Patent Description:
In order to solve the problem of immunosuppression in tumor immunotherapy, the treatment with a cytokine and small molecule inhibitor may play a role in immune regulation. The treatment with a cytokine and small molecule inhibitor may specifically suppress inhibitory cell subsets, of their number or function, or activate an anti-tumor effector cell by immunostimulatory molecules to enhance the anti-tumor immune response of a patient. IL2 (Interleukin-<NUM>) is not only an important mediator in immune response, but also known as one of the most powerful anti-tumor cytokines, which has a wide range of biological activities. IL2 may directly act on a thymic T cell, promote the differentiation of Treg cell, suppress autoimmunity, and can also promote the differentiation of an initial T cell into effector T cell and memory T cell, which can be activated by antigen stimulation. This can protect a human against being infection. However, systemic administration of IL2 may lead to a strong adverse reaction, which limits the therapeutic dose of IL2 [<NUM>-<NUM>]. In addition, the plasma half-life of IL2 is short (about <NUM>-<NUM> hours) [<NUM>], so that the optimal dose in local microenvironment of tumor cannot be achieved. This also limits its clinical application.

In the family of cytokines, IL2, IL15 and IL21 all belong to a common cytokine receptor γ chain family. They can all promote the activity of T cell and NK cell and enhance their lethal effect on target cell. These features make them be an attractive cytokine in tumor immunotherapy study [<NUM>-<NUM>].

IL21 (Interleukin-<NUM>), a type I cytokine found in <NUM>, belongs to the γc family, which consists of <NUM> amino acids. It is mainly secreted by CD4+T cell subset, such as TH17 cell and TFH (T follicular helper) cell. NKT cell can also secrete a high level of IL21 [<NUM>-<NUM>]. It has been reported that IL21 receptor (IL21R) is expressed on the surface of T cell, B cell, NK cell, dendritic cell and monocyte/macrophage, and IL21 is a cytokine with a wide range of immunomodulatory effects.

Many preclinical tests suggested that IL21 resists tumor by various mechanisms, including the innate immune system and the acquired immune system [<NUM>]. IL21 has been approved to enter a human clinical trial to treat melanoma and renal cancer, demonstrating a good therapeutic effect [<NUM>-<NUM>]; IL21 can enhance the response of NK cell to pancreatic cancer cell pre-coated with cetuximab [<NUM>]; A fusion protein constructed by IL21 and monoclonal antibody Rituximab (anti-CD20) was shown to directly induce apoptosis or kill non-Hodgkin's lymphoma (NHL) through effector cell in an animal experiment [<NUM>].

Our previous studies have found that IL21 can significantly improve the tumor-killing effect of the antibody (Herceptin) of breast cancer by transforming M2 macrophage (which increases the drug resistance and malignancy of tumor) into M1 macrophage (which has anti-tumor function) in a mouse breast cancer model [<NUM>].

Meanwhile, many clinical application studies on IL21 suggested that the toxic side effects of IL21 are less than those of IL2 or IFN-α at same dose, which are mainly manifested as a mild symptom, such as fever, fatigue, headache, rash etc., and also manifested as a serious symptom, such as abdominal pain, thrombocytopenia, hypophosphatemia, liver function damage, etc. Nevertheless, at a usual dose of <NUM>/kg, the incidence of various toxic side effects is still up to <NUM>% [<NUM>]. This problem is necessarily to be handled carefully in systemic administration. In fact, although the clinical application of IL21 has been studied for many years, the trials are still in the preclinical phase I and phase II. An important reason is that there is a problem of high incidence of toxic side effects.

In order to increase the effective concentration of cytokine in local area around tumor to avoid systemic toxic reaction, some researchers try to deliver cytokine taking advantage of the targeting ability of an antibody. The combination of the immunotherapeutic ability of cytokine and the targeted anti-tumor reaction of antibody may result high concentration of cytokines in local area around tumor, so as to effectively stimulate a cellular immune response. The combination of a cytokine and a cell-specific antibody are named as an immunocytokine [<NUM>].

However, some clinical studies showed that IL21 has a short plasma half-life, which is only about <NUM> hours [<NUM>]. Even though the fusion protein constructed by IL21 and monoclonal antibody has a molecular weight of up to about <NUM> kD, it still has a short plasma half-life. The fusion protein constructed by IL21 and Rituximab (anti-CD20) has a plasma half-life of only about <NUM> hours in an animal experiment [<NUM>], and still has a low druggability. Therefore, it is required to improve the stability of IL21 to increase its half-life and improve its druggability.

Structural analysis showed that IL21 protein has two conformations, a stable conformation and an unstable conformation, and this is a reason why IL21 has a short half-life. It has been reported that a chimeric IL21/<NUM> may be constructed by substituting the unstable region in the protein structure of IL21 with the homologous region of IL4 (interleukin-<NUM>). The results showed that the chimeric IL21/<NUM> has a unique and stable protein conformation, and has an improved biological activity [<NUM>]. Further IL21 mutants have been described [<NUM>-<NUM>].

In our previous studies, we have established a stable mammalian cell surface protein display system for in vitro control of protein conformation (see <CIT>, <CIT>), which can be used to screen and identify a rationally designed protein or random mutation. In the present invention, we will use this system to stably display interleukin-<NUM> (IL21), and optimize and obtain a group of mutant IL21 proteins through the design based on protein structure analysis.

The invention firstly relates to an interleukin-<NUM> protein (IL21) mutant, as shown in SEQ ID NO: <NUM>, which is: with ILE at position <NUM> and SER at position <NUM> of the amino acid sequence of the wild-type IL21 both mutating into CYS, and a disulfide bond forming between the two mutated CYSs, wherein the amino acid sequence of the wild-type IL21 is shown in SEQ ID NO.

The invention also relates to a mutant of interleukin-<NUM> and interleukin-<NUM> chimeric protein (IL21/<NUM>) as shown in SEQ ID NO: <NUM>, which is: with ILE at position <NUM> and SER at position <NUM> of the amino acid sequences of the interleukin-<NUM> and interleukin-<NUM> chimera (IL21/<NUM>) both mutating into CYS, and a disulfide bond forming between the two mutated CYSs, wherein the amino acid sequence of the interleukin-<NUM> and interleukin-<NUM> chimeric protein (IL21/<NUM>) is shown in SEQ ID NO.

The invention also relates to a nucleotide sequence encoding the IL21 mutant or the IL21/<NUM> mutant.

The invention also relates to a fusion protein comprising the IL21 mutant or the IL21/<NUM> mutant, wherein the fusion protein comprises:.

and a connecting domain that connects the different functional fragments.

The invention also relates to the IL21 mutant or the IL21/<NUM> mutant for use in treating tumor.

The invention also relates to the fusion protein comprising the IL21 mutant or the IL21/<NUM> mutant for use in treating tumor.

The invention also relates to a use of the IL21 mutant or the IL21/<NUM> mutant in promoting in vitro the differentiation and proliferation of B cell, the differentiation and proliferation of T cell, the differentiation and proliferation of NK cell.

The invention also relates to a use of the fusion protein comprising the IL21 mutant or the IL21/<NUM> mutant in promoting in vitro the differentiation and proliferation of B cell, the differentiation and proliferation of T cell, the differentiation and proliferation of NK cell.

The invention also relates to a medicine or pharmaceutical composition with the IL21 mutant or the IL21/<NUM> mutant as an active ingredient, which comprises a therapeutically effective amount of the IL21 mutant or the IL21/<NUM> mutant and a necessary pharmaceutical excipient.

The invention also relates to a medicine or pharmaceutical composition with the fusion protein comprising the IL21 mutant or the IL21/<NUM> mutant as an active ingredient, which comprises a therapeutically effective amount of the fusion protein and a necessary pharmaceutical excipient.

The left panel shows the negative control group of CHO working cell, and the right panel shows the experimental group after transfection. The ordinate presents the display rate of Herceptin HC after binding with the constant region of Herceptin heavy chain. The p3 region shows the display rate of IL21/<NUM>-Herceptin after successful substitution, which was about <NUM>%.

<FIG> shows the display rate of IL21/<NUM>-Herceptin after FRT-IL21/<NUM>-Herceptin plasmid being substituted into a CHO working cell, which was sorted and enriched;
The left panel shows the negative control group of CHO working cell, and the right panel shows the experimental group after transfection. The abscissa presents the display rate after binding to the extracellular domain of IL21 receptor IL21Rα, and the ordinate presents the display rate after binding to the extracellular domain of IL21 receptor γc. The experimental group showed a display rate of <NUM>% after binding to the extracellular domain of IL21Rα, and a display rate of less than <NUM>% after binding to the extracellular domain of γc.

<FIG> shows the display rate after IL21/<NUM>-Herceptin cell binding to Mouse Anti-human IgG-APC antibody.

The left panel shows the negative control group of CHO working cell, the right panel shows the experimental group after transfection. The abscissa presents the display rate after binding to the extracellular domain of IL21 receptor γc; the ordinate presents the display rate of Herceptin HC after binding to the constant region of Herceptin heavy chain. The results showed that the display rate of Herceptin after binding to the fusion protein was <NUM>%.

<FIG> shows the display rate of IL21/<NUM>-Herceptin detected after mutating the disulfide bond.

The shown display rate results were from CHO cells after enrichment, which displayed 16c IL21/<NUM>-Herceptin, 17c IL21/<NUM>-Herceptin and 4c IL21/<NUM>-Herceptin protein. The abscissa presents the display rate after binding to the extracellular domain of IL21 receptor IL21Rα; the ordinate presents the display rate after the constant region of Herceptin heavy chain binding to the labeled antibody, Mouse Anti-human IgG-APC antibody. It shows that the display rate of 16cIL21/<NUM>-Herceptin and 4cIL21/<NUM>-Herceptin group after binding to the constant region of Herceptin heavy chain was significantly higher than that before enrichment; while the display rate of 17cIL21/<NUM>-Herceptin group after binding to the constant region of Herceptin heavy chain is only <NUM>% after enrichment. It shows that the display rate of 16cIL21/<NUM>-Herceptin and 4cIL21/<NUM>-Herceptin groups after binding to the extracellular domain of receptor IL21Rα was significantly higher than that before enrichment; while there was no obvious binding between the 17cIL21/<NUM>-Herceptin group and the extracellular domain of receptor IL21Rα, as similar to that of the control group.

<FIG> shows the construction of expression plasmids of various mutant proteins IL21, 16cIL21 and 16c IL21/<NUM> (structure graph). The gene encoding each mutant protein was inserted into a PCEP4 vector to construct an expression plasmid, in which a His tag was added.

<FIG> shows the SDS-PAGE gel electrophoresis results of IL21 and each of the mutant proteins.

<FIG> shows the mass spectrogram of 16cIL21 and 16cIL21/<NUM> mutant proteins after enzymatic hydrolysis.

<FIG> shows the melting temperatures (Tm) of IL21, 16cIL21 and 16cIL21/<NUM> to determine the thermal stability of IL21 and each of the mutant proteins.

<FIG> shows the proliferation of KOB cell stimulated by IL21, 16cIL21 and 16cIL21/<NUM> proteins.

<FIG> shows the proliferation of CD8+T cells stimulated in vivo by fusion protein 16cIL21/<NUM>-Herceptin and Herceptin in mouse.

<FIG> shows the anti-tumor test results of the fusion protein 16clL21/<NUM>-herceptin in vitro. In the figure, Control represents the control group injected with PBS; Herceptin represents the experimental group injected with Herceptin alone; IL-<NUM>mutant-Herceptin represents the experimental group with fusion protein of 16cIL21/<NUM> and Herceptin; and IL-<NUM>mutant+Herceptin represents the experimental group with mixed solution of 16cIL21/<NUM> and Herceptin, instead of a fusion protein.

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.

CHO/dhFr- (Dihydrofolate reductase-deficient Chinese hamster ovary cells): The cells were cultured with IMDM medium containing <NUM>% calf serum (Hyclone), <NUM> U/mL double antibody, <NUM> hypoxanthine and <NUM> thymine at <NUM> in a <NUM>% CO<NUM> incubator.

293F cells: Human embryonic kidney cells, which were cultured with FreeStyle™ <NUM> Expression Medium in shake flasks at <NUM> rpm and <NUM> in a <NUM>% CO<NUM> incubator.

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 <NUM>% calf serum (Hyclone) and 100U/mL double antibody at <NUM> in a <NUM>% CO<NUM> incubator.

Cell preparation: The cells were seeded in a six-well plate with the amount of <NUM>,<NUM> cells per well one day before transfection, so that, at the time of transfection, they reached <NUM>% confluence, distributed evenly and grew well.

Preparation of transfection complex: <NUM>µl Lipo2000 and <NUM> Opti-MEM serum-free medium were mixed evenly; <NUM>µg target plasmid was mixed with <NUM>µl Opti-MEM serum-free medium evenly. After standing at room temperature for <NUM> minutes, these two mixtures were mixed to prepare the transfection complex, which were gently mixed and placed at room temperature for <NUM> minutes.

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 <NUM>µL of Opti-MEM serum-free medium was added. After <NUM>-<NUM> hours, the medium was changed into the common culture medium to continue the culture.

Transfection of 293F cell (<NUM> culture medium as an example):
Cell preparation: <NUM>×<NUM><NUM>-<NUM>×<NUM><NUM> cells/ml was seeded one day before transfection, and the cell density should be <NUM>×<NUM><NUM> cells/ml at the time of transfection.

Preparation of transfection complex: <NUM>µg of target plasmid was diluted with <NUM> OptiPROTMSFM (Invitrogen) serum-free medium and fully mixed to prepare a DNA diluent. <NUM>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 <NUM> minutes. The transfection complex was added into 293F cell culture medium, and then cultured in shake flasks at <NUM> rpm and <NUM> in a <NUM>% CO<NUM> incubator for <NUM> hours.

After 293F cells being transfected by the plasmid containing the target gene, the cells were continuously cultured in a shaker for <NUM> hours and then centrifuged at <NUM> for <NUM> minutes to remove cell precipitates. The supernatant medium was collected, centrifuged at <NUM> for <NUM> minutes to remove impurities in the culture medium, filtered with a <NUM> filter membrane, centrifuged in a <NUM> kd concentration tube at <NUM> under <NUM> rmp, to concentrate <NUM> times of the volume.

A Sepharose high performance (Amersham Bioscience) chromatographic column filled with nickel sulfate (NiSO<NUM>) was used. Buffers containing <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> imidazole were prepared respectively. Other components in the buffer were <NUM> Tris-HCL, <NUM> NaCl and <NUM>% glycerol.

Washing and balancing the nickel column: the nickel column was firstly washed with <NUM> ddH<NUM>O, and then washed and balanced with <NUM> buffer containing <NUM> imidazole.

Sample loading: The supernatant containing the target protein was dripped into the nickel column, which might be repeated <NUM>-<NUM> times.

Elution: After loading the sample, the column was eluted with <NUM> buffer containing <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> M 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 <NUM>-<NUM> times of the volumes.

Protein preservation: The finally obtained protein was packaged separately, quick-frozen in liquid nitrogen, and preserved at -<NUM>.

A Protein A SefinoseTM-<NUM> (Pre-Packed Gravity Column) chromatographic column was used.

Washing and balancing: the chromatographic column was firstly washed with <NUM> ddH<NUM>O, then washed with <NUM> elution buffer, and then balanced with <NUM> binding buffer.

Sample loading: The supernatant containing a target protein was dripped into the chromatographic column, which might be repeated <NUM>-<NUM> times.

Elution: After loading the sample, the column was washed with <NUM> binding buffer to remove non-specific binding. Ten <NUM> centrifuge tubes were prepared, each being added into <NUM>µl pre-prepared neutralizing buffer. The protein was eluted with <NUM> elution buffer, and collected by <NUM> centrifugal tubes with neutralizing buffer, each collecting <NUM> of the elution.

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 <NUM>/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.

Washing and preservation of chromatographic column: The chromatographic column was washed with <NUM> binding buffer, then washed with <NUM> of ddH<NUM>O, and finally washed with <NUM> <NUM>% ethanol and sealed.

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.

The protein to be determined was firstly adjusted to a concentration of <NUM>-<NUM> and a volume of <NUM>µl.

The Sypro® orange protein (5000x) was used as a dye, which was diluted to 25x with DMSO.

Preparation of <NUM>µl detection system: Each well of the <NUM>-well plate was added with <NUM>µl protein (<NUM>-<NUM>), and then with <NUM>µl Sypro@orang protein (25x), mixed thoroughly, and kept at room temperature away from light.

Stepone software <NUM> in qPCR instrument was used for detection, and operating and parameter setting were according to instructions.

The experimental results were determined and saved. The results were analyzed by the program of Protein Thermal Shift <NUM>.

IL21 has two protein conformations: a stable conformation and an unstable conformation. A chimeric IL21/<NUM> 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/<NUM> 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/<NUM> and Herceptin on the surface of a CHO working cell, it was also beneficial to detect the display efficiency of chimeric IL21/<NUM> through detecting the constant region of Herceptin heavy chain. Therefore, we firstly displayed the fusion protein of chimeric IL21/<NUM> and Herceptin on the surface of a CHO working cell.

In order to obtain an efficient and stable CHO cell line for displaying chimeric IL21/<NUM>, we constructed it using a protein display system established earlier in our laboratory by recombinase-mediated cassette exchange (RMCE) (see <CIT> for details). A recombinant substitution plasmid fragment (FRT-IL21/<NUM>-Herceptin plasmid) was firstly constructed for displaying the fusion protein of IL21/<NUM> 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/<NUM> linked to the N-terminal of the Herceptin heavy chain via a <NUM>(G4S) linker, and the transmembrane region (TM) linked to the C-terminal of the Herceptin heavy chain. By this, the fusion protein of IL21/<NUM> 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 <FIG> (from left to right, the fusion protein comprising: FRT recombination site, the signal peptide of the Herceptin light chain, Herceptin light chain, IRES (internal ribosome entry site) that could bind to ribosome to initiate translation, the signal peptide of the Herceptin heavy chain, IL21/<NUM>, <NUM>(G4S) linker, Herceptin heavy chain, the transmembrane region (TM) that allowed protein molecule anchored and displayed on a cell surface, and LOXp recombination site.

A CHO working cell established in our laboratory (see <CIT> for details), whose genome was inserted with a single copy of recombinant substitution region FRT-puromysin-Loxp, were co-transfected with the FRT-IL21/<NUM>-Herceptin plasmid and the pCI-2A plasmid previously constructed in our laboratory. The fusion protein sequence of Herceptin and IL21/<NUM> between Loxp and FRT sites in FRT-IL21/<NUM>-Herceptin plasmid could be recombined and substituted into the genome of the CHO working cell in single-copy, as shown in <FIG>.

After transfection, the cells were collected on time, and then labeled with Mouse Anti-human IgG-APC antibody. The display rate after IL21/<NUM>-Herceptin substitution was detected by flow cytometry. The results (<FIG>) showed a display rate of <NUM>%, suggesting that the probability of substitution recombination after transfection was very low. Therefore, the cells with successful substitution and positive test results were required to be detected again after enrichment. To avoid a false positive signal, the cells with a strong positive signal in p3 area of the upper left quadrant of the above figure were sorted by flow FACS AriaIII, then were cultured and expanded.

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/<NUM> and Herceptin domains in IL21/<NUM>-Herceptin fusion protein. The results were shown in <FIG> and <FIG>. As shown in <FIG>, the abscissa presents the display rate (<NUM>%) of IL21/<NUM> in IL21/<NUM>-Herceptin fusion protein after binding to the extracellular domain of IL21 receptor IL21Rα; the ordinate presents that the display rate ( < <NUM>%) of IL21/<NUM> in IL21/<NUM>-Herceptin fusion protein after binding to the extracellular domain of IL21 receptor γc. As reported, the affinity of IL21 binding to receptor γc was much lower than that to receptor IL21Rα, which was also confirmed for several times in present experiment.

As shown in <FIG>, the abscissa presents that the display rate of IL21/<NUM> in IL21/<NUM>-Herceptin fusion protein was less than <NUM>% after binding to the extracellular domain of IL21 receptor γc, and the ordinate presents that the display rate of constant region of Herceptin heavy chain in fusion protein was <NUM>% after binding to the labeled antibody, Mouse Anti-human IgG-APC antibody, higher than the display rate (<NUM>%) before enrichment. Combined with the labeling results of IL21R α extracellular domain-linker-GFP fusion protein, it suggested that the affinity of Herceptin heavy chain constant region to the labeled antibody, Mouse Anti-human IgG-APC antibody, was lower than that to IL21R α extracellular domain-linker-GFP.

The above-mentioned CHO cell that stably displayed IL21/<NUM>-Herceptin fusion protein was sorted by flow FACS AriaIII and named as an s0 cell.

The steps for constructing the plasmid that could stably display chimeric IL21/<NUM> on the surface of a CHO cell in the molecular biology experimental process were as follows:.

In order to obtain a CHO cell line that could stably display each of IL21/<NUM> mutants, with FRT-IL21/<NUM>-Herceptin as a template, recombinant substitution plasmids of each of IL21/<NUM> mutants were firstly obtained by point mutation technique, named as 16cIL21/<NUM>-Herceptin, 17cIL21/<NUM>-Herceptin and 4cIL21/<NUM>-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.

As shown in <FIG>, the abscissa represents the display rate of various mutant IL21/<NUM>-Herceptin fusion proteins after binding to the extracellular domain of IL21 receptor IL21Rα. The ordinate represents the display rate of the constant region of Herceptin heavy chain in fusion protein after binding to the labeled antibody, Mouse Anti-human IgG-APC antibody. As shown, the display rate of 16cIL21/<NUM>-Herceptin and 4cIL21/<NUM>-Herceptin were significantly higher than those before enrichment, and bound to the extracellular domain of receptor IL21Rα very well. However, the ordinate of the control group, 17cIL21/<NUM>-Herceptin, represents a display rate of only <NUM>% after binding to the labeled antibody, Mouse Anti-human IgG-APC antibody. There was no obvious binding to the extracellular domain of the receptor IL21Rα. This indicated that the mutant of the control group, 17cIL21/<NUM>-Herceptin, was not displayed.

We had obtained a CHO working cell that could stably display two kinds of IL21/<NUM> mutants on the surface of cell membrane, and then we would do a preliminary stability test of these two IL21/<NUM> mutants.

To test the stability of each of the IL21/<NUM> mutants, the CHO working cells displaying IL21/<NUM>-Herceptin and the CHO working cells displaying mutants 16cIL21/<NUM>-Herceptin and 4cIL21/<NUM>-Herceptin were used as an experimental group. Each group was heated at various temperature gradients of <NUM>-<NUM>, 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.

The particular experimental processes were as follows:.

In order to study the effect of introducing a disulfide bond at the same positions (positions <NUM> and <NUM>) on stability in wild-type IL21 molecule, we constructed two other structures:.

The results showed that 16cIL21/<NUM>-Herceptin and 16cIL21-Herceptin with introduced <NUM>-<NUM> disulfide bond still had a partial display rate after binding to the receptor after being heated to <NUM> , indicating that their stability was higher than that of IL21/<NUM>-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.

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/<NUM> mutant proteins were constructed respectively (<FIG>).

The SDS-PAGE electrophoresis were conducted for IL21, 16cIL21 and 16cIL21/<NUM> proteins, and the results were shown in <FIG>. From the results, the bands of IL21, 16cIL21 and 16cIL21/<NUM> were consistent with their molecular weight in theory. From the molecular weights, these three proteins were the target proteins.

Mass spectrometry was further performed to confirm the formation of the target disulfide bond in 16cIL21/<NUM> protein. The 16cIL21/<NUM> 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.

Analysis of the disulfide bond in the sample 16cIL21/<NUM>:
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 <NUM> Da). The theoretical values of mass-to-charge ratio of the two peptide segments were <NUM> and <NUM>, respectively. As shown in <FIG>, there were obvious signal peaks at <NUM> and <NUM> in the reduced state (with DTT), while the two signal peaks were very low in a non-reduced state. The results suggested that the disulfide bond was broken and two peptide segments were generated in the 16cIL21/<NUM> sample protein when adding DTT. Not adding DTT, the disulfide bond would not be broken, and there was no signal of the two peptide segments. This was consistent with the disulfide bond structure of theoretical prediction.

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 (<NUM>-<NUM>) + (<NUM>-<NUM>)-<NUM>-<NUM>=<NUM> in a non-reduced state. In <FIG>, we could see an obvious peak with a mass-to-charge ratio of <NUM> in the non-reduced state, while the signal of this peak was very poor in a reduced state. This suggested that the disulfide bond of sample protein was not broken when not adding DTT, and thus the enzymolysis product had the signal of a large peptide segment. When adding DTT, the disulfide bond would be broken and the larger peptide segment was split, and thus the signal disappeared.

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 <NUM> and C at position <NUM>.

After the formation of a disulfide bond between cys at positions <NUM> and position <NUM> in 16cIL21/<NUM> protein was confirmed, we measured the melting temperature (Tm) of three proteins, IL21, 16cIL21 and 16cIL21/<NUM>, to determine the thermal stability of IL21 and each of the mutant proteins. The results were shown in <FIG>:
As shown in the results, the Tm values of IL21, 16cIL21 and 16cIL21/<NUM> were as follows: 16cIL21 (<NUM>) > 16cIL21/<NUM> (<NUM>) > IL21 (<NUM>).

The results suggested that the thermal stability of the two mutants 16cIL21 and 16cIL21/<NUM> 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.

We detected whether the biological activities of three proteins, IL21, 16clL21 and 16clL21/<NUM>, 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:.

The results were shown in <FIG>:
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 <NUM> (p<<NUM>), indicating that there was a significant difference between groups. The p value among the three protein groups is above <NUM> (P><NUM>), 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 <NUM> (P><NUM>), indicating that there was no significant difference among the groups with different concentrations of the three proteins.

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 <NUM>µg/ml, <NUM>/ml and <NUM>/ml, there was no significant difference between the groups with different concentrations of protein.

Therefore, the two mutant proteins, 16clL21 and 16clL21/<NUM>, had improved thermal stability and remained the biological activity.

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/<NUM>-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 <NUM>:.

As shown in the results, the highly stable IL21 mutant contained in 16cIL21-Herceptin and 16cl21/<NUM>-Herceptin fusion protein had significantly increased plasma half-life compared with wild-type IL21 in mouse. The 16clL21/<NUM>-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.

The functional activity of 16cIL21/<NUM>-Herceptin fusion protein was detected using BT474 breast cancer-bearing mouse model. <NUM>×<NUM><NUM> human BT474 breast cancer cells were inoculated on the right abdomen or back of a mouse. The fusion protein was administered after <NUM> days. The proliferation of CD8+T cells stimulated by 16cIL21/<NUM>-Herceptin fusion protein and Herceptin in the mouse was detected. The results were shown in <FIG>. In the 16cI21/<NUM>-herceptin fusion protein group, the proportion of CD8+T cells in peripheral blood showed an obvious increase about <NUM> days after tumor inoculation. Meanwhile, T cells in Herceptin group had a tendency of slowly decreasing.

The tumor inhibitory effect of 16cIL21/<NUM>-Herceptin fusion protein was detected using BT474 breast cancer-bearing mouse model. Each mouse was inoculated with <NUM>×<NUM><NUM> BT474 cells (human breast cancer cells). The mice were injected intraperitoneally with PBS (Control), Herceptin (Herceptin alone), 16clL21/<NUM>-Herceptin fusion protein (IL-<NUM>mutant-Herceptin) and the mixed solution of 16cIL21/<NUM> and Herceptin (IL-<NUM>mutant+Herceptin) once every four days. The dosage of each injection was <NUM>µg. The results were shown in <FIG>. Compared with the control group, Herceptin group and mixed solution group, 16clL21/<NUM>-Herceptin fusion protein group significantly reduced tumor volume.

Claim 1:
A mutant of interleukin-<NUM>(IL21), as shown in SEQ ID NO: <NUM>, which is:
with ILE at position <NUM> and SER at position <NUM> of the amino acid sequence of the wild-type IL21 both mutating into CYS, and a disulfide bond form between the two mutated CYSs,
wherein the amino acid sequence of the wild-type IL21 is shown in SEQ ID NO: <NUM>.