibody at 37° C. in a 5% CO.sub.2 incubator.
[0059] Cell Transfection.
[0060] Transfection of CHO Cell.
[0061] Cell preparation: The cells were seeded in a six-well plate with the amount of 200,000 cells per well one day before transfection, so that, at the time of transfection, they reached 80% confluence, distributed evenly and grew well.
[0062] Preparation of transfection complex: 5 μl Lipo2000 and 150 ml Opti-MEM serum-free medium were mixed evenly; 2 μg target plasmid was mixed with 150 μl Opti-MEM serum-free medium evenly. After standing at room temperature for 5 minutes, these two mixtures were mixed to prepare the transfection complex, which were gently mixed and placed at room temperature for 25 minutes.
[0063] The original culture medium was removed from the six-well plate, and wells were washed with Opti-MEM serum-free medium for three times, then the transfection complex was dripped onto the cell surface, and then 500 μL of Opti-MEM serum-free medium was added. After 4-6 hours, the medium was changed into the common culture medium to continue the culture.
[0064] Transfection of 293F cell (50 ml culture medium as an example):
[0065] Cell preparation: 6×10.sup.5-7×10.sup.5 cells/ml was seeded one day before transfection, and the cell density should be 1×10.sup.6 cells/ml at the time of transfection.
[0066] Preparation of transfection complex: 50 μg of target plasmid was diluted with 2 ml OptiPROTMSFM (Invitrogen) serum-free medium and fully mixed to prepare a DNA diluent. 250 μL transfection reagent PEI was added to the DNA diluent to prepare a transfection complex, which was fully mixed and placed at room temperature for 15 minutes. The transfection complex was added into 293F cell culture medium, and then cultured in shake flasks at 125 rpm and 37° C. in a 5% CO.sub.2 incubator for 96 hours.
[0067] Expression and Purification of Protein.
[0068] (1) Expression and Purification of Eukaryotic Cells.
[0069] After 293F cells being transfected by the plasmid containing the target gene, the cells were continuously cultured in a shaker for 96 hours and then centrifuged at 200 g for 3 minutes to remove cell precipitates. The supernatant medium was collected, centrifuged at 10000 g for 15 minutes to remove impurities in the culture medium, filtered with a 0.45 μm filter membrane, centrifuged in a 30 kd concentration tube at 4° C. under 3800 rmp, to concentrate 20 times of the volume.
[0070] (2) Purification of His Tag Protein.
[0071] A Sepharose high performance (Amersham Bioscience) chromatographic column filled with nickel sulfate (NiSO.sub.4) was used. Buffers containing 5 mM, 30 mM, 60 mM, 90 mM, 120 mM and 250 mM imidazole were prepared respectively. Other components in the buffer were 20 mM Tris-HCL, 500 mM NaCl and 10% glycerol.
[0072] Washing and balancing the nickel column: the nickel column was firstly washed with 50 ml ddH.sub.2O, and then washed and balanced with 50 ml buffer containing 5 mM imidazole. Sample loading: The supernatant containing the target protein was dripped into the nickel column, which might be repeated 2-3 times.
[0073] Elution: After loading the sample, the column was eluted with 30 ml buffer containing 5 mM, 30 mM, 60 mM, 90 mM, 120 mM and 250 mM imidazole respectively, and the eluents were collected. Ultrafiltration tubes of different specifications were selected according to the molecular weights of the target proteins. The eluents were concentrated with the ultrafiltration tubes by 50-100 times of the volumes.
[0074] Protein preservation: The finally obtained protein was packaged separately, quick-frozen in liquid nitrogen, and preserved at −80° C.
[0075] (3) Purification of Full-Length Antibody and Full-Length Antibody Fusion Protein.
[0076] A Protein A Sefinose™-5 ml (Pre-Packed Gravity Column) chromatographic column was used.
[0077] Pre-Preparation:
[0078] binding buffer: 0.1 M Na.sub.3PO.sub.4, 0.15 M NaCl, pH7.2;
[0079] elution buffer: 0.1 M citric acid, pH2.7;
[0080] neutralizing buffer: 1 M Tris-HCl, pH9.0;
[0081] Washing and balancing: the chromatographic column was firstly washed with 50 ml ddH.sub.2O, then washed with 50 ml elution buffer, and then balanced with 50 ml binding buffer.
[0082] Sample loading: The supernatant containing a target protein was dripped into the chromatographic column, which might be repeated 2-3 times.
[0083] Elution: After loading the sample, the column was washed with 50 ml binding buffer to remove non-specific binding. Ten 15 ml centrifuge tubes were prepared, each being added into 800 μl pre-prepared neutralizing buffer. The protein was eluted with 30 ml elution buffer, and collected by 15 ml centrifugal tubes with neutralizing buffer, each collecting 3 ml of the elution.
[0084] Fraction collection: After the eluent being collected, the concentration of protein in each tube was detected by nanodrop. The eluent with a concentration of protein below 0.05 mg/ml was discarded. Ultrafiltration tubes of different specifications were selected according to the molecular weight of target protein. The eluent was concentrated by an ultrafiltration tube.
[0085] Protein preservation: The finally obtained protein was packaged separately, quick-frozen in liquid nitrogen, and preserved at −80° C.
[0086] Washing and preservation of chromatographic column: The chromatographic column was washed with 50 ml binding buffer, then washed with 50 ml of ddH.sub.2O, and finally washed with 50 ml 20% ethanol and sealed.
[0087] Determination of Melting Temperature (Tm) of Protein.
[0088] Determination principle of melting temperature (Tm) of protein: When the temperature of protein rose with the ambient temperature and reached the melting temperature (Tm), the conformation of the protein would be destroyed. The hydrophobic core would be opened. The dye could combine with the hydrophobic region to emit a fluorescence that could be detected.
[0089] The protein to be determined was firstly adjusted to a concentration of 20-40 μM and a volume of 24 μl.
[0090] The Sypro® orange protein (5000×) was used as a dye, which was diluted to 25× with DMSO.
[0091] Preparation of 25 μl detection system: Each well of the 96-well plate was added with 24 μl protein (20-40 μM), and then with 1 μl Sypro@orang protein (25×), mixed thoroughly, and kept at room temperature away from light.
[0092] Stepone software 2.1 in qPCR instrument was used for detection, and operating and parameter setting were according to instructions.
[0093] The experimental results were determined and saved. The results were analyzed by the program of Protein Thermal Shift 3.1.
Example 1. Establishment of a CHO Cell Line that can Stably Express Chimeric IL21/4
[0094] IL21 has two protein conformations: a stable conformation and an unstable conformation. A chimeric IL21/4 was constructed by substituting the unstable region in the protein structure of IL21 with the homologous region of IL4. The results showed that the protein conformation of chimeric IL21/4 was unique and stable, with an improved biological activity. Considering that the construction of fusion protein of IL21 mutant and Herceptin was an important purpose of the experiment, after displaying the fusion protein of chimeric IL21/4 and Herceptin on the surface of a CHO working cell, it was also beneficial to detect the display efficiency of chimeric IL21/4 through detecting the constant region of Herceptin heavy chain. Therefore, we firstly displayed the fusion protein of chimeric IL21/4 and Herceptin on the surface of a CHO working cell.
[0095] In order to obtain an efficient and stable CHO cell line for displaying chimeric IL21/4, we constructed it using a protein display system established earlier in our laboratory by recombinase-mediated cassette exchange (RMCE) (see CN 201410803422 for details). A recombinant substitution plasmid fragment (FRT-IL21/4-Herceptin plasmid) was firstly constructed for displaying the fusion protein of IL21/4 and Herceptin. The fragments of the fusion protein were as follows: Herceptin light chain with a signal peptide, IRES (internal ribosome entry site), the signal peptide of the Herceptin heavy chain, IL21/4 linked to the N-terminal of the Herceptin heavy chain via a 3(G4S) linker, and the transmembrane region (TM) linked to the C-terminal of the Herceptin heavy chain. By this, the fusion protein of IL21/4 and Herceptin could be anchored and displayed on the surface of a CHO working cell by the transmembrane region. The sequence structure of the fusion protein was shown in
[0096] A CHO working cell established in our laboratory (see CN 201410803422 for details), whose genome was inserted with a single copy of recombinant substitution region FRT-puromysin-Loxp, were co-transfected with the FRT-IL21/4-Herceptin plasmid and the pCI-2A plasmid previously constructed in our laboratory. The fusion protein sequence of Herceptin and IL21/4 between Loxp and FRT sites in FRT-IL21/4-Herceptin plasmid could be recombined and substituted into the genome of the CHO working cell in single-copy, as shown in
[0097] After transfection, the cells were collected on time, and then labeled with Mouse Anti-human IgG-APC antibody. The display rate after IL21/4-Herceptin substitution was detected by flow cytometry. The results (
[0098] After enrichment, detection was conducted again. This time, we labeled pre-expressed and purified IL21Rα extracellular domain-linker-GFP-his fusion protein, γc extracellular domain-linker-mRFP-his fusion protein and Mouse Anti-human IgG-APC antibody, to simultaneously detect the integrity of IL21/4 and Herceptin domains in IL21/4-Herceptin fusion protein. The results were shown in
[0099] As shown in
[0100] The above-mentioned CHO cell that stably displayed IL21/4-Herceptin fusion protein was sorted by flow FACS AriaIII and named as an s0 cell.
[0101] The steps for constructing the plasmid that could stably display chimeric IL21/4 on the surface of a CHO cell in the molecular biology experimental process were as follows:
[0102] 1. Constructing the pFRT-Herceptin LC-IRES-IL21/4-linker-Herceptin HC-TM-Loxp plasmid and stably displaying the mutant IL21/4 on the surface of a CHO cell: [0103] (1) Primers were designed with IL21 as template, and the IL21/4 fragment was obtained by overlap PCR. [0104] (2) The IL21/4 fragment was linked to the N-terminal of the Herceptin heavy chain HC via a linker by overlap PCR, and then the sp-IL21/4-linker-HerceptinHC fragment was substituted into the heavy chain part of the antibody displaying plasmid pFRT constructed in our laboratory, so as to construct the pFRT-Herceptin LC-IRES-IL21/4-linker-Herceptin HC-TM-Loxp plasmid. The plasmid could stably display the mutant IL21/4 on the surface of a CHO cell.
[0105] 2. Constructing a plasmid that could secrete and express IL21Rα-linker-GFP fusion protein, and secreting and expressing the extracellular domain of IL21 receptor α to examine the functional activity of IL21: [0106] (1) Through querying database, the extracellular domain of IL21 receptor IL21Rα chain was synthesized, and then primers were designed to amplify the GFP fragment by PCR with GFP as a template. [0107] (2) The extracellular domain of IL21Rα chain was linked to the N-terminal of GFP via a linker by overlap PCR, and then the extracellular domain-linker-GFP fragment of sp-IL21 Rα chain was linked into pCEP4, so as to construct a plasmid that could secrete and express IL21Rα-linker-GFP fusion protein.
[0108] 3. Constructing a plasmid that could secrete and express γc-linker-mRFP fusion protein, and secreting and expressing the extracellular domain of IL21 receptor γc to examine the functional activity of IL21:
[0109] Through querying database, the extracellular domain of IL21 receptor γc chain was synthesized, and then, again, primers were designed to amplify the mRFP fragment by PCR with mRFP as a template. Again, the extracellular domain of γc chain was linked to the N-terminal of mRFP via a linker by overlap PCR, and then, again, the extracellular domain-linker-mRFP fragment of sp-γc chain was linked into pCEP4, so as to construct a plasmid that could secrete and express γc-linker-GFP fusion protein.
[0110] 4. Constructing a plasmid that could secrete and express IL21 mutant and fusion protein: [0111] (2) Construction of plasmids that could stably display various mutants of IL21: In order to stably display another mutant of IL21 on the surface of a CHO cell, we used point mutation PCR to construct a plasmid: using pFRT-Herceptin LC-IRES-IL21/4-linker-Herceptin HC-TM-Loxp plasmid as a template to design primers, so as to construct plasmids that could stably display different mutants of IL21. [0112] (3) Construction of plasmids that could secrete and express IL21 mutant protein: Using plasmids that could display various IL21 mutants as templates to design primers, amplifying IL21 mutant fragments by PCR, and then linking them into pCEP4, so as to construct plasmids that could secrete and express IL21 mutant protein.
Example 2. Study on the Stability of IL21/4 by Introducing a Disulfide Bond at Different Sites Through Protein Design
[0113] 1. Through analyzing and designing protein structure, different sites on IL21/4 protein were selected for mutation, and a disulfide bond was introduced to obtain the following groups of mutants. The disulfide bond was introduced as follows: [0114] (1) ILE at position 16 and SER at position 70 were both mutated to CYS, 16-70 disulfide bond was introduced; named as 16cIL21/4; [0115] (2) ILE at position 17 and SER at position 106 were both mutated to CYS, 17-106 disulfide bond was introduced; named as 17cIL21/4; [0116] (2) ILE at position 16, SER at position 70, VAL at position 17 and LYS at position 106 were all mutated to CYS, two groups of disulfide bonds were introduced at the same time; named as 4cIL21/4.
[0117] 2. Obtaining a CHO cell that could stably display each of IL21/4 mutants.
[0118] In order to obtain a CHO cell line that could stably display each of IL21/4 mutants, with FRT-IL21/4-Herceptin as a template, recombinant substitution plasmids of each of IL21/4 mutants were firstly obtained by point mutation technique, named as 16cIL21/4-Herceptin, 17cIL21/4-Herceptin and 4cIL21/4-Herceptin. A CHO working cell was co-transfected with above plasmids and pre-constructed pci-2A plasmid again, and enriched after successful recombination and substitution. The display rate was detected by binding to the labeled antibody, Mouse Anti-human IgG-APC antibody.
[0119] As shown in
[0120] We had obtained a CHO working cell that could stably display two kinds of IL21/4 mutants on the surface of cell membrane, and then we would do a preliminary stability test of these two IL21/4 mutants.
Example 3. Preliminary Stability Test of Each of the IL21/4 Mutants
[0121] To test the stability of each of the IL21/4 mutants, the CHO working cells displaying IL21/4-Herceptin and the CHO working cells displaying mutants 16cIL21/4-Herceptin and 4cIL21/4-Herceptin were used as an experimental group. Each group was heated at various temperature gradients of 4° C.-50° C., and then bound and labeled with IL21 receptor IL21Rα extracellular domain-linker-GFP-his. The display rate of each group was detected by flow cytometry after binding to the receptor at various temperature gradients. As temperature changing, a protein with poor thermal stability would be denatured first, the normal conformation would be destroyed, and the ability of receptor binding would lose. Through this way, the thermal stability of each mutant could be examined.
[0122] The particular experimental processes were as follows: [0123] (1) CHO cells that stably displaying IL21 mutant on the surface of cell membrane were digested with 0.02% EDTA-PBS, eluted with a medium containing serum, and centrifuged at 830 g for 3 min. The supernatant was discarded and the cells were collected. [0124] (2) The cells were re-suspended in 1 ml serum-free Opti-MEM medium precooled at 4° C., centrifuged at 830 g for 3 min. The supernatant was discarded and the cells were collected. The cells were then re-suspended in serum-free Opti-MEM medium precooled at 4° C. again, to about 5×10.sup.6 cells/50 μl. [0125] (3) The cell suspension was added into a PCR tube and heated with temperature gradient set by a PCR instrument. The PCR tube was heated at the preset temperature for 0.5 h. The PCR tube was kept at 4° C. for 15 min, before being removed. [0126] (4) The extracellular domain of IL21 receptor was added in certain proportion, and thoroughly mixed with fluorescent protein fusion protein. The mixture was kept on a shaker at 4° C. for 1 h away from light. [0127] (5) The mixture was centrifuged at 830 g for 3 min, the supernatant was discarded. New Opti-MEM precooled at 4° C. was added to wash the precipitate twice through centrifuging and discarding supernatant. Then, the precipitate was re-suspended with 200 μl OPTI-MEM precooled at 4° C. The labeled cells were detected or sorted by FACSAriaIII(BD) or FACSCalibur.
[0128] The results showed that, [0129] (1) At 4° C., except the negative control group, all groups bound to the extracellular domain of IL21 receptor IL21Rα. The display rate of mutant 16cIL21/4-Herceptin was 20%, which was the highest binding rate. [0130] (2) After heating at 49° C., the binding of IL21/4-Herceptin group and 4cIL21/4-Herceptin group to the extracellular domain of IL21 receptor was substantially lost, while the binding of mutant 16cIL21/4-Herceptin group to the extracellular domain of IL21 receptor were still detectable. This could preliminarily suggest that the mutant 16cIL21/4 had an improved thermal stability.
Example 4. Construction of Wild-Type IL21 Mutant and Preliminary Stability Test
[0131] 1. Detection of Thermal Stability of Mutant by Flow Cytometry.
[0132] In order to study the effect of introducing a disulfide bond at the same positions (positions 16 and 70) on stability in wild-type IL21 molecule, we constructed two other structures:
[0133] IL21-Herceptin: the fusion protein of wild-type IL21 and Herceptin;
[0134] 16cIL21-Herceptin: the fusion protein constructed by Herceptin and IL21, wherein, at the same positions, ILE at position 16—SER at position 70, were mutated to CYS and introduced a disulfide bond;
[0135] They were also displayed on the surface of CHO working cell membrane by transfection and substitution recombination, and then were compared with 16cIL21/4-Herceptin and IL21/4-Herceptin. They were heated at different temperature gradients from 4° C.-55° C., and labeled by binding to IL21 receptor IL21Rα extracellular domain-linker-GFP-his and the labeled antibody, Mouse Anti-human IgG-APC antibody. The display rate of each group was detected by flow cytometry after binding to the receptor at various temperature gradients.
[0136] The results showed that 16cIL21/4-Herceptin and 16cIL21-Herceptin with introduced 16-70 disulfide bond still had a partial display rate after binding to the receptor after being heated to 48° C., indicating that their stability was higher than that of IL21/4-Herceptin and IL21-Herceptin. These four proteins were a fusion protein. The detection by flow cytometry was only a preliminary sorting. It was necessary to express each of IL21 and mutant proteins, and further identify the thermal stability after purification.
[0137] 2. Expression of IL21 and its Mutant Proteins and Detection of Disulfide Bond Structure.
[0138] In order to obtain IL21 and its mutant proteins, a plasmid was firstly constructed. IL21 and mutant coding genes were inserted into PCEP4 vector plasmids respectively, in which a His tag was added. Three plasmids expressing IL21, 16cIL21 and 16cIL21/4 mutant proteins were constructed respectively (
[0139] The SDS-PAGE electrophoresis were conducted for IL21, 16cIL21 and 16cIL21/4 proteins, and the results were shown in
[0140] Mass spectrometry was further performed to confirm the formation of the target disulfide bond in 16cIL21/4 protein. The 16cIL21/4 protein was detected by mass spectrometry, and the disulfide bond should be prevented from being broken during electrophoresis. The sample protein was firstly hydrolyzed by protease and cut into peptide segments of various sizes. The disulfide bond in the protein would affect the result of enzymolysis. Under the condition that the disulfide bond was broken or not, the sample protein would be cut into peptide segments of different sizes and the peptide segments should have different distributions of mass-to-charge ratio. According to the consistency of the detected distribution with the theoretical distribution, it could determine if there was a disulfide bond in the sample protein.
[0141] Analysis of the Disulfide Bond in the Sample 16cIL21/4:
[0142] The amino acid sequences of the two segments that would generate a disulfide bond in theory were IINVCIK and QLIDCVDQLK. When enzymolysis was carried out in a reduced state, the disulfide bond was broken and C would be alkylated (the molecular weight would increase by 57 Da). The theoretical values of mass-to-charge ratio of the two peptide segments were 859.507 and 1231.635, respectively. As shown in
[0143] If enzymolysis was carried out in a non-reduced state, the disulfide bond between the two peptide segments was not broken. We could detect a peptide with a mass-to-charge ratio equal to the sum of that of the two peptide segments. The theoretical mass-to-charge ratio of the sum of the two peptide segments would be (859.587−57)+(1231.769−57)−2−1=1974.356 in a non-reduced state. In
[0144] The above results were also observed during the enzymolysis of 16cIL21 protein, which indicated that a disulfide bond was rightly formed between C at position 16 and C at position 70.
[0145] 3. Determination of Melting Temperature (Tm) of IL21 and Each of the Mutant Proteins.
[0146] After the formation of a disulfide bond between cys at positions 16 and position 70 in 16cIL21/4 protein was confirmed, we measured the melting temperature (Tm) of three proteins, IL21, 16cIL21 and 16cIL21/4, to determine the thermal stability of IL21 and each of the mutant proteins. The results were shown in
[0147] As shown in the results, the Tm values of IL21, 16cIL21 and 16cIL21/4 were as follows: 16cIL21 (65.62° C.)>16cIL21/4 (56.52° C.)>IL21 (48.54° C.).
[0148] The results suggested that the thermal stability of the two mutants 16cIL21 and 16cIL21/4 proteins with a disulfide bond was significantly higher than that of the wild-type IL21 protein. It could be considered that the introduction of disulfide bond would be the reason for improving the thermal stability.
Example 5. Determination of Biological Activities of Various IL21 Mutants
[0149] 1. The Ability of Stimulating the Proliferation of a KOB Cell.
[0150] We detected whether the biological activities of three proteins, IL21, 16cIL21 and 16cIL21/4, were remained while the thermal stability was improved. KOB cells express IL21 receptor and can be stimulated by IL21 to proliferate. The biological activity of various IL21 mutants could be determined by the ability of stimulating the proliferation of KOB cell. The particular processes were as follows: [0151] (1) Preparation of KOB cell: The KOB cells were cultured in RPMI1640 medium containing 10% calf serum (Hyclone) and 100 U/mL double antibody, in 5% CO.sub.2 incubator at 37° C. [0152] (2) Preparation of IL21 and the mutant proteins: The expression plasmids of IL21 and the mutant proteins (with His tag) were constructed and transfected into 293F cells. The steps of culture, expression and purification by nickel column were conducted conventionally. The protein concentration was determined by BCA assay. [0153] (3) Determination of biological activity: KOB cells were seeded in a 96-well plate with 2×10.sup.4 cells/well, and 200 μl culture medium and IL21 or a mutant protein was added into each well. The gradient concentration of IL21 or mutant protein was set to 0.1 ng/ml, 1 ng/ml and 10 ng/ml. The cells were cultured in 5% CO.sub.2 incubator at 37° C., and counted after 96 hours.
[0154] The results were shown in
[0155] According to the results of non-repeated Two-Way Analysis of Variance, the p value between control group and each protein group was less than 0.05 (p<0.05), indicating that there was a significant difference between groups. The p value among the three protein groups is above 0.05 (P>0.05), indicating that there was no significant difference among the three protein groups. The p value among the groups with different concentrations of the three proteins was above 0.05 (P>0.05), indicating that there was no significant difference among the groups with different concentrations of the three proteins.
[0156] From this result, all the three proteins had an effect of stimulating the proliferation of KOB cell and had no significant difference in biological activity. When the gradient concentrations of added protein were 0.04 μg/ml, 0.2 g/ml and 1 g/ml, there was no significant difference between the groups with different concentrations of protein.
[0157] Therefore, the two mutant proteins, 16cIL21 and 16cIL21/4, had improved thermal stability and remained the biological activity.
[0158] 2. Test Results of Plasma Half-Life of the Fusion Protein of Herceptin and IL21 or Each of the Mutants in Mouse
[0159] To determine whether the improved stability of IL21 could increase the plasma half-life in animal, we selected IL21-Herceptin fusion protein, mutated 16cIL21-Herceptin fusion protein and 16cIL21/4-Herceptin fusion protein for comparison test to detect their plasma half-life in mouse. The Herceptin group was used as a control group. The results were shown in Table 4:
TABLE-US-00001 TABLE 4 Half-life of different types of IL21/fusion proteins. IL21-Herceptin 16cIL21-Herceptin 16cIL21/4-Herceptin Herceptin Half-life of 44.14 h 161.18 h 198.02 h — IL21chimeric block Half-life of Herceptin 364.78 h 187.32 h 385.05 h 462.06 h chimeric block
[0160] As shown in the results, the highly stable IL21 mutant contained in 16cIL21-Herceptin and 16cI21/4-Herceptin fusion protein had significantly increased plasma half-life compared with wild-type IL21 in mouse. The 16cIL21/4-Herceptin fusion protein had the longest plasma half-life. This suggested that the improved stability of IL21 was an important reason for increasing plasma half-life.
[0161] 3. Determination of T Cells Proliferation Stimulated by IL21 Fusion Protein.
[0162] The functional activity of 16cIL21/4-Herceptin fusion protein was detected using BT474 breast cancer-bearing mouse model. 1.5×10.sup.7 human BT474 breast cancer cells were inoculated on the right abdomen or back of a mouse. The fusion protein was administered after 35 days. The proliferation of CD8+T cells stimulated by 16cIL21/4-Herceptin fusion protein and Herceptin in the mouse was detected. The results were shown in
[0163] 4. Tumor Inhibitory Effect of IL21 Fusion Protein.
[0164] The tumor inhibitory effect of 16cIL21/4-Herceptin fusion protein was detected using BT474 breast cancer-bearing mouse model. Each mouse was inoculated with 1×10.sup.7 BT474 cells (human breast cancer cells). The mice were injected intraperitoneally with PBS (Control), Herceptin (Herceptin alone), 16cIL21/4-Herceptin fusion protein (IL-21.sub.mutant−Herceptin) and the mixed solution of 16cIL21/4 and Herceptin (IL-21.sub.mutant+Herceptin) once every four days. The dosage of each injection was 500 μg. The results were shown in
[0165] In the end, it should be noted that the above examples are only used to help those skilled in the art understand the essence of the present invention, and should not be used to limit the protection scope of the present invention.