WO2023050694A1

WO2023050694A1 - Combined pharmaceutical use of pd-1 antibody and pseudomonas aeruginosa and pharmaceutical composition thereof

Info

Publication number: WO2023050694A1
Application number: PCT/CN2022/077537
Authority: WO
Inventors: 范星星; 黄敏; 何芳; 李丹; 侯金才; 潘银平; 李晓鹏; 李英丽; 朱洁
Original assignee: 神威药业集团有限公司; 北京万特尔生物制药有限公司; 京津冀联创药物研究（北京）有限公司
Priority date: 2021-09-29
Filing date: 2022-02-24
Publication date: 2023-04-06
Also published as: CN113876946A; CN113876946B

Abstract

Provided in the present invention is the use of a PD-1 antibody and Pseudomonas aeruginosa in the preparation of a tumor drug. The PD-1 antibody and Pseudomonas aeruginosa act together on a tumor, such that the therapeutic effect of a Pseudomonas aeruginosa injection and the PD-1 antibody on the tumor is improved. The method provided in the present invention can effectively inhibit the tumor size and improve the expression of TNF-alpha of CD4+T cells in a mouse and the level of MHC-II in macrophages.

Description

Combination pharmaceutical use and pharmaceutical composition of PD-1 antibody and Pseudomonas aeruginosa

technical field

The invention belongs to the field of biomedicine, and specifically relates to a combination pharmaceutical application and pharmaceutical composition of PD-1 antibody and Pseudomonas aeruginosa.

Background technique

Tumor is one of the major diseases that seriously threaten the survival and health of human beings. The existing technology researches on its treatment methods are very extensive, but the current treatment of tumors has entered a bottleneck stage. When constantly innovating traditional tumor treatment methods, we should actively seek new ones. means of tumor treatment.

Tumor immunotherapy has become the fourth largest treatment mode for tumors alongside surgery, radiotherapy and chemotherapy, and is showing great potential against tumors.

The commonly used methods of tumor immunotherapy are as follows: 1. Immune checkpoint inhibitors, such as pembrolizumab, atezolizumab, and nivolumab for melanoma, non-small cell lung cancer and gastrointestinal tumors Monoclonal antibodies, etc. There have been a large amount of clinical evidence that the efficacy of these immune checkpoint inhibitors is far superior to that of chemotherapy in tumor patients with high expression of PDL-1 or high expression of TMB. Second, CAR-T cell therapy, CAR-T uses in vitro expansion, cell culture, and reinfusion of autologous lymphocytes into the body to kill cancer cells. It is very promising and effective for hematological malignancies Immunotherapy, this treatment method has been used in clinical trials of gastrointestinal tumors. Third, other immunotherapy methods include biological therapy, cell therapy, and so on.

PD-1 monoclonal antibody is a monoclonal antibody against PD-1. PD-1 is programmed death receptor 1 (programmed cell death-1), which is a cellular immune antigen expressed on the surface of tumor cells, which can bind to the PD-L1 ligand on the surface of tumor-specific lymphocytes to induce lymphocyte death, thereby enabling tumor cells to evade the anti-tumor lymphocytes. Attack, which is clinically called immune escape of tumor cells. PD-1 monoclonal antibody can block the combination of PD-1 on the surface of tumor cells and lymphocyte PD-L1, thereby inhibiting the immune escape of tumor cells and enhancing anti-tumor immune function. Immunotherapy represented by PD-1 monoclonal antibody has significantly improved the curative effect of malignant tumors and is currently one of the most popular immunotherapy methods.

technical problem

Chinese patent 201810485125.6 discloses a novel PD-1 tumor immunosuppressant and its drug preparation method. The immunosuppressant is made of the following raw materials in parts by weight: 30-45 parts of PD-1 monoclonal antibody, 2-6 parts of biological Alkali, 4-7 parts of antibiotics, 3-9 parts of alkylating agents, 1-5 parts of platinum agents, 5-9 parts of metabolic antagonists; wherein, alkaloids are one of paclitaxel, vincristine, and docetaxel Composition of one or more kinds; antibiotics are composed of one or more of epirubicin, idarubicin, mitomycin; alkylating agent is one or two of ifosfamide, dacarbazine The platinum agent is one or both of cisplatin and oxaliplatin; the metabolic antagonist is one or more of gemcitabine, cytarabine, and tegafur; the immunosuppressant can block tumor cells The interaction between the PD-L1 molecule expressed on the tumor cell and the receptor on the activated T cell inhibits the apoptosis of the activated T cell and improves the killing ability of tumor cells. However, the pharmaceutical composition provided by the invention is too complicated and the cost is relatively high.

Chinese patent 202011385436.9 discloses a combination based on quinoline derivatives and PD-1 monoclonal antibody and its use in anti-gastrointestinal tumors, urinary system tumors, and neuroendocrine tumors. The drug combination contains tyrosine kinase inhibitors and human PD-1 antibodies, which can be used to treat tumors.

"Pseudomonas aeruginosa", also known as "Pseudomonas aeruginosa" (scientific name: Pseudomonas aeruginosa ), is a Gram-negative, aerobic, rod-shaped bacterium that is aerobic and can produce a variety of Substances related to toxicity, such as endotoxin, exotoxin a, elastase, collagenase, trypsin, etc.

At present, there is no technology to combine Pseudomonas aeruginosa and PD-1 antibody in the treatment of tumors.

technical solution

In the present invention, the term "PD-1" is an expanded CD28/CTLA-4 family T cell regulator, and has the same meaning as the term "programmed death receptor 1".

In the present invention, the term "antibody" refers to a protein with protective effect produced by the body due to the stimulation of an antigen, which can specifically bind to the antigen.

In the present invention, the term "PD-1 antibody" refers to the antibody produced by the body due to the stimulation of PD-1 as an antigen.

In the present invention, the term "anti-tumor" refers to having a certain therapeutic effect on tumors, and tumors include benign tumors and malignant tumors.

In the present invention, "having a certain therapeutic effect" refers to inhibiting the occurrence or development of tumors to achieve the purpose of treatment.

In the present invention, the term "application amount" refers to the amount of drug administered to an individual in the case of combined drug use, which is also the sales volume for treatment.

A "therapeutically effective amount," "effective amount," or "effective dose" is the amount of a therapeutic agent that produces a desired therapeutic effect in a subject, such as preventing or treating the disorder of interest or alleviating symptoms associated with the disorder. A precise therapeutically effective amount is that amount of the composition which produces the most effective results in terms of therapeutic effect in a given subject. This amount will vary depending on a variety of factors including, but not limited to, the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, , type and stage of disease, general physical condition, response to a given dose and type of drug), pharmaceutically acceptable carrier in the formulation or the nature of the carrier, and the route of administration.

In the present invention, the term "antibody or its functional fragment" refers to an immunoglobulin molecule that specifically binds to or immunoreacts with a specific antigen or antigenic determinant, including polyclonal and monoclonal antibodies. The term antibody includes genetically engineered or otherwise modified immunoglobulins such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and hybrid antibodies (e.g., bispecific antibodies, diabodies, Tribodies and Quadrabodies).

In the present invention, "treating" or "treatment" of a condition (such as cancer) can mean preventing the condition, slowing the onset or rate of progression of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition , reducing or terminating symptoms associated with the disease, producing total or partial regression of the disorder, or any combination thereof.

In the present invention, the term "injection" refers to sterile solutions (including emulsions and suspensions) made of drugs for injection into the body, as well as sterile powders or concentrated solutions for making solutions or suspensions before use .

In the present invention, the term "double antibody" refers to the penicillin-streptomycin mixed solution, which is specially used for cell culture and can be directly added to the cell culture medium.

In one aspect, the present invention provides the application of PD-1 antibody and Pseudomonas aeruginosa in the preparation of tumor drugs.

The PD-1 antibody and Pseudomonas aeruginosa act together on the tumor; the PD-1 antibody is the antibody itself or a functional fragment thereof.

Specifically, the application amount of the Pseudomonas aeruginosa is 1×10 ⁹ -1×10 ¹¹ /kg body weight.

The application amount of Pseudomonas aeruginosa is 1×10 ⁹ -2×10 ⁹ /kg body weight, 2×10 ⁹ -3×10 ⁹ /kg body weight, 1×10 ⁹ -1×10 ¹⁰ /kg Body weight, 1×10 ⁹ -2×10 ¹⁰ pieces/kg body weight, 1×10 ⁹ -3×10 ¹⁰ pieces/kg body weight, 1×10 ⁹ -4×10 ¹⁰ pieces/kg body weight, 4×10 ⁹ -4 ×10 ¹⁰ /kg, 4×10 ⁹ -1×10 ¹¹ /kg, 1×10 ⁹ -1.8×10 ¹⁰ /kg, 2×10 ⁹ -1.8×10 ¹⁰ /kg, 1.8×10 ¹⁰ -1×10 ¹¹ cells/kg body weight, 4×10 ⁹ -1.8×10 ¹⁰ cells/kg body weight.

Preferably, the application amount of the Pseudomonas aeruginosa is 4×10 ⁹ -1.8×10 ¹⁰ per kg body weight.

The application amount of Pseudomonas aeruginosa is 4×10 ⁹ -1×10 ¹⁰ /kg body weight, 5×10 ⁹ -1.8×10 ¹⁰ /kg body weight, 5×10 ⁹ -1.2×10 ¹⁰ /kg Body weight, 8×10 ⁹ -1×10 ¹⁰ cells/kg body weight, 9×10 ⁹ -1.5×10 ¹⁰ cells/kg body weight.

Further preferably, the application amount of the Pseudomonas aeruginosa is 1.8×10 ¹⁰ /kg body weight.

Specifically, the application amount of the PD-1 antibody is 10-15 mg/kg body weight.

The application amount of the PD-1 antibody is 11-15mg/kg body weight, 12-15mg/kg body weight, 13-15mg/kg body weight, 14-15mg/kg body weight, 11-12mg/kg body weight, 11-13mg/kg body weight kg body weight, 11-14mg/kg body weight, 12-14mg/kg body weight, 12-13mg/kg body weight.

Preferably, the application amount of the PD-1 antibody is 12-13 mg/kg body weight.

The application amount of the PD-1 antibody is 12.1-12.9 mg/kg body weight, 12.2-12.9 mg/kg body weight, 12.3-12.9 mg/kg body weight, 12.5-12.9 mg/kg body weight, 12.2-12.3 mg/kg body weight , 12.2-12.5mg/kg body weight, 12.5-12.8mg/kg body weight, 12.5-12.7mg/kg body weight, 12.5-12.6mg/kg body weight, 12.6-12.9mg/kg body weight, 12.7-12.9mg/kg body weight, 12.7 -12.8 mg/kg body weight.

Further preferably, the application amount of the PD-1 antibody is 12.5 mg/kg body weight.

Preferably, the application amount of the Pseudomonas aeruginosa is 1×10 ⁹ -1×10 ¹¹ /kg body weight; the application amount of the PD-1 antibody is 10-15 mg/kg body weight; the Pseudomonas aeruginosa The application amount of bacillus is 1×10 ⁹ -2×10 ⁹ /kg body weight, 2×10 ⁹ -3×10 ⁹ /kg body weight, 1×10 ⁹ -1×10 ¹⁰ /kg body weight, 1×10 ⁹ -2×10 ¹⁰ pieces/kg body weight, 1×10 ⁹ -3×10 ¹⁰ pieces/kg body weight, 1×10 ⁹ -4×10 ¹⁰ pieces/kg body weight, 4×10 ⁹ -4×10 ¹⁰ pieces/kg kg body weight, 4×10 ⁹ -1×10 ¹¹ pieces/kg body weight, 1×10 ⁹ -1.8×10 ¹⁰ pieces/kg body weight, 2×10 ⁹ -1.8×10 ¹⁰ pieces/kg body weight, 1.8×10 ¹⁰ - 1×10 ¹¹ /kg body weight, 4×10 ⁹ -1.8×10 ¹⁰ /kg body weight; the application amount of the PD-1 antibody is 11-15 mg/kg body weight, 12-15 mg/kg body weight, 13- 15mg/kg body weight, 14-15mg/kg body weight, 11-12mg/kg body weight, 11-13mg/kg body weight, 11-14mg/kg body weight, 12-14mg/kg body weight, 12-13mg/kg body weight.

Further preferably, the application amount of the Pseudomonas aeruginosa is 4×10 ⁹ -1.8×10 ¹⁰ /kg body weight; the application amount of the PD-1 antibody is 12-13 mg/kg body weight; the green The application amount of Pseudomonas is 4×10 ⁹ -1×10 ¹⁰ /kg body weight, 5×10 ⁹ -1.8× ¹⁰ 10/kg body weight, 5×10 ⁹ -1.2×10 ¹⁰ /kg body weight, 8×10 9 -1.2×10 10/kg body weight, 10 ⁹ -1×10 ¹⁰ cells/kg body weight, 9×10 ⁹ -1.5×10 ¹⁰ cells/kg body weight; the application amount of the PD-1 antibody is 12.1-12.9 mg/kg body weight, 12.2-12.9 mg/kg body weight kg body weight, 12.3-12.9mg/kg body weight, 12.5-12.9mg/kg body weight, 12.2-12.3mg/kg body weight, 12.2-12.5mg/kg body weight, 12.5-12.8mg/kg body weight, 12.5-12.7mg/kg body weight , 12.5-12.6mg/kg body weight, 12.6-12.9mg/kg body weight, 12.7-12.9mg/kg body weight, 12.7-12.8mg/kg body weight.

In some embodiments, the application amount of the Pseudomonas aeruginosa is 4×10 ⁹ -1.8×10 ¹⁰ per kg body weight; the application amount of the PD-1 antibody is 12.5 mg/kg body weight.

The PD-1 antibody can be of human or mouse origin.

Preferably, the PD-1 antibody is InVivoMAb anti-mouse PD-1.

The tumors include but are not limited to: lung cancer, bone cancer, bladder cancer, brain cancer, breast cancer, urinary tract cancer, cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, hepatocellular carcinoma, liver cancer , lymphoma and leukemia, melanoma, ovarian cancer, pancreatic cancer, pituitary cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, testicular cancer, thyroid cancer, and uterine cancer.

The route of administration can be any route of administration known in the art, including, but not limited to, enteral, nasal, transdermal. "Parenteral" refers to routes of administration commonly associated with injection, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, Intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.

Preferably, the administration method is injection.

Preferably, the Pseudomonas aeruginosa is Pseudomonas aeruginosa injection, and the PD-1 antibody is PD-1 antibody injection.

The Pseudomonas aeruginosa and PD-1 antibodies can be used simultaneously or sequentially.

When the Pseudomonas aeruginosa and the PD-1 antibody are used sequentially, the Pseudomonas aeruginosa is used first, and then the PD-1 antibody is used.

When the Pseudomonas aeruginosa and the PD-1 antibody are used sequentially, the PD-1 antibody is used first, and then the Pseudomonas aeruginosa is used.

Described Pseudomonas aeruginosa can be: CCTCC AB 2012006, CCTCC AB 2013115, CCTCC AB 2013246, CGMCC 1.15148, CGMCC 1.10712, CGMCC 1.10612, CGMCC 1.10452, CGMCC 1.10274, CGMCC 1.596, CGMCC 1.12483, CGMCC 1.7418.

The Pseudomonas aeruginosa can also be the finished Pseudomonas aeruginosa injection.

In some embodiments, the therapeutically effective dose of a particular formulation may be equal to or lower than the standard dose. In a preferred embodiment, the therapeutically effective dose is lower than the standard dose. A therapeutically effective dose of a particular formulation used in accordance with the embodiments described herein may be a dose that is a proportion or percentage of the standard dose for that particular formulation. In some aspects, the therapeutically effective dose of a particular formulation may be between about 1% and 99% of the standard dose, between about 1% and 90% of the standard dose, between about 1% and 80% of the standard dose, Between about 1% and 70% of the standard dose, between about 1% and 60% of the standard dose, between about 1% and 50% of the standard dose, between about 1% and 40% of the standard dose between about 1% and 30% of the standard dose, between about 1% and 20% of the standard dose, between about 5% and 20% of the standard dose, between about 1% and 10% of the standard dose % between or about 10% less than the standard dose. In one aspect, the therapeutically effective dose of a particular formulation may be about 10%, about 1% or less than 1% of the standard dose. In yet another aspect, the therapeutically effective dose of a particular formulation may be between about 0.1% and 1% of the standard dose, between about 0.01% and 1% of the standard dose, or between about 0.001% and 1% of the standard dose .

In another aspect, the present invention provides an antitumor drug.

The drugs include PD-1 antibody and Pseudomonas aeruginosa.

The content of the Pseudomonas aeruginosa is 1×10 ⁸ cells/mL-1×10 ¹⁰ cells/mL.

The content of the Pseudomonas aeruginosa is 1×10 ⁸ -2×10 ⁸ /mL, 2×10 ⁸ -3×10 ⁸ /mL, 1×10 ⁸ -1×10 ⁹ /mL, 1× 10 ⁸ -2×10 ⁹ cells/mL, 1×10 ⁸ -3×10 ⁹ cells/mL, 1×10 ⁸ -4×10 ⁹ cells/mL, 4×10 ⁸ -4×10 ⁹ cells/mL, 4×10 ⁸ -1×10 ¹⁰ cells/mL, 1×10 ⁸ -1.8×10 ⁹ cells/mL, 2×10 ⁸ -1.8×10 ⁹ cells/mL, 1.8×10 ⁹ -1×10 ¹⁰ cells/mL mL, 4×10 ⁸ -1.8×10 ⁹ cells/mL.

Preferably, the content of the Pseudomonas aeruginosa is 4×10 ⁸ /mL-1.8×10 ⁹ /mL.

The content of Pseudomonas aeruginosa is 4×10 ⁸ -1×10 ⁹ /mL, 5×10 ⁸ -1.8×10 ⁹ /mL, 5×10 ⁸ -1.2×10 ⁹ /mL, 8× 10 ⁸ -1×10 ⁹ cells/mL, 9×10 ⁸ -1.5×10 ⁹ cells/mL.

Further preferably, the content of the Pseudomonas aeruginosa is 4×10 ⁸ /mL or 1.8×10 ⁹ /mL, further 1.8×10 ⁹ /mL.

The content of the PD-1 antibody is 1-2g/L.

The content of the PD-1 antibody is 1-2g/L, 1-1.8g/L, 1-1.5g/L, 1.2-2g/L, 1.2-1.8g/L, 1.4-1.6g/L.

Preferably, the content of the PD-1 antibody is 1-1.5g/L.

The content of the PD-1 antibody is 1-1.45g/L, 1-1.4g/L, 1-1.35g/L, 1.1-1.5g/L, 1.2-1.5g/L, 1.1-1.3g/L L, 1.1-1.2g/L.

Further preferably, the content of the PD-1 antibody is 1.25g/L.

Preferably, the anti-tumor drug includes 1× ^108-1 × ¹⁰¹⁰ /mL of Pseudomonas aeruginosa and the content of PD-1 antibody is 1-2g/L; the content of Pseudomonas aeruginosa The content is 1×10 ⁸ -2×10 ⁸ /mL, 2×10 ⁸ -3× ¹⁰ 8 /mL, 1×10 ⁸ -1×10 ⁹ /mL, 1×10 ⁸ -2×10 ⁹ pcs/mL, 1×10 ⁸ -3×10 ⁹ pcs/mL, 1×10 ⁸ -4×10 ⁹ pcs/mL, 4×10 ^{8 -4} ×10 ⁹ pcs/mL, 4×10 ⁸ -1× 10 ¹⁰ cells/mL, 1×10 ⁸ -1.8×10 ⁹ cells/mL, 2×10 ⁸ -1.8×10 ⁹ cells/mL, 1.8× ^{10 9} -1×10 ¹⁰ cells/mL, 4×10 ⁸ - 1.8×10 ⁹ cells/mL; the content of the PD-1 antibody is 1-2g/L, 1-1.8g/L, 1-1.5g/L, 1.2-2g/L, 1.2-1.8g/L , 1.4-1.6g/L.

Further preferably, the antitumor drug includes 4×10 ⁸ -1.8×10 ⁹ Pseudomonas aeruginosa/mL and 1-1.5 g/L of PD-1 antibody; the content of Pseudomonas aeruginosa is 4× 10 ⁸ -1×10 ⁹ cells/mL, 5×10 ⁸ -1.8×10 ⁹ cells/mL, 5×10 ⁸ -1.2×10 ⁹ cells/mL, 8×10 ⁸ -1×10 ⁹ cells/mL, 9×10 ⁸ -1.5×10 ⁹ cells/mL; the content of the PD-1 antibody is 1-1.45g/L, 1-1.4g/L, 1-1.35g/L, 1.1-1.5g/L , 1.2-1.5g/L, 1.1-1.3g/L, 1.1-1.2g/L.

In some embodiments, the anti-tumor drugs include 1.8×10 ⁹ Pseudomonas aeruginosa/mL and 1.25 g/L of PD-1 antibody.

In some embodiments, the anti-tumor drugs include 4×10 ⁸ Pseudomonas aeruginosa/mL and 1.25 g/L of PD-1 antibody.

The PD-1 antibody can be of human or mouse origin.

Preferably, the PD-1 antibody is InVivoMAb anti-mouse PD-1.

The antitumor drug also includes other pharmaceutically acceptable carriers or excipients. Examples of suitable drug carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline solution, water, emulsion Such as oil/water emulsions, various types of wetting agents, sterile solutions, etc. Compositions including such carriers can be prepared by well-known conventional methods. These pharmaceutical compositions can be administered to subjects in suitable doses. Administration of suitable compositions can be achieved by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. Inventive compositions may also be administered directly to the target site, such as by ballistic delivery to an external or internal target site. The dosage regimen will be determined by the participating physicians and clinical factors. As is well known in the medical arts, dosage for any patient will depend on many factors including patient size, body surface area, age, the particular compound being administered, sex, time and route of administration, general health and other concomitantly administered drugs.

The antitumor drug may also include an adjuvant, which is used to enhance the effect of the drug, and the adjuvant includes but is not limited to: vitamin C, vitamin A, vitamin E, vitamin B-6, carotenoid carotene and beta carotene, selenium, zinc, flavonoids and bioflavonoids, iron chelators, coenzyme Q10, lysine carnitine, compounds containing glutathione, omega-3 fatty acids, prolactin, growth hormone, Alpha-lipoic acid, lentinan, polysaccharide-K (MC-S), synthetic cytidine phosphate-guanosine (CpG), oligodeoxynucleotides, interleukins (eg, IL-2 or IL-12), tumors Necrosis factor α or β (TNF-α or -β), proline-rich polypeptides, β-glucan, tumor antigens, tumor cell killing therapy, gene therapy vectors expressing cytokines, T cell co-stimulatory molecules or Other suitable immunostimulatory molecules.

The dosage forms of the anti-tumor drugs include but not limited to liquid medicines such as infusions and injections.

The diseases targeted by the antitumor drugs include but are not limited to: lung cancer, bone cancer, bladder cancer, brain cancer, breast cancer, urinary tract cancer, cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, Cancers in the group consisting of hepatocellular carcinoma, liver cancer, lymphoma and leukemia, melanoma, ovarian cancer, pancreatic cancer, pituitary cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, testicular cancer, thyroid cancer, and uterine cancer.

Preferably, the antitumor drug is administered by injection.

Preferably, the Pseudomonas aeruginosa injection is administered every three days, and the PD-1 antibody is administered every other day, for a total of 21 days of treatment.

In some embodiments, when the drug is administered for the first time, it is recorded as D1, and then it is recorded as D2, D3, D4... D1, D4, D7, D10, D13, D16, D19 and Pseudomonas aeruginosa injection is administered every day, D3, D6, D9 , D12, D15, D18, D21 administration of PD-1 antibody.

In another aspect, the present invention provides a preparation method of an antitumor drug.

The preparation method includes adding Pseudomonas aeruginosa and PD-1 antibody.

The added amount of the Pseudomonas aeruginosa is 1×10 ⁸ -1×10 ¹⁰ cells/mL, and the added amount of the PD-1 antibody is 1-2 g/L.

The amount of Pseudomonas aeruginosa added is 1×10 ⁸ -2×10 ⁸ cells/mL, 2×10 ⁸ -3×10 ⁸ cells/mL, 1×10 ⁸ -1×10 ⁹ cells/mL, 1 ×10 ⁸ -2×10 ⁹ cells/mL, 1×10 ⁸ -3× ^{10 9} cells/mL, 1×10 ⁸ -4×10 ⁹ cells/mL, 4×10 ⁸ -4×10 ⁹ cells/mL , 4×10 ⁸ -1×10 ¹⁰ cells/mL, 1×10 ⁸ -1.8×10 ⁹ cells/mL, 2×10 ⁸ -1.8×10 ⁹ cells/mL, 1.8× ^{10 9} -1×10 ¹⁰ cells /mL, 4×10 ⁸ -1.8×10 ⁹ pieces/mL; the added amount of the PD-1 antibody is 1-2g/L, 1-1.8g/L, 1-1.5g/L, 1.2-2g /L, 1.2-1.8g/L, 1.4-1.6g/L.

Preferably, the added amount of Pseudomonas aeruginosa is 4×10 ⁸ cells/mL-1.8 ^{×10 9} cells/mL, and the added amount of the PD-1 antibody is 1-1.5 g/L.

The amount of Pseudomonas aeruginosa added is 4×10 ⁸ -1×10 ⁹ /mL, 5×10 ⁸ -1.8×10 ⁹ /mL, 5×10 ⁸ -1.2×10 ⁹ /mL, 8 ×10 ⁸ -1×10 ⁹ cells/mL, 9×10 ⁸ -1.5×10 ⁹ cells/mL; the added amount of the PD-1 antibody is 1-1.45g/L, 1-1.4g/L, 1-1.35g/L, 1.1-1.5g/L, 1.2-1.5g/L, 1.1-1.3g/L, 1.1-1.2g/L.

In some embodiments, the added amount of Pseudomonas aeruginosa is 1.8×10 ⁹ cells/mL, and the added amount of the PD-1 antibody is 1.25 g/L.

In some embodiments, the added amount of Pseudomonas aeruginosa is 4×10 ⁸ cells/mL, and the added amount of the PD-1 antibody is 1.25 g/L.

The preparation method also includes other necessary steps required in the medicine preparation process.

The necessary steps include, but are not limited to: separation, acid concentration, purification, impurity removal, drug testing, filtration, mixing, subpackaging, and the like.

The preparation method may also include the preparation method of PD-1, the method of preparing it through bioengineering technology, the method of preparing it through vector expression, the method of preparing it through immune reaction, etc.

The preparation method of PD-1 may include vector construction, cell transfection, cell expression and the like.

The construction of the vector is completed by inserting the gene expressing PD-1 into the vector; the vector includes but is not limited to pEGFP-N1 vector, pEGFP-C1 vector, pCMVp-NEO-BAN vector, pBAD vector, pSV plasmid, pEG Vector, pCDNA3.1(+).

The cells in the transfected cells include but not limited to: HEK293T cells, CHO cells.

The preparation method may also include the preparation method of Pseudomonas aeruginosa injection, the method prepared by bioengineering technology, the method prepared by microorganism culture, and the method prepared by natural screening.

The Pseudomonas aeruginosa can be any strain under the species, and the Pseudomonas aeruginosa injection can be an injection prepared by any strain under the species through bioengineering technology.

The preparation method of the Pseudomonas aeruginosa injection may include: a dilute or concentrated method; the dilute method is that the quality of the raw materials is good, and it is made at one time; the concentrated method: first mix the concentrated raw materials and then dilution.

The preparation method of the Pseudomonas aeruginosa injection may include: strain activation, inoculation, expanded cultivation and the like.

The selected Pseudomonas aeruginosa in the described Pseudomonas aeruginosa injection can be: CCTCC AB 2012006, CCTCC AB 2013115, CCTCC AB 2013246, CGMCC 1.15148, CGMCC 1.10712, CGMCC 1.10612, CGMCC 1.10452, CGMCC 1.10274, CGMCC 1.596, CGMCC 1.12483, CGMCC 1.7418.

In yet another aspect, the present invention provides the application of the aforementioned antitumor drugs in the treatment of tumor disorders.

The tumor disorders include but not limited to: lung cancer, bone cancer, bladder cancer, brain cancer, breast cancer, urinary tract cancer, cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, hepatocellular carcinoma, Cancers in the group consisting of liver cancer, lymphoma and leukemia, melanoma, ovarian cancer, pancreatic cancer, pituitary cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, testicular cancer, thyroid cancer, and uterine cancer.

Specifically, when the antitumor drug is applied: the application amount of Pseudomonas aeruginosa is 1×10 ⁹ -1×10 ¹¹ /kg body weight.

When the anti-tumor drugs are applied: the application amount of Pseudomonas aeruginosa is 1×10 ⁹ -2×10 ⁹ /kg body weight, 2×10 ⁹ -3×10 ⁹ /kg body weight, 1×10 ⁹ - 1×10 ¹⁰ pieces/kg body weight, 1×10 ⁹ -2×10 ¹⁰ pieces/kg body weight, 1×10 ⁹ -3×10 ¹⁰ pieces/kg body weight, 1×10 ⁹ -4×10 ¹⁰ pieces/kg body weight , 4×10 ^{9 -4} ×10 ¹⁰ pieces/kg body weight, 4× ¹⁰ 9 -1× ^{10 11} pieces/kg body weight, 1×10 ⁹ -1.8×10 ¹⁰ pieces/kg body weight, 2×10 ⁹ -1.8× 10 ¹⁰ /kg body weight, 1.8×10 ¹⁰ -1×10 ¹¹ /kg body weight, 4×10 ⁹ -1.8×10 ¹⁰ /kg body weight.

Preferably, when the antitumor drug is applied: the application amount of Pseudomonas aeruginosa is 4×10 ⁹ -1.8×10 ¹⁰ per kg body weight.

When the anti-tumor drugs are applied: the application amount of Pseudomonas aeruginosa is 4×10 ⁹ -1×10 ¹⁰ /kg body weight, 5×10 ⁹ -1.8×10 ¹⁰ /kg body weight, 5×10 ⁹ - 1.2×10 ¹⁰ cells/kg body weight, 8×10 ⁹ -1×10 ¹⁰ cells/kg body weight, 9×10 ⁹ -1.5×10 ¹⁰ cells/kg body weight.

Further preferably, when the antitumor drug is applied: the application amount of Pseudomonas aeruginosa is 1.8×10 ¹⁰ /kg body weight.

Specifically, when the anti-tumor drug is applied: the application amount of the PD-1 antibody is 10-15 mg/kg body weight.

When the anti-tumor drug is applied: the application amount of PD-1 antibody is 11-15mg/kg body weight, 12-15mg/kg body weight, 13-15mg/kg body weight, 14-15mg/kg body weight, 11-12mg/kg body weight kg body weight, 11-13mg/kg body weight, 11-14mg/kg body weight, 12-14mg/kg body weight, 12-13mg/kg body weight.

Preferably, when the anti-tumor drug is applied: the application amount of the PD-1 antibody is 12-13 mg/kg body weight.

When the anti-tumor drug is applied: the application amount of PD-1 antibody is 12.1-12.9 mg/kg body weight, 12.2-12.9 mg/kg body weight, 12.3-12.9 mg/kg body weight, 12.5-12.9 mg/kg body weight, 12.2-12.3mg/kg body weight, 12.2-12.5mg/kg body weight, 12.5-12.8mg/kg body weight, 12.5-12.7mg/kg body weight, 12.5-12.6mg/kg body weight, 12.6-12.9mg/kg body weight, 12.7- 12.9mg/kg body weight, 12.7-12.8mg/kg body weight.

Further preferably, when the anti-tumor drug is applied: the application amount of the PD-1 antibody is 12.5 mg/kg body weight.

Preferably, when the anti-tumor drug is applied: the application amount of the Pseudomonas aeruginosa is 1×10 ⁹ -1×10 ¹¹ /kg body weight; the application amount of the PD-1 antibody 10-15mg/kg body weight; the application amount of Pseudomonas aeruginosa is 1×10 ⁹ -2×10 ⁹ /kg body weight, 2×10 ⁹ -3×10 ⁹ /kg body weight, 1×10 ⁹ -1×10 ¹⁰ pieces/kg body weight, 1×10 ⁹ -2×10 ¹⁰ pieces/kg body weight, 1×10 ⁹ -3×10 ¹⁰ pieces/kg body weight, 1×10 ⁹ -4×10 ¹⁰ pieces/kg Body weight, 4×10 ⁹ -4×10 ¹⁰ pieces/kg body weight, 4× ^{10 9} -1× ^{10 11} pieces/kg body weight, 1×10 ⁹ -1.8×10 ¹⁰ pieces/kg body weight, 2×10 ⁹ -1.8 ×10 ¹⁰ /kg body weight, 1.8×10 ¹⁰ -1×10 ¹¹ /kg body weight, 4×10 ⁹ -1.8×10 ¹⁰ /kg body weight; the application amount of the PD-1 antibody is 11-15mg /kg body weight, 13-15mg/kg body weight, 13-15mg/kg body weight, 14-15mg/kg body weight, 11-12mg/kg body weight, 11-13mg/kg body weight, 11-14mg/kg body weight, 12-14mg/kg body weight kg body weight, 12-13mg/kg body weight.

Further preferably, when the anti-tumor drug is applied: the application amount of Pseudomonas aeruginosa is 4×10 ⁹ -1.8×10 ¹⁰ /kg body weight; the application amount of the PD-1 antibody is 12-13 mg/kg. kg body weight; the application amount of Pseudomonas aeruginosa is 4×10 ⁹ -1×10 ¹⁰ /kg body weight, 5×10 ⁹ -1.8×10 ¹⁰ /kg body weight, 5×10 ⁹ -1.2×10 ¹⁰ Individuals/kg body weight, 8×10 ⁹ -1×10 ¹⁰ individuals/kg body weight, 9×10 ⁹ -1.5×10 ¹⁰ individuals/kg body weight; the application amount of the PD-1 antibody is 12.1-12.9 mg/kg Body weight, 12.2-12.9mg/kg body weight, 12.3-12.9mg/kg body weight, 12.5-12.9mg/kg body weight, 12.2-12.3mg/kg body weight, 12.2-12.5mg/kg body weight, 12.5-12.8mg/kg body weight, 12.5-12.7mg/kg body weight, 12.5-12.6mg/kg body weight, 12.6-12.9mg/kg body weight, 12.7-12.9mg/kg body weight, 12.7-12.8mg/kg body weight.

In some embodiments, when the antitumor drug is applied: the application amount of Pseudomonas aeruginosa is 4×10 ⁹ -1.8×10 ¹⁰ /kg body weight; the application amount of the PD-1 antibody is 12.5 mg/kg body weight; kg body weight.

When the antitumor drug is applied, the administration route can be any administration route known in the art, including but not limited to, enteral, nasal, and transdermal. "Parenteral" refers to routes of administration commonly associated with injection, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, Intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.

Preferably, the administration method is injection.

Preferably, when the anti-tumor drug is applied, Pseudomonas aeruginosa is Pseudomonas aeruginosa injection, and the PD-1 antibody is PD-1 antibody injection.

In some embodiments, when the antitumor drug is applied, the therapeutically effective dose may be equal to or lower than the standard dose. In a preferred embodiment, the therapeutically effective dose is lower than the standard dose. A therapeutically effective dose of a particular formulation used in accordance with the embodiments described herein may be a dose that is a proportion or percentage of the standard dose for that particular formulation. In some aspects, the therapeutically effective dose of a particular formulation may be between about 1% and 99% of the standard dose, between about 1% and 90% of the standard dose, between about 1% and 80% of the standard dose, Between about 1% and 70% of the standard dose, between about 1% and 60% of the standard dose, between about 1% and 50% of the standard dose, between about 1% and 40% of the standard dose between about 1% and 30% of the standard dose, between about 1% and 20% of the standard dose, between about 5% and 20% of the standard dose, between about 1% and 10% of the standard dose % between or about 10% less than the standard dose. In one aspect, the therapeutically effective dose of a particular formulation may be about 10%, about 1% or less than 1% of the standard dose. In yet another aspect, the therapeutically effective dose of a particular formulation may be between about 0.1% and 1% of the standard dose, between about 0.01% and 1% of the standard dose, or between about 0.001% and 1% of the standard dose .

In some aspects of the invention, the subject has not previously received systemic chemotherapy. In some regimens, the subject has previously received surgery, radiation therapy, induction chemotherapy, and/or adjuvant chemotherapy, or the subject has received concurrent chemotherapy. In some embodiments, the subject has not previously received systemic chemotherapy, but has received surgery, radiation therapy, induction chemotherapy, and/or adjuvant chemotherapy or will be receiving concurrent chemotherapy. In some embodiments, the subject relapses after complete remission following surgery, radiation therapy, induction chemotherapy, concurrent chemotherapy, and/or adjuvant chemotherapy. In some embodiments, the subject is not in complete remission or in partial remission following surgery, radiation therapy, induction chemotherapy, concurrent chemotherapy, and/or adjuvant chemotherapy. In some embodiments, the subject has metastatic cancer following surgery, radiation therapy, induction chemotherapy, concurrent chemotherapy, and/or adjuvant chemotherapy.

Beneficial effect

Beneficial effects of the present invention:

1. The present invention provides a combination drug method, which improves the therapeutic effect of Pseudomonas aeruginosa injection and PD-1 antibody against tumors;

2. Combined administration (PD-1 antibody and Pseudomonas aeruginosa injection) can effectively inhibit tumor size, and increase the expression of TNF-α in CD4+ T cells in mice, as well as the level of MHC-II in macrophages .

Description of drawings

Figure 1 is an inverted microscope image of M0 macrophages derived from THP-1 cells co-cultured with Pseudomonas aeruginosa injection or LPS+IFN-γ for 24 hours. A: Control group; B: Pseudomonas aeruginosa injection (10 ⁸ cells/mL) co-culture group; C: LPS (20ng/mL) + IFN-γ (20ng/mL) co-culture group.

Figure 2 is flow cytometry detection of THP-1 cell-derived M0 macrophages with different concentrations of Pseudomonas aeruginosa injection (0, 10 ⁶ , 10 ⁷ , 10 ⁸ /mL) or LPS+IFN-γ ( 20ng/mL) after co-culture for 48h, the expression of CD86 and TNF-α in CD14+ cells. It should be noted that the relevant data in FIG. 2 has been marked, and those skilled in the art can judge the technical effect of the present application according to the marked data.

Figure 3 is the Western-blot method to detect the effect of Pseudomonas aeruginosa injection on the levels of NF-κ-B and p-NF-κB in macrophages derived from THP-1.

Figure 4 is the detection of different concentrations of Pseudomonas aeruginosa injection (0, 10 ⁶ , 10 ⁷ , 10 ⁸ /mL) or LPS+IFN-γ (20ng/mL) and THP-1 cell-derived macrophages detected by MTT method The effect of conditioned medium prepared by co-culture of cells for 48h on the proliferation activity of H1975 cells.

Figure 5 shows that the combination of PD-1 antibody and Pseudomonas aeruginosa injection (abbreviated as PA-MS in the figure) reduces tumor size. A: Tumor growth curve. Grouping and number of experimental mice: Control, n=6; PD-1 antibody (250 μg/rat), n=6; PA-MS-HIGH (1.8×10 ⁹ rats/mL), n=6; PA-MS-LOW (4×10 ⁸ cells/mL), n=6; PD-1 antibody (250 μg/body)+PA-MS-HIGH (1.8×10 ⁹ cells/mL), n=6; PD-1 antibody (250 μg/ only) + PA-MS-LOW (4×10 ⁸ cells/mL), n=6. B: Mouse body weight, grouping and number of experimental mice: Control, n=6; PD-1 antibody (250 μg/mouse), n=6; PA-MS (4×10 ⁸ cells/mL), n=6; PD -1 antibody (250 μg/monkey) + PA-MS (4×10 ⁸ cells/mL), n=6. CD: Tumor body weight and photos of mice after sampling. E: Tumor imaging in living mice, in vivo imaging of LUC-LLC cells in C57 mice treated on day 20. * indicates P <0.05; ** indicates P <0.01; *** indicates P < 0.001.

Figure 6 shows the detection results of tumor-infiltrating immune cells and immune cells in blood and spleen, * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in further detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only used to illustrate the present invention. The experimental methods used in the following examples, if there is no special instructions, the experimental methods that do not indicate the specific conditions in the examples, generally according to the conventional conditions, the materials, reagents, etc. used in the following examples, if there are no special instructions, all Commercially available.

In the present invention, the term "cell line" refers to a population of cells propagated after the first successful passage of a primary cell culture. It also refers to cultured cells that can be continuously passaged for a long time.

In the present invention, the term "culture medium" refers to a nutrient matrix formulated from a combination of different nutrients for the growth and reproduction of microorganisms, plants or animals (or tissues). Generally contain carbohydrates, nitrogenous substances, inorganic salts (including trace elements), vitamins and water and other major categories of substances.

In the present invention, the term "LPS" refers to lipopolysaccharide, which is a component of the outer wall of the cell wall of Gram-negative bacteria, and is a substance (glycolipid) composed of lipid and polysaccharide.

In the present invention, the term "IFN-γ" refers to type II interferon, also known as γ-IFN or immune interferon, which is produced by T lymphocytes stimulated by mitogens. Interferon is a highly effective antiviral biologically active substance and a lymphokine with extensive immune regulation.

In the present invention, the term "Brefeldin A" is a commonly used protein transport inhibitor, which specifically and reversibly blocks protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus (Golgi).

In the present invention, "PMA" is phorbol 12-myristate 13-acetate, which is a PKC activator. PKC (protein kinase C) is a family of protein kinases that phosphorylate The hydroxyl groups of the amino acid residues of serine and threonine are involved in controlling the function of other proteins. PKC enzymes are in turn activated by signals such as increased concentrations of diacylglycerol (DAG) or calcium ions (Ca ²⁺ ). Thus, PKC enzymes play important roles in several signal transduction cascades. PKC is involved in receptor desensitization, regulation of membrane structural events, modulation of transcription, mediation of immune responses, regulation of cell growth, and learning and memory. These functions are achieved through PKC-mediated phosphorylation of other proteins. In this application, PMA regulates the growth of THP-1 cells by activating PKC.

In the present invention, a "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, usually two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or Essentially all FRs correspond to those of human antibodies. A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, eg, a non-human antibody, refers to an antibody that has undergone humanization.

In the present invention, "primary antibody" refers to a protein that can specifically bind to a non-antibody antigen (specific antigen). Classes include monoclonal and polyclonal antibodies.

In the present invention, "secondary antibody" is an antibody capable of binding to an antibody, that is, an antibody whose main function is to detect the existence of the antibody and amplify the signal of the primary antibody. The secondary antibody is to use the antigenic property of the protein, which is a macromolecule, to immunize the xenogeneic animal, and the immunoglobulin produced by the immune system of the xenogeneic animal is directed against this antibody. The primary antibody can specifically bind to the substrate and recognize the detection object. The combination of the primary antibody and the substrate is reflected by the combination of the secondary antibody and the primary antibody. The secondary antibody has a detectable label (such as a fluorescent, radioactive, chemiluminescent or chromogenic group), which is used to detect the primary antibody.

In the context of the present invention, "adjuvant therapy" is also referred to as additional therapy, and is usually given after surgery to destroy any remaining cancer cells in the body. Adjuvant therapy is given to reduce the likelihood of tumor recurrence or spread to other sites. Adjuvant treatment may include radiation therapy, chemotherapy, hormone therapy, or further surgery.

In the present invention, "bioengineering technology" is based on the theory and technology of biology (especially microbiology, genetics, biochemistry and cytology), combined with modern engineering technologies such as chemical industry, machinery, electronic computer, etc., fully Using the latest achievements in molecular biology, consciously manipulate genetic material, modify organisms or their functions in a targeted manner, create new species with ultra-distant traits in a short period of time, and then use appropriate bioreactors to treat such "engineering bacteria" or "Engineering cell lines" is an emerging technology for large-scale culture to produce a large number of useful metabolites or to exert their unique physiological functions. The bioengineering technology in the present invention includes the cultivation of Pseudomonas aeruginosa and the preparation of PD-1. In the present invention, the cultivation of Pseudomonas aeruginosa is completed by the bioengineering technology based on microbiology; the preparation of PD-1 is completed by the bioengineering technology based on molecular biology and cytology.

In the present invention, "macrophages" are also called histocytes, which are differentiated from monocytes in blood after passing out of blood vessels. After monocytes enter the connective tissue, their volume increases, the endoplasmic reticulum and mitochondria proliferate, the lysosomes increase, and the phagocytosis function is enhanced. The lifespan of macrophages varies with the tissues and organs where they are located, and generally can survive for several months or longer. Macrophages can be activated by PA-MSHA, thereby inhibiting tumors.

The "THP-1 cell" in the present invention is widely used in the research of monocyte and macrophage-related mechanism, signaling pathway, nutrition and drug transportation, and the like. Compared with U937, HL-60, ML-2 and other leukemia cell lines, THP-1 has more morphological and functional characteristics (including cell differentiation markers) similar to human primary monocytes. Compared with human peripheral blood mononuclear cells (PBMC), THP-1 is easier to culture and expand in the laboratory, and has a more stable genetic background. There is no problem of individual differences in PBMC, which is conducive to the reproduction of experimental results. Therefore, THP-1 is a commonly used acute monocytic leukemia cell line and an ideal tool for studying immunity and inflammation.

In the present invention, "activation" generally refers to cell activation, and refers to the process in which cells change from a dormant state to an active state under stimulation under certain conditions. PA-MSHA activates macrophages, that is, PA-MSHA enables macrophages to exert a tumor suppressor effect.

In the present invention, "natural killer" refers to the killing function of natural killer cells. Natural killer cells (natural killer cells, NK) are important immune cells in the body, not only related to anti-tumor, anti-viral infection and immune regulation, but also in certain In some cases involved in hypersensitivity reactions and autoimmune diseases. Since the killing activity of NK cells is not restricted by MHC and does not depend on antibodies, it is called natural killer activity.

In the present invention, "T cell activation" refers to T cell activation technology, which belongs to cellular immunity. Cellular immunity (cellular immunity) T cells proliferate, differentiate, and transform into sensitized T cells (also called effector T cells) after being stimulated by antigens. When the same antigen re-enters the cells of the body, the direct killing effect of the sensitized T cells (effector T cells) on the antigen and the synergistic killing effect of the cytokines released by the sensitized T cells are collectively referred to as cellular immunity. T cells are the main cells of cellular immunity.

In the present invention, the "model" can be a cell model or a mouse model, which is specifically determined with reference to the contents of the examples. Wherein, the cell model refers to a cell line used to study the efficacy of a specific drug, such as the THP-1 cell used to study the efficacy of Pseudomonas aeruginosa injection in the present invention. Animal models refer to animals used to study the efficacy of specific drugs. Generally speaking, animal models are processed to make them have the characteristics that can be used as drug efficacy markers, such as injecting LUC-LLC cells and degrowth factor matrigel in the present invention. The tumor mouse model of the mixture is used to study the effect of the combination of PD-1 antibody and Pseudomonas aeruginosa on tumors.

In the present invention, the "model" can be an inflammation model, that is, THP-1 is induced by phorbol ester (PMA) to differentiate into macrophages, and then induces M1 polarization through lipopolysaccharide (LPS) and IFN-γ, and releases TNF -α, IL-6 and other cytokines, which is a typical model of inflammation.

In the present invention, "classical activation" refers to one of the polarization types of macrophages. Macrophages can be divided into two polarized types according to their phenotype and secreted cytokines, namely classically activated M1 macrophages and alternatively activated M2 macrophages. Among them, the M1 type often exhibits an inhibitory effect on tumors, and the M2 type often exhibits a promoting effect on tumors.

In the present invention, "pro-inflammatory response" refers to the process in which macrophages induce M1 polarization through lipopolysaccharide (LPS) and IFN-γ, and release cytokines such as TNF-α and IL-6.

In the present invention, "host" is an organism that can provide nutrients and a place for pathogens, including humans and animals.

In the present invention, "pathogen" refers to microorganisms (including bacteria, viruses, rickettsia, fungi), parasites or other media that can cause human or animal or plant infection diseases (microorganism recombinants include hybrids or mutants) .

In the present invention, "protease inhibitor" refers to a substance that combines with some groups on the active center of the protease molecule to reduce or even eliminate the activity of the protease, but does not denature the enzyme protein.

In the present invention, "10% SDS-PAGE electrophoresis" refers to polyacrylamide gel electrophoresis with an acrylamide concentration of 10%. It is usually used to detect the expression of protein (expression amount, expression distribution), and analyze the purity of the target protein.

In the present invention, "NC membrane" is a nitrocellulose membrane (nitrocellulose filter membrane, referred to as NC membrane), can be used as the place where the immune response occurs.

In the present invention, "incubation" refers to antibody incubation. Under a certain dilution and a certain temperature environment, the incubation reaction between the primary antibody and the secondary antibody, antibody and antigen occurs, that is, the combination of the antibody and the antigen or the combination of the primary antibody and the secondary antibody reaction.

In the present invention, "MTT" means MTT method, also known as MTT colorimetric method, which is a method for detecting cell survival and growth. The detection principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystal formazan (Formazan) and deposit in the cells, while dead cells have no such function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its light absorption value can be measured by enzyme-linked immunosorbent assay, which can indirectly reflect the number of living cells. Within a certain cell number range, the amount of MTT crystal formation is proportional to the cell number. The method is used for activity detection of biologically active factors, large-scale antitumor drug screening, cytotoxicity test and tumor radiosensitivity determination, etc.

In the present invention, "absorbance value" is the light absorption value, also called the absorbance value, which refers to the ratio of the incident light intensity before the light passes through the solution or substance to the transmitted light intensity after the light passes through the solution or a certain substance (I0/I1) The base 10 logarithm (i.e. lg(I0/I1)), where I0 is the incident light intensity, I1 is the transmitted light intensity, the factors that affect it are solvent, concentration, temperature and so on.

In the present invention, "OD (cells in the administration group)" refers to the absorbance value of the cells in the administration group.

In the present invention, "OD (untreated group of cells)" refers to the absorbance value of the untreated group of cells.

In the present invention, "one-way ANOVA" is applied when there are three or more independent groups and only one independent variable. It also tests for the significance of the differences between these group means. The goal of one-way ANOVA is to find out whether the variation between these group means is also due to chance. One-way ANOVA is a way grouped ANOVA, a statistical analysis of data from one-way factorial experiments. Basic Problems in One-Way ANOVA Estimate and compare the means of multiple normal populations with equal variances. An independent random sample used for more than two treatments in a single experimental variable is called a completely random design (one-way). The F test in this design is a one-way analysis of variance.

In the present invention, "Dunnett's test" is a variance analysis test method, which is suitable for multiple comparisons of the mean difference between k-1 experimental groups and a comparison group.

In the present invention, "Student's t test", that is, student's t test (Student's t test) is mainly used for small sample size (such as n < 30), a normal distribution with an unknown population standard deviation σ. The t-test is to use the t-distribution theory to deduce the probability of the difference, so as to compare whether the difference between the two means is significant.

In the present invention, "co-cultivation" refers to cell co-cultivation technology, which is to co-cultivate two or more types of cells in the same environment, which has the advantage of better reflecting the in vivo environment.

In the present invention, "adherence" refers to the property of cells to adhere to the wall, and the adherent growth refers to the growth of cells attached to a certain solid surface. Adhesive growth is a characteristic of certain cells. Anchorage-dependent cells must attach to the wall of the culture (flask) vessel during culture. Once the cells attach to the wall, they spread rapidly, then begin mitosis, and quickly enter logarithmic growth. growth period. Generally, after a few days, the culture surface will be covered and a dense monolayer of cells will be formed. Adherent cells go through a free phase, an adsorption phase, a multiplication phase, and a degeneration phase. Adherent cells can be eluted with protease or the like. The opposite of adherent growth is suspension culture.

In the present invention, the generation of "cell pseudopods" is a sign of cell migration, and cell migration (cell migration) is also called cell crawling, cell movement or cell movement, and refers to the movement of cells when they receive migration signals or feel certain substances. The resulting movement after the gradient. Cell migration is an alternate process in time and space of the elongation of the pseudopodia of the cell head, the establishment of new adhesions, and the contraction of the tail of the cell body. Cell migration is one of the basic functions of normal cells, a physiological process of normal growth and development of the body, and a common form of movement in living cells. Cell migration is involved in processes such as embryonic development, angiogenesis, wound healing, immune response, inflammatory response, atherosclerosis, and cancer metastasis.

In the present invention, "concentration-dependent increase" means that the higher the concentration of Pseudomonas aeruginosa injection, the higher the expression of M1 macrophage marker CD86 and cytokine TNF-α.

In the present invention, the "western-blot" experiment is Western blotting (immunoblotting test), in which cells or biological tissue samples treated by gel electrophoresis are stained by specific antibodies. Information about the expression of specific proteins in the analyzed cells or tissues can be obtained by analyzing the location and depth of staining.

In the present invention, the higher the concentration of "concentration-dependent inhibition" of Pseudomonas aeruginosa injection, the lower the proliferation activity of macrophage conditioned medium on H1975 cells.

In the present invention, "LUC-LLC cells" are mouse lung cancer luciferase-labeled cells, which are injected into healthy mice to construct a mouse tumor model (mouse lung adenocarcinoma model).

In the present invention, "growth factor-free matrigel" refers to matrigel without growth factors. Matrigel is a basement membrane matrix extracted from EHS mouse tumors rich in extracellular matrix proteins, and its main components include laminin, Type IV collagen, nestin, heparan sulfate glycoprotein, growth factors and matrix metalloproteinases, etc.

At room temperature, it can simulate the structure, composition, physical properties and functions of cell basement membranes in vivo, which is beneficial to the culture and differentiation of cells in vitro, and can be used for research on cell morphology, biochemical functions, migration, infection and gene expression.

In the present invention, appropriate light, temperature, and humidity refer to general conditions, ie conventional conditions.

In the present invention, "single cell suspension" refers to the cell suspension obtained after the chopped animal tissue or organ is treated with trypsin.

In the present invention, "FC blocker" is used to block the binding of the FC end of the antibody to the receptor on the cell. The use of fluorescent antibodies uses its specific Fab end to recognize specific antigens (cell surface markers). If the non-specific Fc end also binds to cells, a non-specific signal is generated. FC blockers can reduce non-specific signal generation in flow cytometry.

In the present invention, "tumor-infiltrating immune cells" mainly include four types, tumor-infiltrating lymphocytes, tumor-associated macrophages, dendritic cells and myeloid-derived suppressor cells. Tumor-infiltrating immune cells in the lung cancer microenvironment can regulate tumor growth, metastasis and angiogenesis, and play an important role in regulating tumor development.

In the present invention, "MHC-II" refers to MHC class II molecules (MHC class II), which are mostly located on antigen-presenting cells (APCs), such as macrophages. This kind of supply is the situation outside the cell, such as bacteria in the tissue, after the macrophage swallows it, the bacterial fragments are prompted to helper T cells by MHC to initiate an immune response.

In the examples of the present invention, the weight of mice is 20 g.

The source information of cells and reagents used in the following examples are as follows:

THP-1 cell line, H1975 cell line, and luciferase LLC (LUC-LLC) cell line were purchased from ATCC.

RPMI-1640 medium, DMEM medium, fetal bovine serum and double antibodies (penicillin/streptomycin) were purchased from Gibco (Carlsbad, CA, USA).

Pseudomonas aeruginosa injection (Beijing Wanter Biopharmaceutical Co., Ltd.);

InVivoMAb anti-mouse PD-1 (CD279, BioCell, BE0146);

PMA (Sigma);

LPS (Sigma);

IFN-γ (Sigma);

Brefeldin A (Biolegend);

Matrigel without growth factors (Corning, 354230);

PE-CyTM7 mouse anti-human CD14(BD);

FITC anti-human CD86 antibody (BioLegend);

APC anti-human CD206(MMR) antibody(BioLegend);

PE/DazzleTM594 anti-human TNF-α antibody(BioLegend);

APC-H7 AVI (Biolegend, 117308);

PerCP anti-mouse CD45 (Biolegend, 103130);

PE-Cy7 anti-mouse CD45 (Biolegend, 103114);

APC anti-mouse CD3 (Biolegend, 100204);

PE-CF594 anti-mouse CD4 (Biolegend, 100410);

PE-CY7 anti-mouse CD8 (Biolegend, 100722);

PE-CF594 anti-mouse TNFα (Biolegend, 506346);

PE-CF594 anti-mouse IFN-γ (Biolegend, 505846);

FITC anti-mouse CD86 (Biolegend, 105006);

PerCP anti-mouse CD11B (Biolegend, 101230);

PE-CF594 anti-mouse MHC-‖ (Biolegend, 107648);

PE-Cy5 anti-mouse F4/80 (Biolegend, 123112);

PE anti-mouse CD11C (Biolegend, 117308);

NF-κB p65(D14E12) XP rabbit mAb (CST);

phospho-NF-κB p65(Ser536) (93H1) rabbit mAb (CST);

β-Actin (D6A8) rabbit mAb (CST).

The influence of embodiment 1 Pseudomonas aeruginosa injection on cultivating THP-1 cells

Pseudomonas aeruginosa injection is based on Pseudomonas aeruginosa-mannose sensitive hemagglutination pili strain (Pseudomonas aeruginosa-mannose-sensitive hemagglutinin, PA-MSHA) as carrier, prepared by bioengineering technology. It has good safety and low adverse reactions, can improve the immunity of tumor patients, prevent infection, and reduce the degree of infection. It has been approved by the State Drug Administration for adjuvant treatment of malignant tumors. PA-MSHA can activate Th1-type immune response, activate macrophages, natural killer cells, and dendritic cells, and promote T cell activation, thereby inhibiting tumors.

THP-1 cells are widely used to study the function, mechanism, signaling pathway, nutrition and drug transport of monocytes/macrophages. This cell line is the most commonly used model for studying the activity of monocytes and macrophages.

Classically activated (M1-type) macrophages are involved in pro-inflammatory responses and play a central role in host defense against pathogen infection. In the tumor microenvironment, unlike the M2 tumor-associated macrophages that help tumor growth, M1 macrophages often exhibit an inhibitory effect on tumors.

Follow these steps:

(1) Cell culture and treatment

Both THP-1 cells and H1975 cells were cultured in RPMI 1640 medium (10% FBS, 1% PS) at 37°C in a 5% CO ₂ incubator. THP-1 cells were inoculated at 1× ¹⁰⁶ cells/mL, stimulated with PMA (150nM) for 24h to adhere to the wall, then aspirated off the medium, washed twice with PBS, added fresh medium and rested for 24h, at which time THP-1 cells were induced For M0 cells.

(2) Western blot experiment

For the cells induced to M0, remove the old medium, add fresh medium containing Pseudomonas aeruginosa injection (0/mL, ¹⁰⁸ /mL), use LPS (100ng/mL) as a positive control, and continue to cultivate 48h. Aspirate the culture supernatant, wash 2 times with PBS, and aspirate the supernatant. Cells were lysed with RIPA lysis buffer containing protease inhibitors to extract total protein. The total protein was separated by 10% SDS-PAGE electrophoresis, then transferred to NC membrane and blocked with 5% skim milk for 1 hour. Incubate with NF-κB p65(D14E12) XP rabbit mAb (CST), phospho-NF-κB p65(Ser536) (93H1) rabbit mAb (CST) and β-Actin (D6A8) rabbit mAb diluted 1000 times in 5% BSA overnight. Anti-rabbit IgG diluted 2000 times with 5% BSA, and incubated with HRP-linked antibody (CST) secondary antibody for 1 hour. HRP chemiluminescence substrate development (absin), Amersham Imager 600 exposure.

(3) Flow Cytometry

Remove the old medium from the cells induced to M0, add the fresh medium containing Pseudomonas aeruginosa injection (0/mL, 10 ⁶ /mL, 10 ⁷ /mL, 10 ⁸ /mL), and use LPS (20ng/mL) + IFN-γ (20ng/mL) was used as a positive control and cultured for 48h.

In order to measure the expression of TNF-α, the cells of each group were first treated with the protein transport inhibitor Brefeldin A (5 μg/mL) was treated for 4 hours. Then digest with trypsin, collect the cells by centrifugation, wash with PBS twice, and centrifuge to remove the supernatant. PE-CyTM7 mouse anti-human CD14(BD), FITC anti-human CD86 antibody (BioLegend), APC anti-human CD206(MMR) antibody (BioLegend) was diluted 200 times with PBS and mixed. Take 100 μL of the mixed solution and add it to the cell pellet, mix well, incubate at 4°C in the dark for 30 minutes, centrifuge to remove the supernatant, add 1% PFA to fix for 1 hour, and then centrifuge to remove the supernatant. PE/DazzleTM594 anti-human TNF-α The antibody (BioLegend) was diluted 200 times with the permeabilization solution, added to the cell pellet and mixed evenly, and then incubated at 4°C in the dark for 30 minutes. The data were analyzed by BECKMAN COULTER CytoFLEXTM Flow Cytometer and FlowJo10.6.

(4) Preparation of conditioned medium

THP-1 cells (1× ¹⁰⁶ cells/mL) were inoculated on a 10 cm culture dish, and stimulated with PMA (150nM) for 24h to make the cells adhere to the wall, then aspirated the medium, washed twice with PBS, and added fresh medium to rest for 24h , and then add fresh medium containing different concentrations of Pseudomonas aeruginosa injection (0/mL, 10 ⁶ /mL, 10 ⁷ /mL, 10 ⁸ /mL), with LPS (20ng/mL )+IFN-γ (20ng/mL) fresh medium was used as a positive control, and continued to culture for 48h.

Collect the culture supernatant, centrifuge at 300g for 10min, discard the precipitate, and centrifuge at 2500g for 10min. Transfer the supernatant to a new centrifuge tube, and then filter the supernatant with a 0.2 μm filter (PALL, 4652). And transfer it to an ultrafiltration concentration centrifuge tube (Amicon® Ultra-15 Centrifugal Filter, 100KD, Merck), centrifuge at 5000g for 10min, and finally obtain about 200μL of concentrated solution, which is stored at -80°C for later use.

(5) MTT experiment

H1975 cells (3000 cells/well) were inoculated in 96-well plates, and after adhering to the wall overnight, the original medium was aspirated, and 100 μL of conditioned medium concentrated by ultrafiltration was added, and the culture was continued for 72 hours. Then add 10 μL MTT solution (5 mg/mL), incubate in the incubator for 4 hours, suck off the supernatant, add 100 μL DMSO, shake for 10 minutes to completely dissolve the crystals, use Tecan microplate Reader (Tecan US, Inc., Morrisville, NC, USA) detects the absorbance value at 490nm. Cell viability was calculated by OD (cells in the administration group)/OD (cells in the untreated group)×100%.

All experimental data are presented as mean ± standard deviation (SD) of three individuals. by Graph Pad Prism 6.0 software for statistical analysis. One-way analysis of variance (ANOVA) was used to analyze differences between groups (more than two) and Dunnett's test was chosen to compare different groups. Student's t-test was used to compare two groups. Differences were considered statistically significant *indicates P < 0.05; ** means P < 0.01; *** means P < 0.001.

In vitro test results and analysis:

1. Pseudomonas aeruginosa injection induced THP-1-derived macrophages to polarize to M1 type

In order to determine whether Pseudomonas aeruginosa injection promotes the polarization of THP-1-derived macrophages, after PMA (150nM) induced THP-1 cells to differentiate into M0 cells, Pseudomonas aeruginosa injection and THP-1 Cell-derived M0 cells were co-cultured. As shown in Figure 1, compared with the control group (A in Figure 1), the Pseudomonas aeruginosa injection co-culture group (B in Figure 1) and the LPS+IFN-γ co-culture group (C in Figure 1 ), the cells have better adherence, and there are obvious cell pseudopodia (indicated by arrows). The expression of CD86 and TNF-α, the markers of M1 macrophages, was detected by flow cytometry. The results (Figure 2) showed that after induction by PMA, macrophages derived from CD14+ THP-1 and different concentrations of Pseudomonas aeruginosa injection or After LPS+IFN-γ co-culture, the expression of M1 macrophage marker CD86 and cytokine TNF-α increased in a concentration-dependent manner compared with the non-administered group (PA-MSHA 0).

2. Pseudomonas aeruginosa injection enhances the expression of phosphorylated NF-κB in macrophages derived from THP-1

In order to further explore the signaling pathway activated by Pseudomonas aeruginosa injection to promote the polarization of THP-1-derived macrophages to classically activated (M1 type) macrophages, Pseudomonas aeruginosa injection and THP-1-derived M0 macrophages were co-cultured for 48 hours, and after total protein was extracted, western-blot experiments were performed. The results showed (Figure 3), compared with the non-co-culture group, the Pseudomonas aeruginosa injection (10 ⁸ ) co-culture group and the positive control LPS group (100ng/mL) both significantly enhanced the expression of p-NF-κB.

3. Effect of conditioned medium derived from THP-1 cell-derived macrophages on the proliferation activity of H1975 cells

In order to clarify the effect of macrophages on the proliferation of tumor cells after Pseudomonas aeruginosa injection promoted the polarization of THP-1-derived macrophages, Pseudomonas aeruginosa injection and THP-1-derived macrophages were co-cultured Afterwards, the culture supernatant was collected to prepare a conditioned medium, and the conditioned medium was used to culture H1975 cells. The results of MTT test (Figure 4) showed that compared with the non-conditioned medium culture group, the macrophage conditioned medium prepared by the co-culture of Pseudomonas aeruginosa injection inhibited the proliferation activity of H1975 cells in a concentration-dependent manner.

Example 2 Combined pharmaceutical application of PD-1 antibody and Pseudomonas aeruginosa injection

The present embodiment is the in vivo experiment of combined pharmaceutical use, carried out according to the following steps:

1. LUC-LLC cells were cultured in DMEM medium (10% FBS, 1% PS) in a 37°C, 5% CO ₂ incubator. All animal studies were approved by the Animal Ethics Committee of Macau University of Science and Technology. After C57 (6-8 weeks), the mixture of 1×10 ⁶ /mL LUC-LLC cells and matrigel without growth factors was injected on the right side, and the tumor grew to 100 mm. Around ³ , the mice were randomly divided into four groups: blank group (200 μL PBS), PD-1 antibody (InVivoMAb anti-mouse PD-1, 250 μg/mouse, 200 μL/mouse, once every three days), low-dose Pseudomonas aeruginosa Injection (4×10 ⁸ pieces/mL, 200 μL/piece, once every three days), low-dose combined medication (PD-1 and low-dose Pseudomonas aeruginosa injection once every three days, administered every other day between the two drugs) , high-dose Pseudomonas aeruginosa injection (1.8×10 ⁹ /mL, 200 μL/one, once every three days), high-dose combination drug (PD-1 and high-dose Pseudomonas aeruginosa injection once every three days, administered on alternate days between the two drugs). Mice were treated for 21 days, and tumor size and body weight were measured every three days. The formula for calculating tumor size is: volume = (length × width ² )/2, all mice were kept under appropriate light, temperature, and humidity, and finally blood was collected from the eyeballs of the mice to separate the tumor and spleen.

2. Flow cytometry

To prepare a single cell suspension, cells were incubated with FC blocker for 15 min at 4°C with the following antibodies: live-stained APC-H7 AVI, PerCP anti-mouse CD45, PerCP anti-mouse, PE-Cy7 anti-mouse CD45, APC anti-mouse CD3, PE-CF594 anti-mouse CD4, PE-CY7 anti-mouse CD8, PE-CF594 anti-mouse TNFα, PE-CF594 anti-mouse IFN-γ, FITC anti-mouse CD86, PerCP anti-mouse CD11B, PE-CF594 anti-mouse MHC-‖, PE-Cy5 anti-mouse F4/80, PE anti-mouse CD11C.

3. Live mouse tumor imaging

Tumor size was detected with Bruker in-vivo Xtreme imaging System (BMSE-033). In LUC-LLC C57 mice (n = 4), 150 μL of Luciferin was injected intraperitoneally, and 100 μL of 1% pentobarbital sodium was injected intraperitoneally five minutes later. Imaging detection was performed after anesthesia. A fluorescent signal was acquired to assess tumor size.

All experimental data are presented as mean ± standard deviation (SD) of three individuals. by Graph Pad Prism 6.0 software for statistical analysis. One-way analysis of variance (ANOVA) was used to analyze differences between groups (more than two) and Dunnett's test was chosen to compare different groups. Student's t-test was used to compare two groups. Differences were considered statistically significant*, P < 0.05; **, P < 0.01; *** , P < 0.001.

In vivo experiment results and analysis:

1. Combination therapy of PD-1 antibody and Pseudomonas aeruginosa injection inhibits tumor

In order to test that the combined drug can reduce the tumor size, different doses of Pseudomonas aeruginosa injection and PD-1 antibody were used to treat the mice alone or in combination, and we found that the combined drug can effectively inhibit the tumor size. Interestingly, we noticed that there was no statistically significant difference in tumor size between low and high doses of the combination (A in Figure 5), and there was no difference in body weight between the combination and the control (B in Figure 5), and we then chose the low dose To carry out live tumor mouse imaging and take pictures of tumors and weigh them after taking materials. After taking materials, it is found that the combined drug can effectively inhibit the mouse tumor (C-D in Figure 5), and the tumor fluorescence signal of the combined drug is weaker than that of other groups (Figure 5). e).

2. Combining drugs to improve tumor-infiltrating immune cells

To examine the underlying mechanism of the combination, we examined tumor-infiltrating immune cells separately in both the high-dose and low-dose groups. It has been reported in the literature that macrophages can activate T cells through antigen-presenting cells, and the expression of MHC‖ interacts with T cells. Our data showed that PA-MSHA Pseudomonas aeruginosa injection increased MHC‖ in macrophages. It is worth noting that MHC‖ was significantly increased after combined administration compared with single administration (B in Figure 6). Next, we detected tumor-infiltrating immune cells, CD4+ The expression of TNF-α in T cells was significantly higher than that in the single drug group (A in Figure 6). And there was no statistical significance between the high-dose group and the low-dose group in the combined medication. Taken together, these results suggest that the low-dose group of combination therapy is more advantageous in tumor therapy and both can increase tumor-infiltrating immune cells.

3. Combining drugs to improve blood and spleen immune cells

In order to further confirm that the combined drug can increase the expression of TNF-α in CD4+ T cells, we detected blood cells and splenocytes in the low-dose group. We found that in the blood of mice, not only the expression of TNF-α in CD4+ T cells was increased (C in Figure 6), but the expression of TNF-α in CD8+ T cells and the expression of IFN-γ were also increased (C in Figure 6 D-E). In the mouse spleen, the same results were detected as in the blood. In addition, flow cytometry detected an increase in splenic CD8+ T cells after combined administration (F-H in Figure 6).

According to the records in Example 1 and Example 2: In vitro experiments show that Pseudomonas aeruginosa injection has the potential to induce macrophages to polarize to classically activated (M1 type) macrophages and promote the secretion of tumor-killing cytokines by cells. The induced M1 macrophages had a concentration-dependent inhibitory effect on the proliferation of H1975 cells. In vivo experiments also confirmed that Pseudomonas aeruginosa injection can effectively inhibit tumor growth, and the combined treatment of PD-1 antibody and Pseudomonas aeruginosa injection improved the activation of T cells in mice.

The co-culture group of Pseudomonas aeruginosa injection had better cell adhesion and pseudopodia (Figure 1), and further flow cytometric analysis showed (Figure 2) that the marker CD86, which marks M1 macrophages, was in the The administration group increased, especially the high-dose group was the most obvious. This shows that Pseudomonas aeruginosa injection has the potential to induce macrophages to differentiate into classically activated (M1 type) macrophages.

In this study, the results of flow cytometry analysis (Figure 2) showed that the expression of TNF-α in the co-culture group of Pseudomonas aeruginosa injection was also significantly increased, further implying the potential of the cells to suppress tumors. The results of Western-blot (Figure 3) showed that the level of p-NF-κB in the co-culture group of Pseudomonas aeruginosa injection was significantly increased, indicating the activation of inflammatory pathways, and the M1 type representing the pro-inflammatory phenotype The polarization of macrophages is consistent, but further detection of the expression of related inflammatory cytokines (such as IL-6) is still needed to verify the effect of the macrophage polarization phenotype induced by Pseudomonas aeruginosa injection on inhibiting tumor proliferation. potential.

After co-cultivating macrophages derived from THP-1 with Pseudomonas aeruginosa injection for 48 hours, we collected the cell culture supernatant, concentrated and centrifuged through ultrafiltration, and prepared a conditioned medium in order to observe Pseudomonas aeruginosa Bacteria injection induces secreted factors produced by macrophages derived from THP-1 cells, and its effect on tumor cell proliferation. The results of the MTT experiment showed (Figure 4) that after co-cultivation with H1975 cells using the conditioned medium, the conditioned medium of the Pseudomonas aeruginosa injection treatment group inhibited the proliferation activity of H1975 in a concentration-dependent manner. Further confirmed the inhibition of H1975 by secreted factors produced by macrophages derived from THP-1 cells induced by Pseudomonas aeruginosa injection.

In vivo, we found that both in vivo imaging in mice and actual measurement of tumor size showed the inhibitory effect of combined drugs on tumors. Since there is no dose-dependent effect on the size of mouse tumors between the combination of high concentration and low concentration, and there is no statistical significance in the tumor infiltrating lymphocytes between the two in flow cytometry, so 4×10 ⁸ /mL aeruginosa pseudo-sheets were selected. Bacteria injection was used as the concentration for subsequent detection of in vivo functional studies. Single administration of PD-1 antibody and Pseudomonas aeruginosa injection did not show significant anti-tumor effect, while combined administration (PD-1 antibody and Pseudomonas aeruginosa injection) could effectively inhibit tumor size and Increase the expression of TNF-α in CD4+ T cells of mice and the level of MHC-II in macrophages.

Claims

The application of the PD-1 antibody and Pseudomonas aeruginosa in the preparation of tumor drugs is characterized in that the PD-1 antibody and Pseudomonas aeruginosa act together on the tumor; the PD-1 antibody is the antibody itself or a functional fragment thereof.
According to the application according to claim 1, the application amount of the Pseudomonas aeruginosa is 1×10 9 -1×10 11 /kg body weight.
The application according to claim 2, characterized in that the application amount of the Pseudomonas aeruginosa is 4×10 9 -1.8×10 10 per kg body weight.
The application according to claim 3, characterized in that the application amount of the Pseudomonas aeruginosa is 4×10 9 /kg or 1.8×10 10 /kg body weight.
The application according to claim 1, characterized in that the application amount of the PD-1 antibody is 10-15 mg/kg body weight.
The application according to claim 5, characterized in that the application amount of the PD-1 antibody is 12-13 mg/kg body weight.
The application according to claim 6, characterized in that the application amount of the PD-1 antibody is 12.5 mg/kg body weight.
The application according to claim 1, wherein the application amount of the Pseudomonas aeruginosa is 1×10 9 -1×10 11 /kg body weight; the application amount of the PD-1 antibody It is 10-15mg/kg body weight.
The application according to claim 8, characterized in that, the application amount of the Pseudomonas aeruginosa is 4×10 9 -1.8×10 10 /kg body weight; the application amount of the PD-1 antibody is 12- 13mg/kg body weight.
The application according to claim 9, wherein the application amount of the Pseudomonas aeruginosa is 4× 109 /kg or 1.8× 1010 /kg body weight; the application amount of the PD-1 antibody It is 12.5mg/kg body weight.
The application according to any one of claims 1-10, wherein the tumors include: lung cancer, bone cancer, bladder cancer, brain cancer, breast cancer, urinary tract cancer, cancer, cervical cancer, colon cancer, Esophageal cancer, gastric cancer, head and neck cancer, hepatocellular carcinoma, liver cancer, lymphoma and leukemia, melanoma, ovarian cancer, pancreatic cancer, pituitary cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, testicular cancer, thyroid cancer and Cancer in the group consisting of uterine cancer.
An anti-tumor drug, characterized in that the anti-tumor drug includes PD-1 antibody and Pseudomonas aeruginosa.
The antitumor drug according to claim 12, characterized in that the content of the Pseudomonas aeruginosa is 1×10 8 -1×10 10 cells/mL.
The antitumor drug according to claim 13, characterized in that the content of the Pseudomonas aeruginosa is 4×10 8 -1.8×10 9 cells/mL.
The antitumor drug according to claim 14, characterized in that the content of the Pseudomonas aeruginosa is 4×10 8 /mL or 1.8×10 9 /mL.
The antitumor drug according to claim 12, wherein the content of the PD-1 antibody is 1-2g/L.
The antitumor drug according to claim 16, wherein the content of the PD-1 antibody is 1-1.5g/L.
The antitumor drug according to claim 17, wherein the content of the PD-1 antibody is 1.25g/L.
A preparation method of an anti-tumor drug, characterized in that the preparation method includes adding Pseudomonas aeruginosa and PD-1 antibody.
The preparation method according to claim 19, wherein the addition amount of the Pseudomonas aeruginosa is 1×10 8 -1×10 10 /mL, and the addition amount of the PD-1 antibody is 1- 2g/L.