CN111920948A

CN111920948A - Pharmaceutical composition comprising immune cells for treating cancer

Info

Publication number: CN111920948A
Application number: CN202011018283.4A
Authority: CN
Inventors: 刘欢; 朱小明
Original assignee: Beijing Guangwei Biotechnology Co Ltd
Current assignee: Ankerui (Shanxi) biological cell Co.,Ltd.
Priority date: 2020-09-25
Filing date: 2020-09-25
Publication date: 2020-11-13
Anticipated expiration: 2040-09-25
Also published as: CN111920948B

Abstract

The present invention relates to pharmaceutical compositions comprising immune cells for the treatment of cancer. The monoclonal antibody specifically aiming at PD-L1 is prepared, has a good effect of inhibiting the proliferation of the liver cancer cells, and in addition, immune cells capable of inhibiting the proliferation of the liver cancer cells are obtained through separation and optimization, and the immune cells and the PD-L1 cells are combined for use, so that the liver cancer can be effectively treated, and the monoclonal antibody has a synergistic effect and a good application prospect.

Description

Pharmaceutical composition comprising immune cells for treating cancer

Technical Field

The invention relates to the field of biological pharmacy, in particular to the field of liver cancer treatment, and more particularly relates to a pharmaceutical composition containing immune cells for treating cancer.

Background

Hepatocellular carcinoma (HCC) accounts for 70-80% of primary liver cancer, and is the 6 th most common cancer worldwide, and the death due to postoperative recurrence and metastasis is ranked 3. In asian countries, particularly china and japan, mortality is high within weeks to months after definitive diagnosis, 5-year survival rate is less than 14%, recurrence rate 5 years after surgery is greater than 50%, and 5-year survival rate after surgery is only 30% -40%.

With the development of medical technology, the immune cell therapy directly identifies and kills tumor cells in the blood and lymph of a patient, kills the tumor cells in the blood and lymph of the patient on the premise of not damaging and destroying the immune system and functions of the organism, and recovers and enhances the natural anti-cancer immune system of the organism. The immune cell therapy can relieve the toxic and side effects of radiotherapy and chemotherapy of liver cancer patients, reduce the recurrence rate of the patients, and prolong the non-progression survival time and the overall survival time.

NK (natural killer) cells and NKT cells are important components of innate immune cells, both derived from the bone marrow. NK cells that colonize the liver exert host immunity to effectively prevent cellular tumorigenesis. NK cells regulate the activity of other immune cells by secreting cytokines as well as exert cytotoxicity.

Natural Killer (NK) cells account for 25% -40% of lymphocytes in human liver, and can be divided into two sub-groups of CD56bright and CD56dim according to the expression quantity of CD56 molecules. Wherein the CD56bright subset is capable of being amplified following stimulation by IL-2, expresses about 10% of killer cell immunoglobulin-like receptor (KIR), and secretes synthetic TNF-related apoptosis-inducing ligand (TRAIL); whereas the CD56dim subgroup is insensitive to IL-2 stimulation, 85% of CD56dim are KIR +, secreting synthetic perforin and granzyme B. When HCC occurs, Rae1 is expressed on the surface of liver cancer cells on one hand, and the factor can activate NK cells as a ligand of an NK cell activation receptor NKG2D, so that the factor plays a role in anti-tumor immunity. On the other hand, the immune function of NK cells is limited and the CD56dimNK cell subset in the peripheral blood of HCC patients is significantly less than that of healthy controls. Meanwhile, the tumor area of the HCC patient has less IFN-gamma expression of CD56dimNK cells than the non-tumor area, and in vitro experiments show that the IFN-gamma expression is related to CD4+ CD25+ regulatory T cells (Tregs). When liver cancer occurs, the activity and function of NK cells can be inhibited by the change of extracellular matrix microenvironment, TGF-beta secreted by HSC and the like, and further the monitoring function of the NK cells on the liver cells is weakened. Under normal physiological conditions, NK cells exert their physiological effects by producing "cytolytic granules" which contain perforin, granzyme, tnf-related apoptosis-inducing ligand and INF- γ. NKT cells are a subgroup of T lymphocytes, the cell functions of the NKT cells coincide with those of T cells and NK cells, alpha beta T cell receptors and various NK cell receptors are also expressed, and the NKT cells are important secretion sources of cytokines such as IL-4, IFN-gamma and TNF-alpha. The antitumor capacity of NK cells is closely related to the expression levels of NKG2D and MICA. Chen et al demonstrated that scorpion venom Polypeptide (PESV) can enhance the antitumor ability of NK cells by modulating the MICA-NKG2D pathway. Jiang et al also found that NK cell NKL _ IL15 modified by human IL-15 gene) showed strong tumor-inhibiting activity in HCC tumor-bearing mice. Studies have shown that RFA can effectively activate NK cell activity in peripheral blood of HCC patients.

At present, the existing research shows that the combination of the CIK cells and the bevacizumab has inhibition effects on proliferation, invasion and migration of HepG2 cells in vivo and in vitro, and is superior to that of a single treatment group. However, the application of the antibody is limited to bevacizumab, and the cost is high. With the continuous and deep research of molecular biology, immunology, genetic engineering and the like, the immune cell therapy of tumors is greatly developed and widely applied. However, how to further improve the curative effect is still a problem to be solved urgently by the tumor immune cell therapy. The combination of monoclonal antibody and immune cell shows feasibility of therapeutic method, and provides new means for treating liver cancer. But currently there is less research.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides a method for treating liver cancer by combining immune cells with monoclonal antibodies.

In one aspect, the invention provides a specific PD-L1 monoclonal antibody 4-G3, the heavy chain variable region sequence is:

QSLEESGGRLVVPDETLTITCTVSGIDLSFKFLWWVRQAPGEGLEWIGDVNKFQYTDGGSWTRLTISKPSSTKVDLKITSPTTETTATYFCGRYAFKGSTDDWGPGTLVTVSS。

the light chain variable region sequence of 4-G3 is: AIVMTQTPSPVSAAVGGTVTINCTWSESGYSNNYEDWFQQKPGQPPKLLIYFTSTSAAGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGSKSGRTDGMTFGGGTEVVVR。

In another aspect of the present invention, a method for preparing immune cells is provided, which specifically comprises the following steps:

(a) coating a culture bottle with a stimulating factor; wherein the stimulating factor is composed of 5000IU/mL of IL-2 concentration, 5000IU/mL of IFN-gamma concentration, 2000IU/mL of IL-15 concentration, 2000IU/mL of GM-CSF concentration, 750IU/mL of IL-4 concentration and 1000IU/mL of TNF-alpha concentration in physiological saline.

(b) Separating mononuclear cells from blood by density gradient centrifugation;

(c) adding RPMI1640 culture medium containing linoleic acid 2mg/L, amino acid 20mg/L, vitamin 50mg/L, IL-2 concentration 5000IU/mL and IFN-gamma concentration 5000IU/mL into a culture bottle coated with a stimulating factor, inoculating mononuclear cells into the culture bottle coated with the stimulating factor, and culturing at 37 deg.C and carbon dioxide concentration of 5%, wherein the inoculation concentration of the mononuclear cells is 5 × 10⁶Per ml;

(d) after culturing for 3 days, transferring the mononuclear cells into an empty culture bottle, adding an RPMI1640 culture medium containing 2mg/L linoleic acid, 20mg/L amino acid, 50mg/L, IL-2 concentration of vitamin and 5000IU/mL IFN-gamma concentration to the empty culture bottle, and continuously culturing at 37 ℃ and 5% of carbon dioxide concentration;

(e) after 7 days of culture, the mononuclear cells are transferred into a culture bag, RPMI1640 medium containing 2mg/L linoleic acid, 20mg/L amino acid, 50mg/L, IL-2 vitamin with the concentration of 5000IU/mL and 5000IU/mL IFN-gamma with the concentration of 5000IU/mL is added into the culture bag for culture at 37 ℃ and the concentration of 5 percent carbon dioxide, and the mononuclear cells are collected after 15 days of culture. Detecting the content of NK immune cells (CD3-CD56+) in the mononuclear cells collected in the step (e) by adopting a flow cytometry detection method.

In another aspect of the invention, a pharmaceutical composition is provided comprising an isolated immune cell and the PD-L1 monoclonal antibody.

Further, the anti-PD-L1 antibody or PD-L1-binding functional fragment thereof can be formulated alone as a pharmaceutical composition suitable for administration to a target subject by an intended route of administration, as discussed in more detail below.

Pharmaceutical compositions suitable for use in the methods described herein may include a therapeutically active agent (e.g., an anti-PD-L1 antibody or functional fragment thereof) and a pharmaceutically acceptable carrier or diluent.

The composition can be administered intravenously, subcutaneously, intraperitoneally, intramuscularly, orally, nasally, pulmonarily, ocularly, vaginally or rectally. In some embodiments, the anti-PD-L1 antibody or functional fragment thereof is formulated for intravenous, subcutaneous, intraperitoneal, or intramuscular administration, e.g., as a solution, suspension, emulsion, liposome formulation, or the like. The pharmaceutical compositions may be formulated using techniques known in the art into immediate release compositions, sustained release compositions, delayed release compositions, and the like.

Pharmacologically acceptable carriers for various dosage forms are known in the art. For example, excipients, lubricants, binders and disintegrants for solid formulations are known; solvents, solubilizers, suspending agents, isotonic agents, buffers and soothing agents for liquid preparations are known. In some embodiments, the pharmaceutical composition includes one or more additional components, such as one or more preservatives, antioxidants, stabilizers, and the like.

In addition, the disclosed pharmaceutical compositions may be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. For example, proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In some embodiments, it is preferred to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in combination with one or more of the ingredients enumerated above, as required, in an appropriate solvent followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other components from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution of the active ingredient.

The pharmaceutical compositions of the present disclosure may be administered in combination with other therapeutic agents. For example, the combination therapy can include a pharmaceutical composition comprising at least one disclosed anti-PD-L1 antibody or functional fragment thereof and at least one or more additional therapeutic agents, including, but not limited to, CAR-T cells (e.g., modified T cells expressing anti-CD 19, anti-HER 2, anti-BCMA, anti-CS-1, anti-PSCA, anti-CaIX, anti-IL 13R, or anti-PD-L1 CAR), other tumor-targeting antibodies (e.g., anti-CaIX antibodies), immune response enhancement patterns (e.g., anti-GITR antibodies, anti-OX 40 antibodies, anti-CD 137 antibodies, or TLR agonists), and small molecule drugs (e.g., BTK inhibitors, EGFR inhibitors, BET inhibitors, PI3K inhibitors, BRAF inhibitors, or PARP inhibitors). The pharmaceutical compositions of the present disclosure may also be administered in combination with radiation therapy.

Provided herein are methods of treating cancer, malignant disease, or cancer cell proliferation with the disclosed anti-PD-L1 antibodies. More specifically, the disclosed anti-tumor immunization methods comprise administering a therapeutically effective amount of any of the above anti-PD-L1 antibodies or compositions.

Enhancing T cell function and anti-tumor immunity provides a broad spectrum of approaches to the treatment of cancer, malignant diseases, or cancer cell proliferation. Thus, liver cancer can be treated by administering the disclosed anti-PD-L1 antibodies and functional fragments thereof.

In some embodiments, the cancer treated according to the disclosed methods is a highly immunogenic cancer. And in some embodiments, the cancer treated according to the disclosed methods is a PD-L1-expressing cancer.

The dosing regimen is adjusted to provide the optimal desired response (e.g., a therapeutic response such as tumor regression or remission). For example, in some embodiments, a single bolus may be administered, while in some embodiments, several separate doses may be administered over time, or the doses may be proportionally reduced or increased as the case may be. For example, in some embodiments, the disclosed antibodies or functional fragments can be administered once or twice weekly by subcutaneous injection or intravenous injection. In some embodiments, the disclosed antibodies or functional fragments can be administered once or twice a month by subcutaneous injection. In some embodiments, the disclosed antibodies or functional fragments can be administered once a week, once every other week, once every three weeks, once every four weeks, once every one month, once every three months, once every four months, once every five months, or once every six months.

Exemplary dosages may vary depending on the size and health of the individual being treated and the condition being treated. For example, in some embodiments, the disclosed antibodies or functional fragments can be administered at a dose of 1-100 mg/kg. In some embodiments, the disclosed antibodies and functional fragments can be administered at a dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg/kg.

In addition, the disclosed methods of treatment may further comprise administering a second therapeutic compound in addition to the anti-PD-L1 antibody or functional fragment thereof. For example, in some embodiments, the additional therapeutic compound is a CAR-T cell, a tumor-targeting antibody, an immune response-enhancing modality, or a small molecule drug.

Thus, for purposes of this disclosure, a subject is treated if one or more beneficial or desired results, including a desired clinical result, are obtained. For example, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing one or more symptoms caused by the disease, improving the quality of life of a person suffering from the disease, reducing the dosage of other drugs required to treat the disease, delaying the progression of the disease, and/or prolonging the survival of an individual.

Furthermore, although the subject of the method is usually a cancer patient, the age of the patient is not limited. The disclosed methods can be used to treat cancer, malignant disease or cancer cell proliferation with a variety of different recurrence and prognosis outcomes that span all age groups and populations. Thus, in some embodiments, the subject may be a pediatric subject, while in other embodiments, the subject may be an adult subject.

Advantageous effects

The monoclonal antibody specifically aiming at PD-L1 has a good effect of inhibiting liver cancer cell proliferation, and in addition, immune cells capable of inhibiting liver cancer cell proliferation are obtained through separation and optimization, and the immune cells and the PD-L1 cells are combined for use, so that liver cancer can be effectively treated, and the monoclonal antibody has a synergistic effect and a good application prospect.

Drawings

FIG. 1 is a graph showing the results of ELISA detection of each antibody

FIG. 2 is a diagram showing the inhibition of the proliferation of liver cancer cells by different doses of monoclonal antibodies

FIG. 3 graph of the effect of different drugs on tumor volume

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods in the following examples, in which specific conditions are not specified, were selected according to conventional methods and conditions, or according to the commercial instructions.

EXAMPLE 1 preparation of PD-L1 monoclonal antibody

KLH-coupled carboxy-terminal peptide RLRKGRMMDVKKCGIQDTNSKKQSDTHLEET was synthesized against PD-L1. The synthetic peptide is used as immunogen to immunize mice.

A female BALB/c mouse with the age of 6-8 weeks is selected, 100 mu g of synthetic peptide mixed with Freund complete adjuvant is injected subcutaneously for the first time, then 30 mu g of antigen mixed with Freund incomplete adjuvant is injected intraperitoneally every 3 weeks (boosting immunization), and 20 mu g of antigen is injected intraperitoneally 3 weeks after the 3 rd boosting immunization (impact immunization). After completion of the ballistic immunization, the mice were sacrificed within 96 h, spleens were removed by aseptic manipulation, and B cells were isolated. For complete cell fusion.

The fusion was started 2 weeks before, and myeloma cells were placed in a culture dish containing 100mL/L fetal bovine serum RPMI1640 medium and placed in a 37 ℃ incubator. Changing the solution 1 time 24 h before fusion, ensuring that the solution is fused when1 x 10 less⁸And (4) cells.

Mouse splenocytes and myeloma cells were mixed in a 5: mixing at a ratio of 1, adding 40 mL of RPMI1640 solution, centrifuging at 1000 r/min for 6 min, and discarding the supernatant. Draw 1 mL of PEG and slowly drop into the centrifugal tube, and then stand at 37 ℃ for 5 min. 20 mL of RPMI1640 medium was added dropwise to the tube wall, followed by 50mL to stop the PEG action. Centrifuge 5 rain at 800 r/min and discard the supernatant. The pellet cells were gently suspended in HAT medium, inoculated into a 96-well plate, and the plate was incubated at 37 ℃ with 50mL/L CO₂An incubator. After 5 days, 1/2 medium was changed by HAT culture, HT medium was changed after 10 days, and positive clones were selected by ELISA after 14 days.

Mixing the antigen with NaCO₃-NaHCO₃(pH9.6) and 20. mu.g of synthetic peptide, cTnI and H-FABP per microplate, wherein the coating volume of each well is 100. mu.L, and the temperature is 4 ℃ overnight. Discard the antigen, flap the well liquid of the ELISA plate, block with l0mg/mL BSA at 37 ℃ for 2 h. The well was discarded, patted dry, and 100. mu.L of the fused cell culture supernatant was added. Incubating at 37 ℃ for 1h, discarding the liquid, and patting dry; PBST washing 5min x 3 times, each hole is added with 100u LHRP marked goat anti-mouse IgG, 37 ℃ incubation for 1h, liquid is discarded, TMB is added for color development, the reaction is stopped by 50 u L2 mol/L H2SO4 at the right moment, and the A450 value of each hole is read by a microplate reader. The results are shown in FIG. 1. The two monoclonal antibodies with better effect obtained by screening are respectively 5-D4 and 4-G3. The titer of 5-D4 was 3.2X 10 by titer identification⁶And 4-G3 has a titer of 3.9X 10⁶。

Example 2 characterization of PD-L1 mAb antibodies

The non-competitive enzyme immunoassay determines the Affinity constant (Affinity constant), Ka value. With reference to the method of Raghava, the value of the affinity constant Ka is calculated according to the formula Ka = (n-1)/2 (n [ Ab' ] t- [ Ab ] t). As a result, the affinity constants were 4.3X 10 for 5-D4, respectively⁹And 4-G3 is 1.32X 10⁹。

The 2 monoclonal antibodies Ig class and subclass were determined with SBA mouse mAb typing kit. The results showed that 5-D4 was IgG2b and 4-G3 was IgG 1.

The heavy chain variable region sequence of 4-G3 was identified by light and heavy chain sequence analysis as:

Example 34 determination of the Effect of the G3 monoclonal antibody on HepG2 cell proliferation

Using the MTT method, 100. mu.L of HepG2 cell suspension (2 x 10) was added per well⁴/well) well flat bottom cell culture plates, after 12h of culture, the total composition is two groups: adding 4-G3 monoclonal antibody solution with different concentrations into the experimental group; for the control group, only RPMI1640 culture medium was added. The final volume of each well was 200. mu.L. 50ml/L CO at 37 deg.C₂The culture was continued for 12h, and the antibody-free medium was replaced by centrifugation to give a final volume of 200. mu.L. MTT addition was also done at 20. mu.l/well and incubation continued for 4 h. Centrifuging to remove supernatant, adding 150 μ l DMSO into each well, shaking, and measuring D absorption value with full-automatic enzyme standard instrument at wavelength of 570 nm. Each concentration involved 4 replicates and the experiment was repeated 3 times. The cytostatic rate = (1-experimental D value/control D value) × 100% was calculated. The results are shown in FIG. 2.

4-G3 monoclonal antibody with different doses has inhibition effect on liver cancer cell proliferation, and has obvious dose correlation. The proliferation inhibition rate of the 4-G3 monoclonal antibody on HepG2 cells is about 31% when the concentration of the monoclonal antibody is 0.01mg/L, and the proliferation inhibition rate can approach 79% when the concentration of the monoclonal antibody is 10mg/L, so that the monoclonal antibody has better inhibition efficiency.

Example 4 preparation of immune cells for treating liver cancer

(b) Separating mononuclear cells from blood by density gradient centrifugation;

(c) 2mg/L of linoleic acid, 20mg/L of amino acid, 50mg/L, IL-2 concentration of vitamin and 5000IU/mL of vitamin are added into a culture bottle coated with the stimulating factor,RPMI1640 culture medium with IFN-gamma concentration of 5000IU/mL, inoculating mononuclear cells in culture bottle coated with stimulating factor, and culturing at 37 deg.C and carbon dioxide concentration of 5%, wherein the inoculation concentration of mononuclear cells is 5 × 10⁶Per ml;

(e) after 7 days of culture, the mononuclear cells are transferred into a culture bag, RPMI1640 medium containing 2mg/L linoleic acid, 20mg/L amino acid, 50mg/L, IL-2 vitamin with the concentration of 5000IU/mL and 5000IU/mL IFN-gamma with the concentration of 5000IU/mL is added into the culture bag for culture at 37 ℃ and the concentration of 5 percent carbon dioxide, and the mononuclear cells are collected after 15 days of culture. The number of mononuclear cells after 15 days of culture was 162-fold greater than that upon inoculation. Detecting the content of NK immune cells (CD3-CD56+) in the mononuclear cells collected in the step (e) by adopting a flow cytometry detection method, wherein the detection result is 97.43%.

EXAMPLE 5 combination of immune cells and 4-G3 monoclonal antibody

Collecting HepG2 cells in logarithmic growth phase and the cultured immune cells expanded in example 4;

the HepG2 cells are used for constructing a mouse subcutaneous transplantation tumor model, the volumes of subcutaneous tumors of mice in a 4-G3 antibody treatment group, an immune cell single-drug treatment group and a 4-G3 antibody combined immune cell treatment group are observed and compared, and the sensitivity of the mice to the 4-G3 antibody and immune cell treatment is detected in vivo.

The specific steps of this embodiment are: the HepG2 cell line was digested and washed to adjust its concentration to 1 x 10⁷One/ml of each of the mice was kept ready, and 4-6 weeks old C57BL/6J immunized with 100ul of mice containing 1 x 10⁶A suspension of HepG2 cells. The average volume of subcutaneous tumor reaches about 50-60mm³The treatment was randomized into four groups, and the treatment was performed with 4-G3 antibody (n ═ 10), immunocyte monotherapy, and 4-G3 antibody combination immunocyte therapy (n ═ 10)(ii) a Model group blank (n = 10). Wherein the 4-G3 antibody is administered by daily subcutaneous injection at a dose of 10mg/kg body weight, and the immunocyte is administered as a single drug at a dose of 5 x 10⁹Subcutaneous injection of one/kg body weight dose; the administration mode of the 4-G3 antibody and immune cell combination therapy is that according to the dosage of 5mg/kg body weight of the antibody, the immune cells are according to 1 x 10⁹The doses per kg body weight were administered together by subcutaneous injection; model group blank was given no drug. Tumor volume was measured 2 weeks after dosing.

The results are shown in fig. 3, the 4-G3 antibody single drug therapy and the immune cell single therapy can inhibit the tumor growth in the mice, and the 4-G3 antibody combined with the immune cell therapy can obviously and synergistically inhibit the tumor growth and proliferation. The tumor volume after 2 weeks is only 38mm³About 453mm greater than the model group without treatment³To be reduced far. The results show that the 4-G3 monoclonal antibody combined immune cell therapy of the invention shows obvious tumor inhibition effect superior to that of two single-drug therapy groups, thereby further indicating that the two drugs can be effectively used for treating liver cancer.

Sequence listing

<110> Beijing Guangdong Biotechnology Ltd

<120> pharmaceutical composition comprising immune cells for treating cancer

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Asp Val Asn Lys Phe Gln Tyr Thr Asp Gly Gly Ser Trp Thr Arg Leu

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Thr Ile Ser Lys Pro Ser Ser Thr Lys Val Asp Leu Lys Ile Thr Ser

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Pro Thr Thr Glu Thr Thr Ala Thr Tyr Phe Cys Gly Arg Tyr Ala Phe

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Lys Gly Ser Thr Asp Asp Trp Gly Pro Gly Thr Leu Val Thr Val Ser

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Ser

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Leu Ile Tyr Phe Thr Ser Thr Ser Ala Ala Gly Val Pro Ser Arg Phe

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Claims

1. A pharmaceutical composition comprising a monoclonal antibody and an extracted immune cell, wherein the immune cell is prepared by the following method:

(a) coating a culture bottle with a stimulating factor; wherein the stimulating factor is composed of 5000IU/mL of IL-2 concentration, 5000IU/mL of IFN-gamma concentration, 2000IU/mL of IL-15 concentration, 2000IU/mL of GM-CSF concentration, 750IU/mL of IL-4 concentration and 1000IU/mL of TNF-alpha concentration in physiological saline;

(b) separating mononuclear cells from blood by density gradient centrifugation;

(e) after culturing for 7 days, transferring the mononuclear cells into a culture bag, adding an RPMI1640 culture medium containing 2mg/L linoleic acid, 20mg/L amino acid, 50mg/L, IL-2 vitamin with the concentration of 5000IU/mL and 5000IU/mL IFN-gamma with the concentration of 5000IU/mL into the culture bag, culturing at 37 ℃ and the carbon dioxide concentration of 5%, and after culturing for 15 days, collecting the mononuclear cells, namely the immune cells;

the monoclonal antibody is a specific PD-L1 monoclonal antibody 4-G3,

the heavy chain variable region sequence of 4-G3 is set forth in SEQ ID NO: 1 is shown in the specification;

4-G3 has the light chain variable region sequence set forth in SEQ ID NO: 2, respectively.

2. The pharmaceutical composition of claim 1, wherein: the pharmaceutical composition can be administered intravenously, subcutaneously, intraperitoneally, intramuscularly, orally, nasally, pulmonarily, ocularly, vaginally or rectally.

3. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is in the form of a solution, a suspension, an emulsion, or a liposome preparation.

4. The pharmaceutical composition of claim 1, wherein an isotonic agent selected from the group consisting of sugar, mannitol, sorbitol and sodium chloride is included in the pharmaceutical composition.

5. Use of the monoclonal antibody of claim 1 and immune cells for the preparation of a pharmaceutical composition for the treatment of liver cancer; the immune cells are prepared by the following method:

(b) separating mononuclear cells from blood by density gradient centrifugation;

(c) adding RPMI1640 culture medium containing linoleic acid 2mg/L, amino acid 20mg/L, vitamin 50mg/L, IL-2 concentration 5000IU/mL and IFN-gamma concentration 5000IU/mL into a culture bottle coated with stimulating factors, inoculating mononuclear cells into the culture bottle coated with stimulating factors, culturing at 37 deg.C and carbon dioxide concentration of 5%,wherein the seeding concentration of the mononuclear cells is 5X 10⁶Per ml;

(e) after 7 days of culture, the mononuclear cells are transferred into a culture bag, RPMI1640 medium containing 2mg/L linoleic acid, 20mg/L amino acid, 50mg/L, IL-2 vitamin with the concentration of 5000IU/mL and 5000IU/mL IFN-gamma with the concentration of 5000IU/mL is added into the culture bag for culture at 37 ℃ and the concentration of 5 percent carbon dioxide, and the mononuclear cells are collected after 15 days of culture.

6. A monoclonal antibody that specifically binds to PD-L1, characterized by: the heavy chain variable region sequence is shown as SEQ ID NO: 1, the light chain variable region sequence is shown as SEQ ID NO: 2, respectively.