CN111905101B - Application of CD47 antibody and IL-6 cytokine drug in tumor treatment and method for verifying tumor treatment by using CD47 antibody and IL-6 cytokine drug - Google Patents

Application of CD47 antibody and IL-6 cytokine drug in tumor treatment and method for verifying tumor treatment by using CD47 antibody and IL-6 cytokine drug Download PDF

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CN111905101B
CN111905101B CN202010636864.8A CN202010636864A CN111905101B CN 111905101 B CN111905101 B CN 111905101B CN 202010636864 A CN202010636864 A CN 202010636864A CN 111905101 B CN111905101 B CN 111905101B
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tumor
antibody
cytokine
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nano
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CN111905101A (en
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陈俊
钟诚
黄春柳
许小丁
李森林
王立翔
王馨语
毛承舟
李懿逸
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Sun Yat Sen University
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    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/204—IL-6
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    • A—HUMAN NECESSITIES
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    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A61P35/00—Antineoplastic agents
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    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3

Abstract

An application of the CD47 antibody and IL-6 cytokine in treating tumor and its method for treating tumor are disclosed, which features that the forward action of IL-6 on immune system is used to cooperate with the CD47 antibody to treat tumor, so activating macrophage to kill tumor cells and improving immunity. Can further realize the immune response of tumor cells based on IL-6 activated T cells and the like, improve the response rate of combined application for treating tumors, fully activate the anti-tumor effect of the immune system of an organism, and can obviously reduce adverse reactions compared with other methods such as chemotherapy and the like. The invention also can improve the treatment targeting property by loading the IL-6 cytokine drug on the nano material with the targeted tumor or tumor microenvironment, enrich the IL-6 in the tumor microenvironment, reduce the amount of the IL-6 entering peripheral blood, reduce side effects, more fully exert the amount of the CD47 antibody reaching the tumor, reduce the dosage of the single action thereof and realize complete combined application.

Description

Application of CD47 antibody and IL-6 cytokine drug in tumor treatment and method for verifying tumor treatment by using CD47 antibody and IL-6 cytokine drug
Technical Field
The invention relates to the field of medical treatment, in particular to application of a CD47 antibody and an IL-6 cytokine drug in tumor treatment and a method for verifying tumor treatment by the same.
Background
CD47 is also called integrin-associated protein, and its ligand is the signal regulatory protein alpha chain (SIRP alpha) on the surface of macrophage, and when CD47 binds with SIRP alpha, it can transmit inhibition signal to inhibit phagocytic activity of macrophage. The tumor cells express CD47 and combine with macrophage SIRP alpha to promote the immune escape of the tumor cells, so that the phagocytosis of the huge macrophages cannot be fully exerted, and the influence of the macrophages on the development of the tumor is reduced. On the basis, aiming at achieving the purpose of treating tumors, the prior art has been studied on drugs for blocking a CD 47-SIRPa pathway and targeting CD47, including CD47 antibodies, but most indications of the drugs are concentrated on malignant hematological tumors, few are directed to solid tumors, and the drugs are basically in clinical stage I. In addition, clinical experiments show that the blood toxicity provides challenges for the drug development design and clinical dose tolerance of the target drug, and the selection of an animal model also has a small challenge for evaluating the effectiveness and safety of the drug; therefore, the development of CD 47-targeted antibody drugs is still further advanced.
IL-6 is a multifunctional cytokine produced by macrophages, fibroblasts and T cells, and has the functions of activating T cells, preventing apoptosis, inducing macrophage activation, recruiting immune cells and the like to regulate immune response. The existing research shows that the compound can promote the survival of tumor cells and also is helpful for immunosuppression of CD4 + T cell mediated antitumor effects and high levels of IL-6 and tumorPoor prognosis for patients with tumors is relevant. However, Elisabeth et al have recently found that blocking the TNF pathway significantly enhances the effect of immunotherapy, while blocking the IL-6 pathway significantly inhibits the effect of immunotherapy, after administering anti-CTLA-4 and anti-PD-1 to a mouse tumor model. It is suggested that IL-6 is not the only inhibitor of the immunotherapeutic process.
If tumors could be treated by increasing the appropriate concentration of IL-6 in conjunction with the CD47 antibody, it may be possible to significantly increase the efficacy of the treatment of tumors over the prior art using the CD47 antibody alone.
At present, the report of the combination of IL-6 cytokine and CD47 antibody for resisting tumor is not available.
Disclosure of Invention
The invention aims to overcome at least one defect of the prior art, and provides an application of a CD47 antibody and an IL-6 cytokine drug in tumor treatment and a verification method for treating tumors thereof, wherein the IL-6 cytokine with a proper concentration or the drug for promoting cells to generate the IL-6 cytokine is combined with anti-CD-47 to treat the tumors, so that the ability of macrophages to phagocytose tumor cells can be effectively enhanced, the tumor immunotherapy effect is improved, the life cycle of tumor patients is prolonged, and a new choice is provided for clinical immunotherapy; the method for treating the tumor in a combined manner can further confirm that the CD47 antibody and the IL-6 cytokine drug can effectively improve the immunotherapy effect, so as to provide a new tumor treatment direction based on the effect.
The technical scheme adopted by the invention is as follows:
the invention provides an application of a CD47 antibody and an IL-6 cytokine drug in preparation of antitumor drugs, wherein the IL-6 cytokine drug comprises an IL-6 cytokine and/or an IL-6 cytokine promoting drug, and the basis is the combined use of a CD47 antibody and an IL-6 cytokine. The IL-6 is utilized to treat the tumor by utilizing the positive effect of the IL-6 on an immune system to cooperate with the CD47 antibody, for example, the IL-6 is utilized to promote the polarization effect of a cytoskeleton in the phagocytosis process of macrophages to cooperate with the CD47 antibody to treat the tumor, so that the macrophage is fully activated to kill tumor cells, and the immunotherapy effect of the CD47 antibody is improved as the main factor. And immune cells such as T cells activated based on IL-6 can further promote the immune response to tumor cells, improve the response rate of combined application for treating tumors, fully activate the anti-tumor effect of the immune system of an organism, and obviously reduce adverse reactions compared with other methods such as chemotherapy and the like. The improvement of the tumor treatment effect of the CD47 antibody and the IL-6 cytokine can prolong the survival time of tumor patients and improve the survival rate of the tumor patients. And treatment based on the body's own immune system also helps to reduce the possibility of recurrence and metastasis. The IL-6 cytokine is used in combination with the CD47 antibody at a specific concentration, and IL-6 is used in combination with the CD47 antibody at a higher and moderate concentration, since an excessively high concentration may cause an adverse reaction of the immune system to itself. The invention can directly use IL-6 cytokine and CD47 antibody to be used together, can use IL-6 cytokine medicine and CD47 antibody to be used together, and can also use IL-6 cytokine, IL-6 cytokine medicine and CD47 antibody to be used together. Because the IL-6 cytokine-promoting drug can promote the production of IL-6 cytokines by cells including phagocytes, the drug can play at least the same role as the IL-6 cytokines, and the IL-6 cytokine-promoting drug can also be helpful to take advantage of the effects of the IL-6 cytokine-promoting drug in combination with IL-6 to further resist tumors.
Further, the IL-6 cytokine-promoting drugs comprise BayK8644 reagent and/or Etoposide (VP-16) and the like, and the BayK8644 can induce the IL-6 expression of macrophages, so that the enrichment of IL-6 cytokines is facilitated. Etoposide (VP-16) is a common anti-tumor chemotherapeutic agent, can promote IL-6 cytokine production, and can also cooperate with CD47 antibody and IL-6 produced to resist tumor together by using the characteristics of Etoposide, thereby obviously improving the anti-tumor effect.
Further, the IL-6 cytokine drug is loaded on the nano material which is targeted to the tumor or the tumor microenvironment. Compared with a single tumor cell, the absorption capacity of the single macrophage for the nano material is thousands of times of the absorption capacity of the single macrophage, namely the macrophage can be used as a 'reservoir' of the nano material to enrich the nano material in a tumor microenvironment. The appropriate amount of the IL-6 cytokine drug is modified on the nano material, so that the IL-6 cytokine drug is enriched in a tumor microenvironment, particularly macrophages in the tumor microenvironment, and the high concentration of the IL-6 cytokine drug and the CD47 antibody jointly act on tumor tissues, the capability of the macrophages for phagocytizing tumor cells is enhanced, and the immune response of an immune system to the tumor cells is promoted. Thereby effectively treating the tumor, prolonging the life cycle of a patient and providing a new choice for the treatment of the clinical tumor (such as colon cancer) at present. In addition, the nano material can freely pass through gaps of tissues and capillaries in a human body, and even can pass through a blood brain barrier, so that the targeted transportation of an IL-6 cytokine-carrying drug to a tumor or a tumor microenvironment is facilitated, the targeted therapy is realized, and the therapeutic effect is further improved; and the targeted delivery of IL-6 cytokine drugs also helps to reduce IL-6 concentration in peripheral blood, thereby reducing side effects caused by high IL-6 concentrations.
Further, the antineoplastic drug is administered in combination with at least one anticancer therapy selected from the group consisting of immunotherapy, chemotherapy, radiation therapy, angiogenesis inhibitor therapy, biologic therapy, bone marrow transplantation, peripheral blood stem cell transplantation, thermotherapy, laser therapy, photodynamic therapy, cancer-targeted therapy. In addition to the combination of an IL-6 cytokine drug and an antibody to CD47 to achieve and enhance immunotherapeutic effects, the combination can be used in combination with other therapies or drugs to further enhance the therapeutic effects of tumors.
Furthermore, in the using process of the anti-tumor drug, the IL-6 cytokine drug is enriched in a tumor microenvironment, and then the CD47 antibody acts on the tumor. The enrichment of IL-6 in a tumor microenvironment can be realized before the CD47 antibody reaches a tumor tissue, the concentration of the antibody which works together with the CD47 antibody is improved in a tumor microenvironment enrichment mode, the CD47 antibody is taken as the main factor to be rapidly combined with the tumor, and the partial dosage of the CD47 antibody is prevented from acting on the tumor when the concentration of the IL-6 does not reach the adaptive concentration, so that the treatment effect of combining the IL-6 cytokine and the CD47 antibody is further improved. Compared with the simultaneous administration with the CD47 antibody, the method relaxes the requirement on the concentration of the IL-6 cytokine drug during administration, and is beneficial to simplifying dosage and operation and achieving the same technical effect.
Further, the tumor is a hematological tumor or a solid tumor. Because of the specificity of IL-6 cytokines, particularly as an inflammatory cytokine, which recruits immune cells, at least tumor-targeted immune cells, including solid tumors, can be recruited by tumor-targeted IL-6 to achieve anti-tumor. And both the CD47 antibody and the IL-6 cytokine can activate macrophages, so that the combination of the CD47 antibody and the IL-6 cytokine can obviously improve the immune function of the macrophages when being applied to solid tumors, thereby realizing the effective treatment of the solid tumors. Meanwhile, the CD47 antibody is an effective blood tumor treatment substance, and the combination of the CD47 antibody and IL-6 cytokines can also realize improvement of the treatment effect on blood tumors.
Further, the tumor is colon cancer. Colon cancer is the most common malignancy of the digestive tract, with the third in global tumor incidence and the second in mortality. At present, the incidence rate of the Chinese medicine is always in an increasing trend in China. In the prior art, the treatment means for colon cancer mainly comprises three traditional treatment methods of operation, radiotherapy and chemotherapy, but the recurrence and metastasis after treatment are still obvious, and the toxic and side effects are not negligible. The invention is characterized in that IL-6 cell factor with properly increased concentration is combined with CD47 antibody to be administered, IL-6 cell factor belongs to the material of immune system, and the combined administration of IL-6 cell factor and anti-CD47 antibody has improved immunotherapy effect compared with the administration of CD47 antibody alone, therefore, when the same therapy effect is achieved, the combined administration can reduce the dosage of CD47 antibody, thereby reducing the adverse reaction caused by CD47 antibody acting on normal cells. The treatment of colon cancer based on the IL-6 cytokine + CD47 antibody is helpful for providing a basis for the clinical application of the subsequent research and providing a direction for treating colon cancer.
A kit comprising a CD47 antibody and an IL-6 cytokine drug, said IL-6 cytokine drug comprising an IL-6 cytokine and/or a pro-IL-6 cytokine drug. The kit is applied to tumor treatment, and further applied to colon cancer treatment. The CD47 antibody and the IL-6 cytokine in the kit are prepared into pharmaceutically acceptable reagents by pharmaceutically acceptable pharmaceutical carriers. The kit can realize the combination of the CD47 antibody and the IL-6 cytokine drug, namely the combination of the CD47 antibody and the IL-6 cytokine, thereby improving the effect of activating macrophages based on the IL-6 and CD47 antibodies, promoting the activation of the macrophages and killing tumor cells, promoting the response of immune cells such as T cells and the like, and fully playing the anti-tumor effect of the immune system of an organism.
Further, the tumor cell material also comprises a nano material for loading IL-6 cytokine drugs to target tumors or tumor microenvironment. The nano material loaded with the IL-6 cytokine drug is beneficial to targeted transportation, so that the IL-6 cytokine is directly concentrated in a tumor microenvironment or indirectly concentrated in the tumor microenvironment through the IL-6 cytokine drug, on one hand, adverse reaction caused by concentration of the IL-6 in normal tissues can be avoided, on the other hand, the IL-6 can be concentrated in macrophages in the tumor microenvironment, and therefore, the concentration of the IL-6 is high, and the IL-6 can jointly act on tumors when the CD47 antibody arrives, so that an effective anti-tumor effect is realized.
Further, an IL-6 cytokine drug is loaded on the nanomaterial and administered prior to the CD47 antibody; alternatively, the IL-6 cytokine drug is loaded on a nanomaterial and administered concomitantly with the CD47 antibody. When the IL-6 cytokine drug is loaded on the nano material and is administered before the CD47 antibody, the targeted enrichment of IL-6 can be realized before the CD47 antibody reaches the tumor tissue, the concentration of the antibody which interacts with the CD47 antibody is increased by the enrichment in the tumor microenvironment, the CD47 antibody is used as the main factor to quickly combine and act on the tumor, and the partial dosage of the CD47 antibody can be prevented from acting on the tumor when the IL-6 concentration does not reach the adaptive concentration. In addition to loading the IL-6 cytokine drug on the nanomaterial prior to administration of the CD47 antibody, the IL-6 cytokine drug can be concomitantly administered with the CD47 antibody, but it is necessary to administer a specific high concentration of the IL-6 cytokine drug prior to administration so that it has or produces a concentration of IL-6 cytokine adapted to act in combination with the CD47 antibody when targeted to the tumor, in a manner that also achieves the effect of treating the tumor with the combination, but requires precise control of the amount compared to the manner in which the IL-6 cytokine drug is loaded on the nanomaterial and administered first.
Another objective of the present invention is to provide a method for verifying the combined use of a CD47 antibody and an IL-6 cytokine drug for the effective treatment of tumors, comprising the steps of:
s1, detecting phagocytosis of phagocytes in vitro, and detecting the influence of the IL-6 cytokine drug on the phagocytosis of the CD47 antibody on the phagocytes by comparing the influence of the independent CD47 antibody and the CD47 antibody in combination with the IL-6 cytokine drug on the process of phagocytes for phagocyting tumor cells;
s2, animal tumor model-based tumor therapy testing, the effect of IL-6 cytokine drugs on the anti-tumor effect of CD47 was verified by comparing the effect of CD47 antibody alone and CD47 antibody in combination with IL-6 cytokine drugs on tumor development.
Compared with the prior art, the invention has the beneficial effects that: the IL-6 is utilized to treat the tumor by utilizing the positive effect of the IL-6 on an immune system to cooperate with the CD47 antibody, for example, the IL-6 is utilized to promote the polarization effect of a cytoskeleton in the phagocytosis process of macrophages to cooperate with the CD47 antibody to treat the tumor, so that the macrophage is fully activated to kill tumor cells, and the immunotherapy effect of the CD47 antibody is improved as the main factor. Can also further realize the immune response of tumor cells based on IL-6 activated T cells and the like, improve the response rate of combined application for treating tumors, and fully activate the anti-tumor effect of the immune system of the organism. Compared with other methods such as chemotherapy and the like, the method can obviously reduce adverse reactions based on immunotherapy, more importantly, the invention also loads IL-6 cytokine drugs through the nano material with the targeted tumor or tumor microenvironment, can improve the targeting of the therapy, can enrich IL-6 in the tumor microenvironment, can reduce the amount of IL-6 entering peripheral blood in the transportation process, reduce side effects, can fully exert the dosage of CD47 antibodies of the reached tumor based on the enriched IL-6, reduce the dosage of CD47 acting on the tumor alone and realize complete combined application. And the IL-6 cytokine drug can be fully absorbed and enriched by macrophages based on the nano material, and can directly act on the macrophages to improve the activity and the efficiency of the macrophages. Meanwhile, the IL-6 cytokine drug and the CD47 antibody are combined for immunotherapy to treat solid tumors including colon cancer, and the restriction that the CD47 antibody in the prior art is difficult to realize effective treatment effect on the solid tumors is broken through; provides a new research direction for immunotherapy and colon cancer therapy in the prior art and promotes the development of the field of tumor therapy.
Drawings
FIG. 1 is a schematic diagram of the addition of 24-well plate material in the in vitro phagocytosis assay of phagocytes according to the present invention.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
The following examples relate to practices and apparatus including: RAG-1KO mouse (offered by Zhan institute of medicine, Zhongshan university, Yonghan institute of Okinawa, Biotech, Inc.), C57BL/6 mouse (purchased from Jiangsu Jiejiaokang Biotech, Inc.), MC38 cell line (offered by Xurui institute of Ohwi, Inc., affiliated with Zhongshan university, Okinawa's research group), Anti-CD47 (purchased from Bioxcell, BE0270, clone No.: MIAP301), Anti-CSF-1R (purchased from Bioxcell, BE0213, clone No.: AFS98), Anti-CD8 α (purchased from Bioxcell, BE0061, clone No.: 2.43), nanomaterial, CFSE (C34554; Life Technologies, Burleore, Ontario, Canada), IL-6(216-16, PeproTech, Rocky Hicky, USA), cell culture medium (Hybc, serum (USA, USA), flow cytometry, USA, Biotech, USA, Fisher 2), and Nippo, USA 32).
Example 1
In vitro macrophage phagocytosis assay of CD47 antibody alone and CD47 antibody in combination with IL-6 cytokine drug or with IL-6 cytokine drug loaded on nanomaterials
S1, obtaining and culturing macrophages in vitro; in this example, bone marrow was removed from the leg bone of a mouse, and macrophages were cultured in vitro. And macrophages were taken out to 24-well plates for overnight culture at 5X 10 per well 4 And macrophages are formed.
S2, and in-vitro macrophage phagocytosis colon cancer cell detection based on a CD47 antibody, a CD47 antibody and an IL-6 cytokine drug, and a CD47 antibody and the IL-6 cytokine drug loaded on a nanometer material.
In this example, the macrophage cell culture medium was replaced with a serum-free medium at 37 ℃ with 5% CO 2 Incubating for 2h in an incubator;
then 2 is taken out 5 After washing each colon cancer cell line (MC38) in PBS solution, it was stained with CFSE, and then 10ug/mL lgG or CD47 antibody was added according to the treatment group, and PBS or 100ng/mL IL-6 or 100ng/mL nano-IL-6, which is nano material, was added, and the nano-IL-6 was IL-6 cytokine loaded on the nano material. The 100ng/mL IL-6 may be replaced with 5. mu.g/mL BayK8644 or Etoposide (VP-16) in a specified amount or other chemical capable of inducing IL-6 production in a specified amount, the BayK8644 or Etoposide (VP-16) or other chemical capable of inducing IL6 being capable of inducing IL-6 expression by macrophages, increasing IL-6 concentration in the tumor microenvironment.
Specifically, as shown in FIG. 1, the substances and the corresponding wells are combined with each other to obtain the phagocytic result of colon cancer cells phagocytized by phagocytes corresponding to lgG, lgG + PBS, lgG + IL-6, lgG + nano-IL-6, anti-CD47, anti-CD47+ PBS, anti-CD47+ IL-6, anti-CD47+ nano, anti-CD47+ nano-IL-6, which are not added with any substance, respectively, wherein the anti-CD47 is CD47 antibody. Thus, the anti-CD47+ nano-IL-6 has specificity compared with any other combination, and has the obvious effect of promoting macrophages to phagocytose colon cancer cells.
Specifically, in this example, after floating tumor cells were washed out with PBS in each well, 5 fields were arbitrarily photographed with a fluorescence microscope, and the phagocytosis rate was calculated. The phagocytosis rate is the number of tumor cells phagocytosed/(number of all macrophages in the field x 100), and was repeated three times per experiment.
The experimental results show that: the phagocytosis rate of the anti-CD47+ nano-IL-6 group is > anti-CD47+ IL-6 group phagocytosis rate > other groups phagocytosis rate, the phagocytosis rate of the anti-CD47+ nano-IL-6 group is highest, the anti-CD47 and IL-6 can promote macrophages to phagocytose colon cancer cells, and especially when IL-6 is loaded on a nano material for drug application, the effect is more remarkable.
Example 2
Treatment of colon cancer in RAG-1KO mouse subcutaneous transplantation tumor model by using CD47 antibody alone and CD47 antibody in combination with IL-6 cytokine drug or in combination with IL-6 cytokine drug loaded on nano material
S1, the colon cancer cell line is transplanted to the right side of a RAG-1KO mouse subcutaneously, and a transplantation tumor model is constructed. In this example, 5X 10 5 A colon cancer cell line (MC38) was implanted subcutaneously on the right side of RAG-1KO mice.
S2, comparing the effect of colon cancer treatment with CD47 antibody alone, CD47 antibody in combination with IL-6 cytokine drug based on the transplanted tumor model, wherein the CD47 antibody in combination with IL-6 cytokine drug comprises CD47 antibody in combination with IL-6 cytokine drug loaded on nano material. In this example, mice were divided into eight different treatment groups, each treatment group consisting of 5 mice, each treatment group consisting of lgG + PBS, anti-CD47+ PBS, lgG + IL-6, anti-CD47+ IL-6, lgG + nano, anti-CD47+ nano, lgG + nano-IL-6, and anti-CD47+ nano-IL-6. The IL-6 may be replaced with specific amounts of BayK8644 or Etoposide (VP-16) or other chemical agent capable of inducing IL-6, the nano-IL-6 may be replaced with specific amounts of nano-BayK8644 or nano-Etoposide (VP-16) or nano-other chemical agent capable of inducing IL6, and other embodiments are equally capable of IL-6 or nano-IL-6 replacement based thereon.
After 7 days of injection, when the tumor volume grows to 50-100mm 3 When the injection is carried out, the corresponding groups are respectively matched and injected with 200ug anti-CD47, 200ug lgG, 500ng or 1ug IL-6 (or 150ug or 200ug BayK8644 intraperitoneally or specific amount of Etoposide (VP-16) or other chemical drugs capable of inducing IL-6), 500ng or 1ug nano-IL-6 intravenously or 150ug or 200ug nano-BayK8644 or specific amount of nano-Etoposide (VP-16) or nano-other chemical drugs capable of inducing IL-6) and PBS, and the injection is carried out once every three days for 4 times. And mice tumors were examined every three days for length (L), width (W) and height (H), and tumor volume was calculated as L W H/2. When the tumor volume of the mice in any one of the four groups of mice exceeds 1000mm 3 All treatment groups are sacrificed immediatelyMice, finally analyzing the growth trend of the tumor volume and the tumor microenvironment.
The results show that: the anti-CD47+ nano-IL-6 group tumor size is smaller than that of an anti-CD47+ IL-6 group tumor size is smaller than that of other groups tumor size, animal experiments prove that the anti-CD47 and IL-6 can effectively treat tumors, and the IL-6 is loaded by the nano material, so that the treatment effect is more obvious.
S3, comparing the effect of colon cancer treatment with CD47 antibody alone, CD47 antibody in combination with IL-6 cytokine drug, CD47 antibody in combination with IL-6 cytokine drug and CSF-1R based on the transplanted tumor model.
Take 5X 10 5 A colon cancer cell line (MC38) was implanted subcutaneously on the right side of RAG-1KO mice. Mice were divided into three different treatment groups, 5 mice each: three groups are injected with lgG + PBS, anti-CD47+ nano-IL-6, anti-CD47+ nano-IL-6+ CSF-1R antibody respectively. After 7 days, when the tumor volume grows to 50-100mm 3 When in use, the corresponding matched injections of the corresponding groups are performed based on lgG, PBS, anti-CD47, nano-IL-6 and CSF-1R anti body, and the injections are performed once every three days for four times in total. Mice were examined every three days for length (L), width (W) and height (H) of tumors, tumor volume was calculated as L W H/2, and tumor volume growth trends and tumor microenvironment were analyzed. Similarly, the IL-6 may be replaced with specific amounts of BayK8644 or Etoposide (VP-16) or other chemical capable of inducing IL-6, and the nano-IL-6 may be replaced with specific amounts of nano-BayK8644 or nano-Etoposide (VP-16) or nano-other chemical capable of inducing IL 6.
The results show that: the anti-CD47+ nano-IL-6 group tumor size is less than anti-CD47+ nano-IL-6+ CSF-1Rantibody group tumor size is less than lgG + PBS group tumor size, which indicates that the anti-tumor effect of nano-IL-6 on promoting anti-CD47 is mediated by macrophages.
Example 3
Treatment of colon cancer in C57BL/6 mouse subcutaneous transplantation tumor model by using CD47 antibody alone and CD47 antibody in combination with IL-6 cytokine drug or in combination with IL-6 cytokine drug loaded on nanometer material
S1, subcutaneously transplanting the colon cancer cell line to the right side of a C57BL/6 mouse to construct a transplantTumor model. In this example, 1X 10 6 A colon cancer cell line (MC38) was implanted subcutaneously into the right side of C57BL/6 mice.
S2, comparing the colon cancer treatment effect of the CD47 antibody and the CD47 antibody in combination with the IL-6 cytokine drug based on the transplantation tumor model, wherein the CD47 antibody in combination with the IL-6 cytokine drug comprises the CD47 antibody in combination with the IL-6 cytokine drug loaded on the nanometer material. In this example, mice were divided into eight different treatment groups, each treatment group consisting of 5 mice, each treatment group consisting of lgG + PBS, anti-CD47+ PBS, lgG + IL-6, anti-CD47+ IL-6, lgG + nano, anti-CD47+ nano, lgG + nano-IL-6, and anti-CD47+ nano-IL-6. After 7 days of injection, when the tumor volume grows to 50-100mm 3 200ug anti-CD47, 200ug lgG, 500ng or 1ug IL-6 intratumorally (or 150ug or 200ug BayK8644 intraperitoneally or Etoposide (VP-16) in a specific amount or other chemical drug capable of inducing IL-6), 500ng or 1ug nano-IL-6 intravenously (or 150ug or 200ug nano-BayK8644 or nano-Etoposide (VP-16) in a specific amount or nano-other chemical drug capable of inducing IL-6), and PBS (PBS) were injected in a corresponding combination according to the groups, once every three days and 4 times in total. And detecting the length (L), width (W) and height (H) of the mouse tumor every three days, calculating the tumor volume (L) W H/2, and finally analyzing the growth trend of the tumor volume and the tumor microenvironment. The IL-6 may be replaced with a specific amount of BayK8644, and the nano-IL-6 may be replaced with a specific amount of nano-BayK 8644.
The result is similar to that of example 2, the anti-CD47+ nano-IL-6 group tumor size is less than that of the anti-CD47+ IL-6 group tumor size is less than that of other group tumors, animal experiments prove that the anti-CD47 and the IL-6 can effectively treat tumors, and the treatment effect is more obvious by loading the IL-6 on the nano material.
S3, comparing the effect of treating colon cancer with CD47 antibody, CD47 antibody in combination with IL-6 cytokine drug, CD47 antibody in combination with IL-6 cytokine drug and CSF-1R, CD47 antibody in combination with IL-6 cytokine drug and CD8 alpha based on the transplanted tumor model.
Take 1X 10 6 A colon cancer cell line (MC38) was implanted subcutaneously into the right side of C57BL/6 mice. Dividing the mice intoFour different treatment groups, 5 mice per treatment group: four groups are respectively injected with lgG + PBS, anti-CD47+ nano-IL-6, anti-CD47+ nano-IL-6+ CSF-1R antibody, anti-CD47+ nano-IL-6+ CD8 + T cell deletion antibody. After 7 days, when the tumor volume grows to 50-100mm 3 Based on lgG, PBS, anti-CD47, nano-IL-6, CSF-1R anti body, CD8 + And (3) carrying out corresponding matched injection on the corresponding groups by the T cell deletion antibody, wherein the injection is carried out once every three days and is totally carried out for four times. Mice were examined every three days for length (L), width (W) and height (H) of tumors, tumor volume was calculated as L W H/2, and tumor volume growth trends and tumor microenvironment were analyzed.
The results show that: anti-CD47+ nano-IL-6+ CSF-1 ramtibody group and anti-CD47+ nano-IL-6+ CD8 + The size of the tumors in the Tcellplementitiody group is larger than that in the anti-CD47+ nano-IL-6 group, which indicates that the nano-IL-6 promotes the anti-tumor effect of anti-CD47 by macrophages and CD8 + T cell mediated.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the claims of the present invention should be included in the protection scope of the claims of the present invention.

Claims (7)

  1. The application of the CD47 antibody and IL-6 cytokine in preparing anti-colon cancer drugs.
  2. 2. The use according to claim 1, wherein the IL-6 cytokine is supported on a nanomaterial targeted to the tumor or tumor microenvironment.
  3. 3. The use according to claim 1, wherein the anti-colon cancer medicament is administered in combination with at least one anti-cancer treatment selected from the group consisting of immunotherapy, chemotherapy, radiotherapy, angiogenesis inhibitor therapy, biologic therapy, bone marrow transplantation, peripheral blood stem cell transplantation, thermotherapy, laser therapy, photodynamic therapy, cancer-targeted therapy.
  4. 4. The use of claim 1, wherein the anti-colon cancer drug is administered by enriching the tumor microenvironment with IL-6 cytokine prior to administration of the CD47 antibody to the tumor.
  5. 5. Use of a kit for the manufacture of a medicament for the treatment of colon cancer, wherein the kit comprises a CD47 antibody and an IL-6 cytokine.
  6. 6. The use of claim 5, further comprising nanomaterials for loading IL-6 cytokines to target a tumor or tumor microenvironment.
  7. 7. The use according to claim 5, wherein the IL-6 cytokine is loaded on the nanomaterial and administered prior to the CD47 antibody; alternatively, the IL-6 cytokine is loaded on a nanomaterial and administered concomitantly with the CD47 antibody.
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