CN113599395B

CN113599395B - Pharmaceutical composition for treating cancer comprising NK cells

Info

Publication number: CN113599395B
Application number: CN202111105925.9A
Authority: CN
Inventors: 张克礼; 顾超
Original assignee: Henan Yuanchuang Life Stem Cell Bank Technology Co ltd; Zhengzhou Bain Biotechnology Co ltd; Zhengzhou Revo Gene Industry Co ltd
Current assignee: Henan Yuanchuang Life Stem Cell Bank Technology Co ltd; Zhengzhou Bain Biotechnology Co ltd; Zhengzhou Yuanchuang Gene Technology Co ltd
Priority date: 2021-09-22
Filing date: 2021-09-22
Publication date: 2022-08-12
Anticipated expiration: 2041-09-22
Also published as: CN113599395A

Abstract

The present invention relates to a pharmaceutical composition for treating cancer, which contains NK cells. The high-activity TNF-alpha mutant protein is obtained by screening high-activity mutant site aiming at TNF-alpha, the TNF-alpha protein and IL-15 are co-expressed in NK cells to remarkably promote the proliferation and killing activity of the NK cells, and the TNF-alpha protein and IL-15 can be used for preparing a pharmaceutical composition for treating cancers and have excellent application prospect.

Description

Pharmaceutical composition for treating cancer containing NK cells

Technical Field

The present invention relates to the field of biology, more specifically to the use of transgenic NK cells for the preparation of a pharmaceutical composition for the treatment of cancer.

Background

NK cells are lymphoid cells with multiple immunological functions. Human NK cells were positive for CD56 and CD16, negative for CD3, CIM and CD 19. A small fraction of NK cells may be CD8 positive. Mouse NK cells were positive for NK1.1 and ASGM1, and CD3, CIM and cr18 negative NK cells killed tumor cells without MHC restriction and without prior exposure to antigen or any memory response. The killing of tumors by NK cells has the advantages of directly dissolving and secreting cytokines, and the tumors can be killed by perforin and Fas ligand. NK cells can produce TNF-a, IFN-gamma and IL-1, and these cytokines play a very important role in NK cell anti-cancer response. NK cells have the ability to rapidly reject allogeneic bone marrow, but do not mediate transplant rejection of solid tissues.

The NK cell has 4 receptors including a killer cell activating receptor (KAR), a killer cell inhibiting receptor (KIR), a killer lectin-like receptor (KLR) and an Fc gamma receptor (CD16), the first 3 receptors can inhibit or activate the NK cell to enable the NK cell to recognize the capability of self-tissue cells and in-vivo abnormal tissue cells, the 4 th receptor mainly recognizes IgG1 and IgG3 markers on the surface of the NK cell and mediates the NK cell to recognize target cells coated by the antibody, so that antibody-dependent cell-mediated cytotoxicity (ADCC) is exerted, and further tumors specifically bound by the IgG and the antibody are killed. NK cells have a wide antitumor spectrum, can kill homologous, homologous or heterologous tumor cells, and can kill target cells by a mechanism that (i) perforin and granzyme are released to cause necrosis or apoptosis of the target cells. Induction of apoptosis of target cells by death receptor modulation. ③ secreting a plurality of effector cytokines to resist the metastatic tumors. And fourthly, stimulating the immune effect of the secondary tumor.

NK cells can be activated by IL-2, IL-12, IL-15, etc., wherein IL-15 and Fh-3 ligands are particularly important in NK cell differentiation. Activated NK cells produce a large number of cytokines including IL-1, TNF-a, IFNG-CSF and GM-CSF. These cytokines are most likely involved in the positive and negative regulation of hematopoiesis. When the activated NK cells are transfused into a body, the hematopoietic recovery process of mouse syngeneic bone marrow transplantation is obviously accelerated, especially the recovery of granulocytes and platelets is accelerated, and a certain application prospect is shown.

Studies have shown that cytokine gene modification can enhance NK cell function. The cytokine IL-12 can enhance the proliferation capacity and the effector function of NK cells, Goding and the like modify mouse primary NK cells depending on IL-2 activation by using mouse IL-12 genes, so that the requirement on exogenous IL-2 is reduced. The study used an adenovirus vector to achieve transient overexpression of the GFP/luciferase/beta-gal and mlL-12 genes, which resulted in the expression of green fluorescent protein in up to 80% of the cells, which peaked IL-12 secretion 48h after transfer, and then declined due to the transient nature of the adenovirus vector expression. The research also shows that the survival period of a B16 lung metastatic tumor mouse treated by using NK cells expressing mlL-12 can be remarkably improved while exogenous IL-2 injection is received, and the survival period is related to the enhancement of mlL-12-NK cell functions and the accumulation of tumor tissues. Another study shows that the adoptive transfer of mlL-12 modified NK cells can further enhance the functions of unmodified NK cells, and shows that the secretion of IL-12 by NK cells can activate adjacent cells, so that surrounding immune cells are activated, and thus, the immune response is improved.

Stem Cell Factor (SCF) combined with IL-2 and IL-15 can remarkably improve the proliferation capacity of NK cells and promote the NK cells to evolve Zhang and the like, SCF carriers are transfected into the NK-92 cells by using liposomes and cultured in the presence of IL-2 or IL-l5, and the proliferation capacity of the cells is remarkably enhanced; under the same culture conditions, the killing capacity of the SCF modified NK-92 cells on various tumor cells is remarkably enhanced compared with that of an unmodified control group. This may be associated with increased expression of killing-related molecules such as perforin and FasL, among others. This mode of genetic modification had no significant effect on the phenotype of NK cells and their ability to secrete IFN, but at low doses of IL-2, NK-92-SCF cells responded relatively poorly. Therefore, clinically, the use of SCF-modified NK cells for therapy is still dependent on the presence of IL-2.

However, studies have found room for improvement in wild SCF activity, and improvement in NK cell proliferation and self-activation efficiency is the major direction of the studies.

Disclosure of Invention

The present invention overcomes the deficiencies of the prior art and provides an improved transgenic NK cell and its use for cancer.

The present invention provides modified NK cells expressing TNF- α.

Optionally, the modified NK cell also expresses IL-15.

Optionally, the NK cell may also be an NK-92 cell.

Further, SEQ ID NO: 3 with the T2A linker and the 89V/H mutant of SEQ ID NO: 2 to obtain the amino acid sequence of SEQ ID NO: 5 into NK cells.

Still further, the TNF- α is a mutated protein, the mutation corresponding to SEQ ID NO: 2, i.e. V at position 89 is mutated to H. The mutation is based on the biological characteristics of the bioinformatics analysis tumor necrosis factor, and the factor is found to contain a death domain on the structural analysis, and forms a helix-loop-helix-like structure to interact with the intracellular death domain of TNFR 1. Under the condition of ensuring TNF-alpha three-dimensional binding activity, a plurality of mutation sites with expected improvement on activity are designed, and mutation is obtained through identification, wherein the mutation is obtained by increasing a polarity effect through a basic amino acid side chain which often contains protonatable basic chemical groups so as to promote binding speed and binding effect.

Further, in SEQ ID NO: 2, conservative substitutions can be carried out on the basis of 89V/H mutation of the amino acid sequence. Where one class of amino acid is substituted with another of the same class of amino acid, it is within the scope of the disclosed variants, so long as the substitution does not substantially alter the activity of the polypeptide. Conservative substitutions are well known to those skilled in the art.

In some embodiments, the sequence of the construct used to transform the NK cells is codon optimized to maximize the efficiency of gene expression in the system. Codon optimization is typically performed by modifying the nucleic acid sequence by replacing at least one, more than one, or a significant number of codons in the native sequence with codons that are used more frequently or most frequently in the genes of the expression system. Codon optimization can be used for translation rate or to produce recombinant RNA transcripts with desired properties, such as longer half-life, as compared to transcripts produced using non-optimized sequences. Methods of codon optimization are readily available from Thermo Fisher Scientific (Waltham, MA), such as Geneart @; optimizers, freely accessible at http genes, urv. es/optimizer, and Genegps expression optimization techniques from DNA2.0(Newark, CA).

To generate modified NK-92 cells expressing TNF- α and IL-15, plasmids are introduced into NK cells, for example, by electroporation. Transformed NK-92 cells were grown in IL-2 free medium and individual clones were selected from transformed NK-92 cells by limiting dilution cloning. Suitable clones may also express surface markers, such as CD3, CD16, CD54, CD56, NKG2D and/or NKP30, at substantially similar levels as NK cells. Optionally, Whole Genome Sequencing (WGS) is performed to determine the transgene integration site. Clones meeting one or more of these criteria can be selected for further development and used for clinical treatment of patients.

Further, the present invention discloses a pharmaceutical composition comprising transgenic NK cells.

The pharmaceutical compositions of the present invention may also include pharmaceutically acceptable carriers, excipients or diluents commonly used in the preparation of pharmaceutical compositions, and such carriers may include non-naturally occurring carriers. Examples of the carrier, excipient and diluent include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, ethylparaben, propylparaben, talc, magnesium stearate and mineral oil.

In addition, the pharmaceutical composition may be formulated into tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, transdermal absorbents, gels, lotions, ointments, creams, patches, cataplasms, pastes, sprays, skin emulsions, skin suspensions, transdermal delivery patches, medicated bandages or suppositories according to conventional methods. In particular, when formulated, the pharmaceutical compositions may be prepared using diluents or excipients such as commonly used fillers, weighting agents, binders, wetting agents, disintegrants and surfactants. Solid materials for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules. Such solid materials may be prepared by mixing at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, gelatin, and the like.

In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc may be used. In addition to the liquid and liquid paraffin for oral administration, various excipients such as wetting agents, sweeteners, flavors, preservatives and the like may be added. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used as the nonaqueous solvent and suspending agent. WITEPSOL, MACROGOL, TWEEN 61, cacao butter, lauryl alcohol, glycerogelatin, etc. can be used as the base of suppositories.

The pharmaceutical compositions of the present disclosure are administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used above means an amount sufficient to treat the disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective amount level, age, sex, pharmaceutical activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration may be determined according to factors including the type and severity of the subjectTime duration, and concomitant use of drugs, as well as other factors well known in the medical arts. For example, the pharmaceutical composition is about 1X 10 ⁸ To about 1X 10 ¹¹ The subject is administered at a concentration of/mL. In some embodiments, the present disclosure provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a composition comprising a plurality of the above-described modified NK-92 cells. In some embodiments, the cancer is selected from melanoma, breast cancer, ovarian cancer, gastric cancer, prostate cancer, squamous cell carcinoma, head and neck cancer, colon cancer, pancreatic cancer, uterine cancer, renal cell carcinoma, glioblastoma, medulloblastoma, sarcoma, and lung cancer. In some embodiments, the cells are administered intravenously. In some embodiments, the cells are administered intratumorally.

The term "cancer" refers to all types of cancers, tumors or malignancies found in mammals, including leukemias, carcinomas and sarcomas. Exemplary cancers include brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, gastric cancer, uterine cancer, and myeloblastomas. Additional examples include, hodgkin's disease, non-hodgkin's lymphoma, multiple myeloma, neuroblastoma, ovarian cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, primary brain tumor, carcinoma, malignant islet tumor, malignant carcinoid, bladder cancer, malignant pre-cutaneous lesion, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortex cancer, endocrine and pancreatic exocrine tumors, prostate cancer.

Advantageous effects

The high-activity TNF-alpha mutant protein is obtained by screening high-activity mutant site aiming at TNF-alpha, the TNF-alpha protein and IL-15 are co-expressed in NK cells to remarkably promote the proliferation and killing activity of the NK cells, and the TNF-alpha protein and IL-15 can be used for preparing a pharmaceutical composition for treating cancers and have excellent application prospect.

Drawings

FIG. 1 is a graph of the results of the activity of mutant TNF-alpha

FIG. 2 fusion scheme

FIG. 3 is a graph showing the results of relative expression levels of genes

FIG. 4 results of proliferation of transgenic NK cells

FIG. 5NK cell killing Effect

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto: materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation and Activity verification of TNF-alpha high Activity mutant

Based on the bioinformatics analysis of the biological characteristics of tumor necrosis factor, it was found that the factor contains a death domain, forming a helix-loop-helix like structure that interacts with the intracellular death domain of TNFR 1. Under the condition of ensuring TNF-alpha three-dimensional binding activity, 6 mutation sites with expected activity improvement are designed, and the polarity effect is increased by the fact that the side chain of basic amino acid often contains basic chemical groups capable of being protonated, so that the binding speed and the binding effect are promoted. Three of them include those in which the nucleotide sequence of SEQ ID NO: 1, the mutations of which correspond to the amino acid sequences shown in SEQ ID NO: 2, 83T/R, 89V/H, 94A/K (A/K indicates the wild type substitution of A for K). Specifically, an upstream primer and a downstream primer are designed according to a wild TNF-alpha gene sequence, and the specific upstream primer sequence is as follows: ATGAGCACTGAAAGCATGAT, and the sequence of the downstream primer is TCACAGGGCAATGATCCCAA. The upstream primer is added with an EcoR I recognition sequence, and the downstream primer is added with a BamH I recognition sequence. Human DNA was used as a template, and the upstream and downstream primers were 1.25pmol each. The reaction parameters are 94 ℃ for 5 min; 30 cycles of 94 ℃ for 30s,52 ℃ for 35s,72 ℃ for 55 s; 72 deg.C, 5 min. Mutant TNF-alpha is amplified using primers with mutant sites and then constructed using overlapping PCR as is conventional in the art.

The PCR product was ligated with pUCm2T (from Shanghai Producer) and transformed into JM109 competent cells, corresponding controls were set up and cultured overnight at 37 ℃. EcoRI/BamHI double digestion and PCR identify the correct clone for sequencing. The pUCm2T/TNF alpha (wild type and 3 mutant TNF alpha of 83T/R, 89V/H and 94A/K) with correct sequence were respectively cloned into an expression vector pBV220 and transformed into Escherichia coli BL21(DE3), and positive recombinant clones were identified and screened again. And (3) carrying out amplification culture on the positive bacterial colonies according to a ratio of 1: 50 (30 ℃), taking a small amount of bacteria as a control when the OD600 reaches 0.4-0.6, carrying out induction expression on the residual bacteria liquid (42 ℃ and 5 hours), collecting the bacteria, cracking and breaking cells, collecting inclusion bodies, carrying out protein renaturation by adopting a dilution method, and adjusting the protein concentration to be 1 mg/mL.

The cytotoxic activity was determined by MTT, i.e.harvesting L929 cells in the logarithmic growth phase, the number of cells being adjusted to 1X 10 ⁵ ·mL ^-1 Inoculating to 96-well flat-bottom culture plate, incubating for 24 hr, discarding supernatant of each well, adding 100 μ L diluted mutant and wild type proteins, respectively, adding 10 μ L and 0.01 mol/L of each well after culturing for 24 hr, and adding blank and no protein ^-1 MTT incubation was continued for 4 h. 150 μ L of supernatant was removed from each well, 150 μ L of DMSO was added to dissolve purple crystals, and absorbance was read at 570 nm. The results of the mutant type are shown in FIG. 1, with the wild type protein as the relative basis.

As can be seen from the results in FIG. 1, the 89V/H mutant TNF-. alpha.has a better enhanced protein activity. While neither 83T/R nor 94A/K showed an increasing effect, but on the contrary there was a considerable reduction in activity, which also indicates that not any mutation was able to increase the activity of the protein.

Example 2 IL-15 coupling TNF-alpha transfected NK cells

Converting SEQ ID NO: 3 with the T2A linker and the 89V/H mutant of SEQ ID NO: 2, and the expression form is shown in figure 2. Specifically, the 89V/H mutant plasmid prepared in example 1 was digested with the amplified sequence of SEQ ID NO: 4, performing sequence splicing on the IL-15 by adopting an overlapping PCR technology through a T2A joint to obtain a full-length sequence IL-15-T2A-89V/H TNF-alpha, wherein the nucleotide sequence is shown as SEQ ID NO: 5, respectively.

Carrying out double enzyme digestion on the fusion gene and a pCDH-CMV vector together, then adopting T4 DNA Ligase for connection, taking 8 mu L of the connected reaction products, adding the obtained product into 50 mu L of DH5 alpha competent cells for plasmid transformation, and then adopting colony PCR to identify a positive strain. Extracting plasmids of positive strains, performing activation culture on 293T cells, carrying out passage on the 293T cells within 24h before transfection, adjusting the cell density according to the cell growth density and state, wherein the cell density during transfection needs to reach 70-80%, the adherence is uniform, and the growth state is good. Preparing plasmid mixed solution according to the proportion of 7.5 mu g of PXPAX, 5 mu g of

PMD2G

5 and 10 mu g of plasmid, slightly swirling, and standing at room temperature for 5 min; according to the DNA: preparing a PEI (polyetherimide) diluent for a transfection reagent according to the proportion of 1:5, carrying out vortex oscillation, and standing for 5min at room temperature; 5) mixing the plasmid mixed solution with PEI diluent by using a 1.5ml EP tube, slowly adding PEI diluent in equal proportion into the plasmid mixed solution, immediately vortex and shake to fully mix the solution uniformly, and standing and polymerizing for 15min at room temperature; slowly dripping the mixed solution into the starvation-treated 293T cells, gently shaking the culture dish to uniformly mix the transfection solution and the culture supernatant, placing the mixture in a carbon dioxide incubator, and transfecting for 12 hours at 37 ℃; discarding all supernatants, adding 8ml DMEM complete culture medium, continuing culturing for 72h, collecting cell culture supernatant, wherein the supernatant is target virus solution, and concentrating virus with PEG 8000.

And (3) infecting NK92 cells with good growth state, wherein the specific infection conditions are that the number of NK92 cells is 1 multiplied by 106. mL < -1 >: virus 5X 106TU, supplemented with NK cell medium to 6mL, was cultured in culture flasks with 75% MEM α (composition: containing 2mM L-glutamine, 1.5g/L sodium bicarbonate, no ribonucleotides and no deoxyribonucleotides), + 12.5% heat-inactivated horse serum + 12.5% imported fetal bovine + 1% diabody +0.2mM myo-inositol +0.02mM folic acid +0.1mM β -mercaptoethanol +200U/mL IL-2. And (3) after the cells are cultured for 48 hours, subculturing liquid, simultaneously screening the cells, and adding puromycin into a culture medium to kill the cells without infection success so as to stably proliferate the cells into positive cells.

Through 3 weeks of screening, the cell positive rate is stabilized to be more than 98%, and the usable NK transgenic cells are obtained, which indicates that the gene is stably chimeric in the cells. Finally, NK92-IL-15-T2A-89V/H TNF-alpha of stably expressing exogenous genes is obtained. In addition, control NK92-89V/H TNF-alpha and NK92-IL-15 cell lines were prepared using preparation methods conventional in the art. The relative expression amount of the three cell lines relative to each transgene in NK92 cells is shown in figure 3 and is over-expressed, wherein the relative expression amount of TNF-alpha of NK 92-IL-15-T2A-89V/HTNF-alpha reaches 3.22, and IL-15 reaches 2.89.

EXAMPLE 3 proliferation assay of NK cells

Respectively adjusting NK92-IL-15-T2A-89V/H TNF-alpha, NK92-89V/H TNF-alpha, NK92-IL-15 cell and NK92 cell strains to 1 × 10 ⁵ The cells were incubated in 24-well plates at 37 ℃ and counted 1 time on

day

3 and 7, respectively, half a second day, and incubated for 7 days. The results are shown in FIG. 4.

FIG. 4 shows the effect of increasing NK92-IL-15-T2A-89V/H TNF- α, NK92-89V/H TNF- α, NK92-IL-15 and NK92 cell lines under culture conditions. As can be seen, the proliferation capacities of NK92-IL-15-T2A-89V/H TNF-alpha are all obviously higher than that of NK92-89V/H TNF-alpha, NK92-IL-15 cells and NK92 cell strains.

Example 4 killing Activity assay of transgenic NK cells

The killing activity of NK cells was measured by MTT method. Respectively taking NK92-IL-15-T2A-89V/H TNF-alpha, NK92-89V/H TNF-alpha, NK92-IL-15 cells and NK92 cell strains as effector cells and BT474 cancer cells as target cells, wherein the effective to target ratio is 10: 1, adding the effector cells and the BT474 cancer cells into a 96-well plate, setting 4 multiple wells in each effective target ratio, setting the total volume of each well to be 200 mu l, and setting a complete culture medium as a zero-adjusting well. 37 ℃ and 5% CO ₂ The culture was continued for 24h, and the culture was continued for 4h after addition of MTT. After centrifugation, 100. mu.l of the supernatant was removed, and 100. mu.l of 10% SDS was added, followed by incubation at 37 ℃ overnight. OD at 570nm was measured against cancer cell wells treated without NK cells. NK cell killing activity was calculated according to a calculation method conventional in the art, and the result is shown in FIG. 5.

As can be seen from FIG. 5, NK92-IL-15-T2A-89V/H TNF-alpha cells had the strongest killing activity, which could reach 98.1%, NK92-89V/H TNF-alpha cells could reach 90.0%, while NK92-IL-15 cells and NK92 cells did not differ much.

It should be understood that the above describes only some embodiments of the present invention and that various other changes and modifications may be affected therein by one of ordinary skill in the related art without departing from the scope or spirit of the invention.

Sequence listing

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Claims

1. Use of a transgenic NK cell for the preparation of a pharmaceutical composition for the treatment of cancer; wherein, the NK cells contain genes with sequences shown in SEQ ID NO: 5.

2. The use according to claim 1, characterized in that said NK cells are NK-92 cells.

3. Use according to claim 2, characterized in that the cancer is breast cancer.

4. Use according to claim 1, characterized in that the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient or diluent normally used for the preparation of pharmaceutical compositions.

5. Use according to claim 1, characterized in that the pharmaceutical composition is in the form of an injection.