Disclosure of Invention
The invention overcomes the defects of the prior art and provides an enhanced IL-7 mutant and a CIK cell containing the mutant.
In one aspect, the present invention provides an improved enhanced IL-7 mutant, the gene sequence of which is shown below (SEQ ID NO: 2).
Further, the invention also provides a transgenic CIK cell transfected with the nucleotide sequence shown in SEQ ID NO: 2 under the control of a control gene.
Further, the present invention provides a pharmaceutical composition comprising the modified CIK cells described herein and a pharmaceutically acceptable carrier or excipient.
The present application also provides methods of inhibiting cancer cells by administering to a subject in need thereof an effective cancer cell inhibiting amount of a modified CIK cell of the invention or a pharmaceutical composition of the invention. Without being bound by any particular theory, it is believed that the modified CIK cells inhibit cancer cells by one or more CIK cell functions: enhance cytotoxicity, stimulate proliferation of cancer-specific T lymphocytes or IFN-secretion.
Routes of administration of the pharmaceutical composition or modified CIK cells of the invention include, but are not limited to, intravenous, intramuscular, subcutaneous, oral, topical, intradermal, transdermal, subcutaneous, parenteral, rectal, spinal or epidermal administration. In one embodiment, the modified CIK cells are administered by intravenous injection or infusion.
The pharmaceutical compositions of the present application may be prepared as injectables, liquid solutions or suspensions, or solid forms suitable for dissolution in, or suspension in, liquid carriers prior to injection.
The modified CIK cells of the invention are formulated into pharmaceutical compositions for delivery to a mammalian subject. The pharmaceutical composition of CIK is administered alone and/or in admixture with a pharmaceutically acceptable carrier, excipient or vehicle. Suitable carriers for CIK are, for example, saline (e.g., physiological saline), glucose, glycerol, Platelet Rich Plasma (PRP), and the like, and combinations thereof. CIK in addition, the carrier may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents or adjuvants. The CIK pharmaceutically acceptable carrier may comprise a physiologically acceptable compound that functions, for example, to stabilize, or increase or decrease the rate of absorption or clearance of the pharmaceutical composition of the present application. The CIK physiologically acceptable compound may include, for example, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, detergents, liposome carriers, or excipients or other stabilizers and/or buffers. Other physiologically acceptable compounds of CIK include wetting agents, emulsifying agents, dispersing agents or preservatives. The pharmaceutical compositions of the present application may also include auxiliary substances, such as pharmacological agents, cytokines or other biological response modifiers.
The modified CIK cells or pharmaceutical composition of the invention may be administered as a single dose treatment or as a multiple dose treatment over a period of suitable age as planned, the weight and condition of the CIK subject, the particular composition used for CIK, CIK and route of administration, CIK whether modified CIK cells or pharmaceutical composition of the invention are used for prophylactic or therapeutic purposes, e.g., CIK in one embodiment, CIK is administered once a month for the modified CIK cells or pharmaceutical composition of the present application, CIK twice a month, CIK three times a month, CIK every other week, CIK once a week, CIK 2 times a week, 3 times a week, 4 times a week, 5 times a week, 6 times a week, every other day, daily), 2 times a day, 3 times a day or 4 times a day.
The pharmaceutical composition is formulated to comprise an effective amount of the modified CIK cells of the invention, wherein the amount depends on the animal to be treated and the condition to be treated. The specific dose level for any particular subject will depend upon a variety of factors including the activity of the specifically modified CIK cells, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy. An exemplary, non-limiting range of therapeutically or prophylactically effective amounts of the modified CIK cells of the present application is at least about 1X 10 3 About 1X 10 per dose 9 Per cell per dose of cells. Other dosages are also possible, including but not limited to 1 × 10 4 ,1×10 5 ,1×10 6 ,1×10 7 ,1×10 8 Or 1X 10 9 Per dose of cells.
The pharmaceutical compositions of the present invention may also be administered in combination therapy, i.e., in combination with other agents. For example, a combination therapy may comprise a composition of the invention in combination with at least one anti-inflammatory agent or at least one immunosuppressive agent. In one embodiment, such therapeutic agents include one or more anti-inflammatory agents, such as a steroidal drug or NSAID (non-steroidal anti-inflammatory drug). Preferred agents include, for example, aspirin and other salicylates, COX-2 inhibitors, such as rofecoxib (Vioxx) and celecoxib (Celebrex), NSAIDs such as ibuprofen (Motrin, Advil), fenoprofen (Nalfon), naproxen (Naprosyn), sulindac (Clinoril), diclofenac (Voltaren), piroxicam (Feldene), ketoprofen (oridis), diflunisal (doluninsal), nabumetone (Relafen), etodolac (Lodine), oxaprozin (Daypro) and indomethacin (Indocin).
In yet another embodiment, the pharmaceutical composition further comprises that such therapeutic agents include one or more chemotherapeutic agents, such as paclitaxel derivatives, docetaxel, gemcitabine, 5-fluorouracil, doxorubicin (adriamycin), cisplatin (platinum alcohol), cyclophosphamide (cytotoxin, protocytotoxin, Neosar). In another embodiment, the antibodies of the invention may be administered in combination with a chemotherapeutic agent, preferably one that exhibits therapeutic efficacy in patients with breast, lung, stomach, prostate and/or ovarian cancer or other types of cancer.
Other cancers classified by histological type may be suitable targets for therapeutic compositions according to the present application, including but not limited to tumor tumors; cancer (NOS) not otherwise specified; carcinomas, undifferentiated, macrophage and spindle cell carcinomas; small cell carcinoma, papillary carcinoma, squamous cell carcinoma, lymphoepithelial carcinoma; basal cell carcinoma, hairy basal carcinoma; transitional cell carcinoma head transitional cell carcinoma; adenocarcinoma, gastrinoma; bile duct cancer; hepatocellular carcinoma, combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis, escherichia coli; solid cancer, carcinoid tumor; bronchoalveolar carcinoma; papillary adenocarcinoma, anachoric carcinoma; eosinophilic carcinoma; hydrophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma, granular cell carcinoma; follicular adenocarcinoma, papillary and follicular adenocarcinoma; non-encapsulated hardened cancers; adrenocortical carcinoma; endometrial cancer; skin adnexal cancer; adenocarcinoma of the major gland; sebaceous gland cancer; cervical adenocarcinoma; mucosal epidermoid carcinoma; bladder cancer, papillary serous bladder cancer; mucinous bladder cancer, mucinous adenocarcinoma; seal ring cell carcinoma; invasive ductal carcinoma; bone marrow cancer, lobular cancer; inflammatory cancer; paget's disease, breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma W/squamous metaplasia; thymoma; ovarian stromal tumors; sheath tumor; granulocytoma; a fibroblast tumor; sertoli cell carcinoma; a stromal cell tumor; lipocytoma; paragangliomas; external paraganglioma of mammary gland; pheochromocytoma; angiosarcoma; malignant melanoma, melanoma; superficial diffuse melanoma; melanoma in giant pigmented nevi; epithelial-like cell melanoma; blue nevi; sarcoma, fibrosarcoma; myxosarcoma; liposarcoma, leiomyoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma, mixed tumor, mullite mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma, mesenchymal tumor; brenner tumors; phyllomas; synovial sarcoma, mesothelioma; poor germ development; embryonal carcinoma, teratoma ovarian cyst; choriocarcinoma; mesothelioma; angiosarcoma; vascular endothelioma; kaposi's sarcoma; hemangioma; lymphangioleiomyosarcoma; osteosarcoma, paracortical osteosarcoma; chondrosarcoma, chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumors; fibroblast odontoclastic sarcoma; an ameloblastic tumor; fibroblast fibrosarcoma; a cancellous tumor; a chord tumor; glioma; ependymoma, astrocytoma, protoplasmic astrocytoma; a fibrillar astrocytoma; a fibroblast tumor; glioblastoma, oligodendroglioma; primitive neuroectoderm; cerebellar sarcoma, ganglionic neuroblastoma; neuroblastoma, retinoblastoma, olfactory neurogenic tumor; meningioma; neurofibrosarcoma; neuroma; granulocytoma; malignant lymphoma, hodgkin's disease; accessory granulomatous lymphoma, small lymphocytes; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular, mycosis; other specific non-hodgkin lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia, lymphoid leukemia, plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia, basophilic leukemia; eosinophilic leukemia; monocytic leukemia, mast cell leukemia; megakaryocytic leukemia; myeloid sarcoma; and hairy cell leukemia.
Advantageous effects
The invention obtains the specific high-activity IL-7 mutant by analyzing and screening the space structure of IL-7, can obviously enhance the tumor killing effect after the mutant gene is introduced into CIK cells, and has excellent application effect.
Detailed Description
To further illustrate the objects, aspects and advantages of the present invention, we shall now describe the invention with reference to the following specific examples, which are only for better illustrating the patent of the present invention and are not intended to limit the scope of the present invention. All other embodiments that can be obtained by a person skilled in the art without making any inventive step based on the examples of the present invention belong to the protection scope of the present invention.
Example 1 preparation of IL-7 mutation and Activity identification
From the protein sequence of IL-7, protein machine learning was used, and the core structure sequence was analyzed based on the structural information of IL-7, and from the structure shown in FIG. 1, the IL-7 molecule has a main conformation of α -helical structure, and disulfide bonds contained in the structure are indispensable for the bioactivity of the IL-7 molecule. Since IL-7 must bind to its receptor IL-7R before exhibiting a biological effect, it was found by computer modeling that increasing the proportion of basic amino acids at the central long-helical position of the protein increases the efficiency of binding of IL-7 to IL-7R for the spatial structural site of the binding. In particular in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 2-3 two mutants of hIL-7mut1 and hIL-7mut 2. The sequences were biosynthesized with BglII cleavage sites added upstream and NotI cleavage sites added downstream.
Converting the amino acid sequence of SEQ ID NO: 1-3 and pET32a (+) vector were digested with restriction enzymes Bgl II and Not I, respectively, to obtain the target gene and vector with complementary cohesive ends, ligated with T4 DNA ligase, and transferred to DH5a, and spread on solid LB medium (containing Amp resistance). After positive colonies are selected and verified by plasmid PCR, plasmids which are verified to be correct are selected for sequencing (Shanghai Producer Co., Ltd.), the construction success of recombinant plasmids pET32 a-hIL-7, pET32 a-hIL-7 mut1 and pET32 a-hIL-7 mut2 is determined by sequencing, and then the recombinant plasmids are transferred into escherichia coli BL21(DE3), and engineering bacteria containing recombinant plasmids pET32 a-hIL-7, pET32 a-hIL-7 mut1 and pET32 a-hIL-7 mut2 are obtained through screening.
The engineering bacteria containing recombinant plasmids pET32 a-hIL-7, pET32 a-hIL-7 mut1 and pET32 a-hIL-7 mut2 were inoculated into LB liquid medium with ampicillin resistance, and cultured overnight at 37 ℃ with a shaker rotation speed of 180 rpm. After the overnight culture was completed, colonies were: transferring the culture medium V into a culture medium added with antibiotics according to the proportion of 1:100, continuing to culture under the same conditions, adding 0.8mmol/L IPTG inducer when detecting that the OD600 value of a colony reaches about 0.6, and carrying out shake culture in a 37 ℃ incubator for 4 h. Taking a small amount of thalli, and suspending the sediment in 50mmol/L PB, pH 8.0; ultrasonic cracking, centrifuging, and respectively performing electrophoresis on the supernatant and the precipitate, wherein the three proteins are expressed in the form of inclusion bodies.
Ultrasonically breaking the bacteria to obtain an inclusion body, washing the inclusion body by using bufferA (50mmol/L PB; 2mol/L guanidine hydrochloride pH8.0), dissolving the inclusion body in bufferB (6mol/L guanidine hydrochloride, 50mmol/L PB, 100mmol/L mercaptoethanol (B-ME) pH8.0) lg: 10ml, centrifuging 20000 Xg for 30min after the inclusion body is completely dissolved, discarding the precipitate to obtain protein denatured liquid, transferring the protein denatured liquid into a dialysis bag, changing the protein denatured liquid for 1 time after 4 hours, performing gradient dialysis, finally, transferring the protein denatured liquid into 50mmol/L PB and pH8.0 buffer solution for dialysis twice, centrifuging 20000g for lh, and discarding the precipitate to obtain a renaturated protein solution.
The renatured protein solution was subjected to nickel column purification. Nickel column balance: equilibrium solution (50mmol/L PB, 150mmol/L NaC1, pH 8.0); loading the recombinant protein; and (3) elution: eluting hetero protein with eluting solution 1(20mmol/L PB, 150mmol/L NaC1, 50mmol/L imidazole, pH8.0), eluting target protein with eluting solution 2(20mmol/L PB, 150mmol/L NaC 1200 mmol/L imidazole, pH8.0), filtering and sterilizing.
2E8 cell proliferation assay for IL-7 biological Activity: hIL-7, hIL-7mut1 and hIL-7mut2 prepared as described above were diluted to 1. mu.g/mL with the buffer RPMI 1640+ 20% FBS +0.05mM 2-mercaptoethanol and then subjected to gradient dilution in 96-well cell culture plates, and hIL-7 standard was purchased from Invivogen and diluted under the same conditions; transfer to a new 96-well cell culture plate at 100 μ Ι/well after dilution is complete; centrifuging to collect 2E8 cells, washing the cells for 2 times by PBS, after counting, resuspending to a certain concentration by buffer RPMI 1640+ 20% FBS +0.05mM 2-mercaptoethanol, adding 50 mu L/hole into a 96-hole cell culture plate filled with IL-7 standard substances or samples to be detected with different concentrations, culturing the cells for 48h under the conditions of 37 ℃ and 5% CO2, and adding 5mg/mL MTT solution into 20 holes; standing at 37 ℃ for 4h, adding 15O mu L/hole of MTT solution, blowing the liquid in the hole uniformly by using a multi-channel pipette, placing the hole into an enzyme-linked immunosorbent assay, measuring an OD570 value by taking 630nm as a reference wavelength, taking the average value of 2 multiple holes as a result, and performing curve fitting and sample activity calculation by four-factor regression by using softMaxpro5.2 software. The results of the biological activity of the samples are shown in FIG. 2.
From the results in FIG. 2, it can be seen that hIL-7 activity is slightly weaker than that of the standard, while hIL-7mut1 mutant can significantly improve the growth promoting rate of 2E8, and OD570nm reaches about 1.71 at 1000pg concentration, while hIL-7mut2 mutant does not exhibit a good growth promoting effect, but rather the activity is reduced, which fully indicates that any mutation can not improve the corresponding activity.
EXAMPLE 2 preparation of CIK cells
Taking 30ml of healthy peripheral blood, centrifuging with human lymphocyte separating medium density gradient (2000 Xg, 20min), gently sucking off-white layer mononuclear cells, placing in a 15ml centrifuge tube, and washing 3 times with normal saline. Suspending the cells in CIK medium (containing low concentrations of IL-2, IFN-. gamma., IL-12 and anti-CD3) to adjust the cell density to 1X 10 5 Perml, the cells were placed in a 12-well plate and incubated at 37 ℃ in a 5% CO2 incubator. Culturing for 36h, collecting cells, washingAfter washing and centrifugation, the culture was continued for 15 days by adding 10. mu.g/m 1 anti-CD3, 800U/m1 IL-1a and 1000U/m1 IL-2.
After 15 days of cell culture, a certain number of cells were washed and centrifuged, and the cell density was adjusted to 1X 10 with PBS 6 Adding the solution into a flow cytometry detection tube, namely a 200-mu-l tube; different fluorescently-labeled anti-human CD3, CD16 and CD56 antibodies are respectively added, the mixture is placed in a dark place and labeled for 20min at 4 ℃, PBS is used for washing for 2 times, and the detection result is carried out by a flow cytometer, and the result is shown in Table 1.
TABLE 1 CIK cell phenotype
As can be seen from Table 1, the isolated CIK cells were mainly CD3+ or CD3+ CD16+ CD56+ cells, indicating that the cultured cells were CIK cells.
Example 3 preparation of transgenic CIK cells
Converting the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2 were inserted into the pcDNA3.0 plasmids, respectively, with the empty plasmid pcDNA3.0 as a control. Coli DH 5. alpha. was transformed and then confirmed by sequencing, and the plasmid was extracted with the Endo-free Maxiplasmid Ex.
Gene transfection was performed using a square wave electroporator. CIK cells in logarithmic growth phase prepared in example 2, cultured to day 15, were washed twice with serum-free RPMI1640 medium and adjusted to a cell density of 5 × l0 with transfection buffer 6 And (3) ml. And adding the plasmid according to 20 mu g/ml, fully and uniformly mixing, transferring the mixture into an electric shock cup with a gap of 2mm, setting the transfection parameter to be 160V and the time to be 70 mu s, carrying out gene transfection under the condition of single pulse, and standing the mixture for 8 minutes at room temperature after transfection, wherein 400 mu l of the mixture is used in each cup. Then washing twice with serum-free 1640 culture solution, culturing in IL-2-containing 1640 culture solution at 37 deg.C in 5% CO2 incubator for 5 days to obtain cells called CIK-hIL-7 cell and CIK-hIL-7mut1 cell, and simultaneously using empty plasmid pcDN A3.0 as a control, the procedure was as above.
Detecting IL-7 gene transcription transfection by RT-PCR, collecting CIK cells, extracting total RNA by Trizol reagent, performing electrophoresis identification and quantification, taking 8 mu g of RNA to perform reverse transcription reaction for 1 hour at 42%, taking 5 mu l of reverse transcription product as a template, and performing PCR amplification by using IL-7 upstream and downstream primers under the specific reaction conditions of 94 ℃ and 55 s; 57 ℃ for 50 s; 72 ℃,60 seconds, thermal cycling 32 times, and final extension at 72 ℃ for 1O min. The products were identified by agarose gel electrophoresis, based on the relative expression of the control, and the results are shown in FIG. 3.
RT-PCR detection proves that IL-7mRNA transcription does exist in CIK-hIL-7mut1 and CIK-hIL-7 cells (figure 3), and the expression amount is improved by more than 4 times compared with a control.
Example 4 killing experiment of CIK cells
Culture of lung adenocarcinoma cell line SPC-A-1 frozen in liquid nitrogen is recovered, cultured with culture medium containing 10% IMDM to vigorous growth stage, digested with 0.25% pancreatin, washed with IMDM culture medium, and adjusted to 5 × 10 cell number 4 Perml, seeded into 96-well cell culture plates, 100. mu.l/well. The cell number of CIK cells is 5 × 10 6 Perml, 100 l/well of 96-well cell culture plates inoculated with SPC-A-1, with 6 wells per set, was added as follows.
Group 1: SPC-A-1 control;
group 2: CIK-hIL-7+ SPC-A-1 (50: 1);
group 3: CIK-hIL-7mut1+ SPC-A-1 (50: 1);
group 4: CIK (empty vector) + SPC-A-1 (50: 1); culturing for 48 h. MTT was added 4h before the end of the incubation, and the OD was measured at 292nm with a microplate reader. Killing activity ═ 1 one experimental set of OD value-effector cell OD value)/target cell OD value × 100. The results are shown in Table 2.
TABLE 2 killing Activity of CIK cells against SPC-A-1 cells
Experiment grouping
|
Killing rate (%)
|
Group 1
|
-
|
Group 2
|
70.56±4.23
|
Group 3
|
83.23±5.19
|
Group 4
|
63.21±2.57 |
As can be seen from the results in Table 2, the killing rate of CIK cells to cancer cells is enhanced after introducing hIL-7, and particularly, the killing rate is remarkably improved to (83.23 +/-5.19)%, after introducing mutant hIL-7mut 1. Since CIK cells (cytokine-induced killers) have the non-specific killing effect of NK cells, the killing property of the CIK cells has the characteristic of no MHC restriction, and the anti-tumor activity of strong T lymphocytes is also called as NK cell-like T lymphocytes, the CIK cells play an important role in the aspect of tumor cell immunity. Particularly, the CIK cell transferred with hIL-7mut1 has better application prospect in tumor treatment.
Sequence listing
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