CN114377118A

CN114377118A - NK immune cell drug composition and application

Info

Publication number: CN114377118A
Application number: CN202111650731.7A
Authority: CN
Inventors: 熊学清; 黄金花; 熊子铭
Original assignee: Guangdong Hanshi Stem Cell Biotechnology Co ltd
Current assignee: Guangdong Hanshi Stem Cell Biotechnology Co ltd
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2022-04-22

Abstract

The invention discloses an NK immune cell drug combination and application, wherein the mAb is used as a targeting agent for tumor treatment, different types of antibody molecules are combined with enzyme, chemical drugs, radioactive isotopes, biotoxin and the like, and cytotoxic substances are limited in tumors by virtue of the guiding effect of the antibody, so that the specificity can be enhanced, and the side effects can be reduced; kaminski et al 1 treated solid non-Hodgkin's lymphoma patients with murine labeled B (anti-CD 20 antibody), more than 75% of which were therapeutic; jurcic et al, using a humanized anti-CD 33mAM195 antibody to treat acute cytologic leukemia (AML) and acute promyelocytic leukemia, have achieved very good therapeutic effect; currently, radiolabeled antibodies, such as anti-CD 45 antibodies, have become an integral part of BMT treatment regimens for AML or acute monocytic leukemia; in addition, the application of m marked by radioactive isotope or chemical toxin in the treatment of tumors such as colon cancer, breast cancer, liver cancer, kidney cancer and the like has also achieved obvious curative effect, and the components have synergistic effect, so that stronger immune response can be stimulated, and higher proportion of tumor-specific CD4 and CD8T cells can be induced.

Description

NK immune cell drug composition and application

Technical Field

The invention relates to the technical field of cell therapy, in particular to an NK immune cell drug combination and application.

Background

Cell engineering is an important component of bioengineering, has been widely applied to many fields such as medicine, animal husbandry, environmental protection and the like, and obtains better social benefit and economic benefit.

As one applied science and engineering technology, the history of cell engineering development is still short, and the definition is not yet fully complete. The present disclosure is generally related to cell engineering, and it is intended to design and operate in a targeted manner by applying cell biology, molecular biology and other theories and techniques according to the will of people, so as to change some genetic characteristics of cells, thereby improving or producing new species, and increasing or recovering the ability of cells to produce a specific product, so as to culture, proliferate and extract a large amount of useful products from human under in vitro conditions. It includes theories and techniques such as gene recombination, introduction, amplification and expression, cell fusion, cell microinjection production of transgenic animals, transplantation of plant cell organelles, especially cell nucleus, chromosome modification, in vitro fertilization, embryo transplantation, cell mass culture, and physical and biochemical necessary for extracting relevant cell products.

Researches show that the tumor patients have the characteristics of reduced DC number and functional defects, and the number and the function of tumor tissues and DC infiltrated around the tumor tissues have close relation with the occurrence, the development, the metastasis and the prognosis of tumors. The tumor with DC dense infiltration has high differentiation degree and better prognosis. Whereas tumors that are mildly infiltrated by DCs are often associated with low differentiation and malignant progression. Tumor cells have high-level Fas (protein receptor molecules on the cell surface) expression, can induce apoptosis of lymphocytes expressed by FasL (Fas natural ligand), and can secrete immunosuppressive cytokines such as TGF-beta, IL-10 and the like, so that the antigen presenting capability is reduced, and immune attack is avoided.

In recent years, it has become clear that the immune system does recognize tumor antigens, but that T cells remain quiescent despite the presence of tumor antigens. Based on this phenomenon, there is a hypothesis that: antigen presenting cells in the patient, which fail to correctly recognize the tumor antigen, present it to T lymphocytes, causing a tumor-specific immune response. In recent years, increasing the number of antigen presenting cells, and improving the ability of antigen presenting cells, especially DC cells, to take up, transport, present antigen, and stimulate T cells, are a major focus in the current tumor immunization research.

Based on the discovery of the proliferation, differentiation and function of cells of the immune system, they are regulated by a series of cytokines, among which interleukin-12 (IL-12) and interleukin-15 (IL-15) having structural homology play an important role. In preclinical models, IL-15 has been shown to enhance the anti-tumor immunity of CD8+ T cells. One phase of clinical trials, evaluating the safety, dose and antitumor efficacy of IL-15 in patients with metastatic melanoma and renal cell carcinoma (renal carcinoma), has begun to enroll patients in the National Institutes of Health (NIH). Two types of interleukin-15 superagonists are currently available. One approach has been to combine IL-15 and IL-15R α -Fc (R & DSystems) in vitro to produce a complex, termed IL-15 SA. Another IL-15 superagonist complex (provided by altrbioscience) called ALT-803. Currently, in addition to boosting immunotherapy and vaccination, IL-15SA is also being evaluated for its antiviral and anticancer activity. ALT-803 is an IL-15 superagonist complex comprising an IL-15 mutant (IL-15N72D) fused to the IL-15 receptor alpha/igg 1fc fusion protein. ALT-803 was quickly followed by the U.S. Food and Drug Administration (FDA) in 2017, when a third stage trial of bladder cancer was being prepared. In addition, based on the important role of IL-12 in anti-tumor immunity and anti-infection immunity, it is expected that its clinical application is very promising, especially IL-12 can promote the generation of Cytotoxic T Lymphocyte (CTL) and lymphokine activated killer cell (LAK) in cooperation with IL-2, indicating that the combination of IL-12 and IL-2 is expected to constitute a more effective tumor immunotherapy method.

In recent years, there have been a number of reports of tumor treatment using antigen-loaded DC vaccines, and from the data reported so far, DC vaccines appear to represent a new and very promising approach for improved tumor immunotherapy. At present, Nivolumab is a PD-1/PD-L1 blocking agent on the market, the tumor treatment strategy mainly comprises comprehensive treatment including operation, radiotherapy, systemic chemotherapy, targeted therapy and minimally invasive interventional therapy, but the bottlenecks of high metastasis rate and high recurrence rate after tumor treatment are still difficult to break through. It has been shown that the combination of IL-12 and sPD-1 effectively down-regulates the immunosuppressive cytokine IL-10 and enhances anti-tumor immunity. The PD-1/PD-L1 blocking agent was shown in early clinical studies to demonstrate that monotherapy has been proven safe and controlled, with some patients developing early sustained responses. The PD-1/PD-L1 blocking agent on the market has many side effects, and part of patients stop treatment due to the fact that the patients cannot tolerate the toxicity of the medicine. Recently, several immunodetection point vaccine drugs targeting the PD-1 receptor and its ligands PD-L1 as well as CTLA-4 have been approved for sale. The immune checkpoint inhibitor has good clinical efficacy in patients with various tumors such as melanoma, renal cancer, colorectal cancer, non-small cell lung cancer, liver cancer and the like.

However, many current clinical studies show that clinical response rates are low with immune checkpoint inhibitors such as PD-1/PD-L1 antibody and CTLA-4 antibody, such as in tumor patients, only about 15% of patients can receive clinical benefit with PD-1 antibody; in a plurality of clinical experiments for treating melanoma by using the CTLA-4 antibody, the effective rate is 5-22% before using the CTLA-4 monoclonal antibody ipilimumab alone or in combination. In more recent studies, the antitumor effect of combination with PD-1 pathway immunodetection point inhibitors has been evaluated in clinical models. The current tumor research shows that the single treatment means is difficult to solve the immune suppression of the tumor. Similarly, the use of autologous DC cell reinfusion infected with a recombinant adenovirus encoding human IL-12 (AFIL12) has been reported to treat solid tumors. A total of 17 patients with metastatic pancreatic cancer, colorectal cancer and primary liver cancer received different doses of cell-back therapy. Of the 11 patients who received the complete treatment, only one patient (metastatic pancreatic cancer) showed a Partial Response (PR), 2 patients with metastatic hepatocellular carcinoma had Stable Disease (SD), and the remaining 8 patients showed tumor Progression (PD), although after treatment, the tumor-infiltrating lymphocytes were significantly increased over before treatment. Description of the use of IL12, [ MazzoliniGD, AlfaroC, SangroB, et al.1134. Intratumorlistation of DendriticC wells Engineered Secretienterlite-12 by recombinant Adenoviral in Patientswmet specific magnetic cations [ J ]. journal of clinical Oncologel of journal of clinical Oncolology, 2005,23(5): 999-.

Current clinical trials show that the response rate of DC therapeutic vaccines rarely exceeds 15%, the overall response rate is low, and the use of DC vaccines alone generally does not result in the expected improvement of immunotherapeutic effects and does not result in satisfactory clinical results.

Therefore, the technical personnel in the field provide an NK immune cell drug combination and application to solve the problems in the background technology.

Disclosure of Invention

The invention aims to provide an NK immune cell drug combination and application thereof, so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme:

an NK immune cell medicine combination and a vaccine preparation scheme applied.

Tumor antigen is presented by Antigen Presenting Cells (APC), activated B cells and Dendritic Cells (DC) are all strong antigen presenting cells in vivo, and at present, tumor antigen peptide, tumor cell lysate, tumor cell RNA are used for impacting APC in vitro or tumor associated antigen (TAT) gene is transduced into APC or even the tumor cells and APC cells are fused.

Exposing the self dendritic cells to tumor cells to enable the self dendritic cells to carry tumor antigens; the melanoma vaccine applies four synthetic peptides derived from 100 and acid chromoprotein to enable the peptides to be exposed on the surface of dendritic cells; the possibility of replacing dendritic cells with newly discovered antigen presenting cell fibroblasts by recombinant prostate specific membrane anti-prostate vaccines; fusing the dendritic cells with the whole foot cells.

Cytokine

The development of molecular biology and genetic engineering technology enables many cytokines to be artificially synthesized, and the cytokines become い which is another major product IN cell engineering, wherein EPO, CM-CSF, csr, IN, L2 and the like are applied to clinic to obtain good curative effect, and L-3, L-6, -11, TPO, NGF, EGF, LIra, TNF, MCSF and the like show good prospects through animal tests and clinical application.

Products for direct therapeutic use

The anti-tumor effect of interferon is mainly to cause tumor cell lysis by prolonging cell cycle of each stage and single or synergistic effect of several IN, and can also enhance the killing to tumor cells by the cytotoxic effect of leucocytes, and enhance the tumor cell lysis by antibody-mediated cytotoxicity and complement; DN is currently widely used in the treatment of hairy cell leukemia (response rate up to 8090%), Multiple Myeloma (MM), Chronic Myelogenous Leukemia (CML), non-hodgkin's lymph and other entities such as gastric cancer, basal cell carcinoma of the skin, etc.

IL2 can induce activation and increase of ML, LAK and CL cells in vivo, and induce TNF and DN gamma release, so it has anti-swelling effect; 2 alone has little anti-tumor effect, but IL-2/LAK therapy has better curative effect; the R is injected with IL-2(1-6) x10u/(Kg.d), after 1wK, peripheral lymphocytes are separated by 5x10-5x10 degrees, the peripheral lymphocytes are cultured with the IL-2 in vitro for 3-4 days and then are infused back to a patient, and IL-2 is given for intravenous injection, and the effective rates of renal cancer, pigment, colorectal cancer and NHL are 31 percent, 18 percent, 11 percent and 75 percent respectively. M-CSF can support the growth of CFU-macrophages, and induce hematopoietic cells to differentiate and mature, and can cause giant cells to proliferate in the presence of other colony stimulating factors; the M-M-CSF can enhance the anti-tumor capability of a tumor patient by improving the number and the function of effector cells with the effect of killing tumors; the application of MCSF (magnetic resonance particle) by Bagorin and the like to improve the number of mononuclear macrophages and ADCC (advanced cellular cytotoxicity) effect and obtain a certain curative effect on treating the melanoma; the combination therapy of M-M-CSF with an anti-gall antibody is currently on trial.

Cytokine product for adjuvant therapy of tumor

Erythropoietin (EPO) can stimulate the transformation of various stem cells to erythroid systems, promote the proliferation of protoerythroid cells and the self-synthesis of hemoglobin, shorten the maturation time of the erythrocyte, the EPO is used for treating the tumorous anemia at present, granulocyte and granulocyte colony stimulating factor can increase the neutrophilic granulocytes in blood and bones, the EPO is used for treating the neutrophilic granulocytic deficiency in radiotherapy and chemotherapy, a good treatment result is obtained, IL-3 can stimulate hematopoietic stem cells, so that the progenitor cells of the erythroid systems, the granulocyte systems and the megakaryocyte systems can proliferate and differentiate, the EPO is used for inhibiting bone and treating bone failure diseases such as AA after late tumors and chemotherapy, a certain treatment effect is obtained, IL-6 can promote the proliferation of the bone hematopoietic cells, and has obvious stimulation effects on the maturation of megakaryocyte iron and the generation of straight platelets.

mAb 17-1A, used directly in tumor therapy, for the adjuvant treatment of colon cancer after surgery, used alone or in combination with chemotherapy, is a humanized mAb for the treatment of acute leukemia, anti-B1 mAb, which acts on CD20 antigen of B cell lymphoma, and is used in combination with high dose anti-B1 bone transplantation for the treatment of B cell lymphoma; the anti-Her 2/neu (ani-H2/ne) mAb is used for the treatment of advanced breast cancer with over-expression of this protein, and some mAb products against other tumors are also in clinical trials.

Compared with the prior art, the invention has the beneficial effects that:

the mAb used as a targeting agent for tumor treatment combines different types of antibody molecules with enzyme, chemical drugs, radioactive isotopes, biotoxins and the like, and enables cytotoxic substances to be limited to tumors by virtue of the guiding effect of the antibody, so that the specificity can be enhanced, and the side effects can be reduced; kaminski et al 1 treated solid non-Hodgkin's lymphoma patients with murine labeled B (anti-CD 20 antibody), of which more than 75% were effective. (ii) a Jurcic et al, using a humanized anti-CD 33mAM195 antibody to treat acute cytologic leukemia (AML) and acute promyelocytic leukemia, have achieved very good therapeutic effect; currently, radiolabeled antibodies, such as anti-CD 45 antibodies, have become an integral part of BMT treatment regimens for AML or acute monocytic leukemia; in addition, the application of m marked by radioactive isotope or chemical toxin in the treatment of tumors such as colon cancer, breast cancer, liver cancer, kidney cancer and the like has also achieved obvious curative effect, and the components have synergistic effect, so that stronger immune response can be stimulated, and higher proportion of tumor-specific CD4 and CD8T cells can be induced. The invention is expected to solve the defects that the immunosuppression and the clinical effect of the tumor are not ideal by a single treatment means.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments. In the description of the present invention, it should be noted that the terms "upper", "lower", "inner", "outer", "front", "rear", "both ends", "one end", "the other end", and the like indicate orientations or positional relationships only for convenience in describing the present invention and simplifying the description, but do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance. In the description of the present invention, it is to be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "disposed," "connected," and the like are to be construed broadly, such as "connected," which may be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.

In the embodiment of the invention, an NK immune cell medicine composition and application thereof,

Cytokine

Products for direct therapeutic use

Cytokine product for adjuvant therapy of tumor

The mAb used as a targeting agent for tumor treatment combines different types of antibody molecules with enzyme, chemical drugs, radioactive isotopes, biotoxins and the like, and enables cytotoxic substances to be limited to tumors by virtue of the guiding effect of the antibody, so that the specificity can be enhanced, and the side effects can be reduced; kaminski et al 1 treated solid non-Hodgkin's lymphoma patients with murine labeled B (anti-CD 20 antibody), of which more than 75% were effective. (ii) a Jurcic et al, using a humanized anti-CD 33mAM195 antibody to treat acute cytologic leukemia (AML) and acute promyelocytic leukemia, have achieved very good therapeutic effect; currently, radiolabeled antibodies, such as anti-CD 45 antibodies, have become an integral part of BMT treatment regimens for AML or acute monocytic leukemia; in addition, the application of m marked by radioactive isotope or chemical toxin in the treatment of tumors such as colon cancer, breast cancer, liver cancer, kidney cancer and the like has also achieved obvious curative effect.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims

1. A NK immune cell medicine combination and application are characterized in that an Antigen Presenting Cell (APC) is used for presenting a tumor antigen, activated B cells and Dendritic Cells (DC) are all in vivo strong antigen presenting cells, and tumor antigen peptides, tumor cell lysate and tumor cell RNA are used for impacting the APC in vitro or tumor-associated antigen (TAT) genes are transduced into the APC or even the tumor cell and the APC are fused at present.

2. The NK immune cell pharmaceutical composition and use according to claim 1, wherein the method comprises exposing autologous dendritic cells to tumor cells to make them carry tumor antigens; the melanoma vaccine applies four synthetic peptides derived from 100 and acid chromoprotein to enable the peptides to be exposed on the surface of dendritic cells; the possibility of replacing dendritic cells with newly discovered antigen presenting cell fibroblasts by recombinant prostate specific membrane anti-prostate vaccines; fusing the dendritic cells with the whole foot cells.

3. The NK immune cell drug combination and use according to claim 1, comprising the following components (a) to (d):

(a) interleukin-15, a mutant or functional fragment thereof, or a nucleic acid encoding same;

(b) interleukin-15 receptor alpha, a mutant or functional fragment thereof, or a nucleic acid encoding same;

(c) interleukin-12, a mutant or functional fragment thereof, or a nucleic acid encoding same;

(d) PD1-CD80 fusion protein, a mutant or functional fragment thereof, or a nucleic acid encoding the same.

In certain embodiments, the molar ratio of component (a) to component (b) of the composition for enhancing T lymphocyte immunity is (0.8-1.2);

in certain embodiments, components (a) and (b) of the composition for enhancing T lymphocyte immunity are separate proteins, and components (a) and (b) are produced by expression of the same nucleic acid molecule.

In certain embodiments, the molar ratio of component (a) to (b) to (c) to (d) of the composition for enhancing T lymphocyte immunity is 1:1 (0.8-1.2) to (0.8-1.2). In a second aspect of the invention, there is provided an immune cell comprising the composition of the first aspect.

In certain embodiments, the immune cell is a dendritic cell.

In certain embodiments, the immune cell further comprises an antigen or a nucleic acid encoding the same.

There is provided a pharmaceutical composition comprising a composition or immune cell according to above, and optionally a pharmaceutically acceptable carrier.

There is provided a method for improving the properties of T lymphocytes, comprising the step of contacting said T lymphocytes with said composition or said immune cells according to said composition, thereby improving the properties thereof.