CN115772497A - Dendritic cell vaccine targeting G12V mutant KRAS - Google Patents

Abstract

The invention discloses a dendritic cell vaccine targeting G12V mutant Kras protein, and simultaneously provides a dendritic cell vaccine targeting G12V mutant Kras protein. According to the invention, the related antigen of the target G12V mutation KRAS protein receptor is synthesized in the dendritic cells and then presented to the T lymphocytes, so that the problems of large side effect of small molecule chemotherapy, high CAR-T treatment cost, cytokine storm and the like are solved.

Description

Dendritic cell vaccine targeting G12V mutant KRAS

Technical Field

The invention relates to the field of immunology, in particular to a novel dendritic cell for expressing G12V mutant Kras protein and application thereof.

Background

Dendritic Cells (DCs) are the most powerful professional antigen-presenting cells in the human body, and can activate CD8+ cytotoxic T Cells (CTLs) and CD4+ helper T cells (Th), playing an important role in immune response processes such as anti-tumor and anti-virus. Most studies indicate that tumors in vivo inhibit DC maturation rather than directly inhibit DC function. The research shows that the number and the function of dendritic cells in malignant tumor tissues are inversely related to the infiltration degree of tumor cells to peripheral tissues in primary foci and metastatic foci of the tumor tissues and the clinical stage of patients, and the larger the number of the infiltrating dendritic cells in most solid tumors, the better the prognosis of patients (J Surg Oncol,2007,95 (2): 123-128). Dendritic cells in tumor patients have differentiation and maturation obstacles, which cause functional defects of different degrees in aspects of expressing cytokines, surface antigens, activating T cell proliferation, inducing CTL generation and the like, including increase of CD8+ T cells, reduction of CD4+/CD8+ ratio, reduction of the number of natural killer cells and the like. Therefore, the in vitro induced functional DC has important clinical application value for immunotherapy.

DCs are widely distributed in various parts of the human body, such as blood, interstitial tissues of liver, spleen, lymph nodes, lung, kidney, gastrointestinal tract and the like, and account for 0.5-1% of the total mononuclear cells of peripheral blood. The DC subpopulations are complex to classify, with myeloid-derived DCs (mdcs) and lymphoid-derived DCs (pdcs) being the two major types of peripheral blood DC. DCs can be generated via monocyte differentiation, in vitro culture of monocytes in isolated PBMCs, GM-CSF and IL-4 induced to generate immature DCs (Curr Protoc Immunol 2005. Immature DCs can be differentiated to mature by different maturation-inducing components, and the maturation state of DCs is a key factor that determines the effectiveness of DC vaccines. Immature DCs have limited capacity to prime T cells and may also induce T cell tolerance. In addition, the mature DC has stronger migration capability and can migrate to the T cell area of the secondary lymphoid tissue more effectively so as to induce immune response.

ras oncogene is involved in regulation of cell growth and differentiation, and in formation and development of various tumors, kras is a member of ras gene family, and two other ras genes related to human tumors are Hras and Nras. The Kras protein consists of 188 (type b) or 189 (type a) amino acids. When Kras is not mutated, like a molecular switch, the Kras can normally control the pathway of regulating cell growth. However, mutation of Kras results in abnormal cell proliferation and disorganization of intracellular cell signaling, leading to canceration. Mutations of Kras proteins occur mainly at positions 12, 13, 62 or 146 (Swiss Med Wkly,2010,140, ww13112) of the amino acid sequence, and mutations at positions 12 or 13 are relatively common, with the highest percentage of mutations at position 12 accounting for about 85%, including G12D, G12V, G12C, G12A, G12S and G12R, etc., while mutations at G12D account for about 35% of Kras mutations. Kras protein mutations are common in colorectal, pancreatic, lung, biliary, ovarian, and endometrial cancers. Wherein the number of Kras protein mutation in colorectal cancer patients accounts for 30% -60%.

For The treatment of Kras mutations, existing studies include inhibition of Kras activation by small molecule inhibitors (The New England joarnal of medicine,2020, 383. However, no method for achieving therapeutic purposes by using a Kras mutant antigen-loaded dendritic cell vaccine has been reported.

Disclosure of Invention

The present invention provides dendritic cell vaccines that target G12V mutant KRAS protein in response to the deficiencies of the above-described treatment methods or to provide one more option for KRAS gene mutant cancer patients. The invention synthesizes related antigen targeting G12V mutant KRAS protein receptor in dendritic cell, and then presents the antigen to T lymphocyte. Thus, the problems of large side effects of small molecule chemotherapy, high CAR-T treatment cost, cytokine storm and the like can be avoided.

Accordingly, one aspect of the present invention provides dendritic cells capable of expressing a G12V mutant Kras protein.

Another aspect of the invention provides a prophylactic and/or therapeutic vaccine targeting G12V mutant Kras protein positive tumors. Illustratively, the prophylactic and/or therapeutic vaccine according to the invention comprises the dendritic cell capable of expressing the G12V mutant Kras protein. Preferably, the dendritic cells are infected by lentivirus to express the G12V mutant KRAS protein, so that the epitope of the G12V mutant KRAS protein is loaded into the dendritic cells.

In a third aspect of the invention there is provided a method of treating or preventing a cancer that expresses a G12V mutant Kras protein comprising administering to a subject a therapeutically or prophylactically effective amount of dendritic cells capable of expressing a G12V mutant Kras protein or a vaccine comprising the same.

In a fourth aspect, the invention provides the use of a dendritic cell capable of expressing a G12V mutant Kras protein in the manufacture of a vaccine for the treatment or prevention of a cancer expressing a G12V mutant Kras protein.

In some cancer species, the G12V mutant KRAS protein receptor is overexpressed on the surface of cancerous cells. Thus, the cancer in the present invention may be, for example, colorectal cancer, pancreatic cancer, lung cancer, bile duct cancer, ovarian cancer, endometrial cancer, and the like.

The invention also provides a method for stimulating and activating dendritic cells, which comprises the step of contacting a vector capable of expressing the G12V mutant Kras protein with dendritic cells to be activated, so that the dendritic cells are loaded with the tumor-specific G12V mutant Kras protein.

Illustratively, the dendritic cells provided herein are isolated activated dendritic cells that present on the surface a G12V mutant KRAS protein receptor, a G12V mutant KRAS protein-HLA complex or a polypeptide-MHC complex. Illustratively, the isolated dendritic cells are obtained by contacting and culturing a vector (e.g., a viral vector, such as a lentivirus) expressing the G12V mutant Kras protein of the present invention with dendritic cells to be activated.

The invention also provides a T cell activated by a dendritic cell of the invention.

The invention also provides the use of a vector (e.g., a viral vector, such as a lentivirus) capable of expressing the G12V mutant Kras protein for activating dendritic cells.

The invention also provides a carrier capable of expressing the G12V mutant Kras protein, an activated dendritic cell or an activated T cell, and application of the carrier, the activated dendritic cell or the activated T cell, such as preparation of a medicament for preventing or treating cancer.

The invention also provides a pharmaceutical composition or a medicament, which comprises a pharmaceutically acceptable carrier, the carrier capable of expressing the G12V mutant Kras protein, and an isolated activated cell (such as a dendritic cell or a T cell).

Illustratively, the medicament or the pharmaceutical composition is a vaccine.

The present invention also provides a method for preventing or treating a disease (e.g., cancer) comprising administering to a subject in need thereof an effective amount of the vector capable of expressing the G12V mutant Kras protein, isolated activated cells (e.g., dendritic cells or T cells) of the present invention.

The invention also provides a cytotoxic T Cell (CTL) specific for the cancer expressing the G12V mutant Kras protein, which comprises the step of co-culturing the dendritic cell loaded with the G12V mutant KRAS protein and the T cell to obtain the cytotoxic T Cell (CTL) specifically targeting the cancer expressing the G12V mutant Kras protein.

The invention also provides a cellular composition comprising an activated dendritic cell of the invention and a T cell or CTL cell.

The invention also provides another cell composition comprising the activated dendritic cells and CIK cells of the invention.

Since the activated dendritic cells of the present invention are capable of presenting G12V mutant KRAS protein molecular targets, they can be used as therapeutic or prophylactic vaccines against cancers that express G12V mutant KRAS protein. Accordingly, the present invention also provides a therapeutic or prophylactic vaccine against a cancer expressing a G12V mutant Kras protein comprising isolated cells (e.g. activated dendritic cells) of a peptide according to the present invention, and optionally an adjuvant.

Further, the present invention also provides a method of preventing or treating a cancer expressing a G12V mutant Kras protein comprising administering the vaccine of the invention into a subject.

Further, the present invention also provides a method of stimulating and activating dendritic cells comprising the step of expressing a G12V mutant KRAS protein in a dendritic cell to be activated, thereby loading the dendritic cell with the expressed G12V mutant KRAS protein.

The present invention also provides an activated dendritic cell obtained by contacting and culturing a vector (e.g., viral vector, such as lentivirus) expressing the G12V mutant Kras protein of the present invention with a dendritic cell to be activated.

The invention also provides a cytotoxic T Cell (CTL) specifically aiming at the cancer expressing the G12V mutant KRAS protein, which comprises the step of co-culturing the dendritic cell loaded with the G12V mutant KRAS protein molecular target and lymphocytes to further obtain the cytotoxic T cell specifically targeting the G12V mutant KRAS protein.

The invention also provides a cellular composition comprising the activated dendritic cells of the invention and an immune cell.

The invention also provides another cell composition comprising the activated dendritic cells and CIK cells of the invention.

Likewise, since the activated dendritic cells of the present invention are capable of presenting cancer-specific molecular targets expressing G12V mutant Kras protein, they can be used as therapeutic or prophylactic vaccines against cancers expressing G12V mutant Kras protein. Accordingly, the present invention also provides a therapeutic or prophylactic vaccine against a cancer expressing a G12V mutant Kras protein comprising activated dendritic cells of the invention.

Further, the present invention also provides a method of preventing or treating a cancer expressing a G12V mutant Kras protein comprising administering to a subject an activated dendritic cell of the invention or a cell composition of the invention.

In a specific embodiment of the invention, the activated DC cells of the invention have a high positive loading rate when tested. Further, the activated DC cells express CD80, CD86, CD83, HLA-DR, and present T lymphocytes, which become CTL cells specifically recognizing cancer cells expressing G12V mutant Kras protein. Ex vivo experimental data indicate that CTL stimulated and matured by the dendritic cells of the present invention can significantly inhibit tumor cells expressing G12V mutant Kras protein (relative to tumor cells not expressing G12V mutant Kras protein).

Furthermore, the invention also provides a preparation method of the dendritic cells, which comprises the step of infecting the dendritic cells with lentiviruses for expressing the G12V mutant Kras protein so that the dendritic cells can express the G12V mutant Kras protein.

Further, the method for preparing dendritic cells according to the present invention further comprises a step of maturing the prepared dendritic cells. Specifically, immature dendritic cells are subjected to induction culture in the presence of IFN-. Gamma.and LPS to promote differentiation and maturation of DCs. Illustratively, the IFN-gamma and LPS content is 10-100ng/mL and 10-100EU/mL, respectively, preferably, the IFN-gamma and LPS content is 50ng/mL and 50EU/mL, respectively.

Further, in the maturation culture, a low-temperature standing is introduced, for example, a standing at-4 ℃ to 10 ℃ (for example, -4 ℃ to 4 ℃, or-4 ℃ to-2 ℃) and then an ordinary-temperature culture is performed, for example, a standing for 0.5 to 4.0 hours, and then the temperature is gradually raised to 37 ℃, and the ordinary-temperature culture is continued.

The invention also provides a CTL (cytotoxic T lymphocyte) specifically aiming at the G12V mutant KRAS protein, which is prepared by the following method:

the matured CD cells of the invention are co-cultured with T lymphocytes to produce CTLs specific for the G12V mutant KRAS protein. Illustratively, the T cell is a CD3+ T cell.

The therapeutic or prophylactic vaccine of the invention can be administered by instillation or injection, such as intratumoral or intravenous or intramuscular injection, and further acts on the immune system to enhance the immune response of the body, thereby finally prolonging the life of the patient and improving the quality of life.

Therapeutic vaccines: in organisms infected with pathogenic microorganisms or suffering from certain diseases, natural, artificially synthesized or expressed by gene recombination technology for treating or preventing disease progression is achieved by inducing specific immune responses.

CTL (Cytotoxic T Lymphocyte) Cytotoxic T cell

DC (Dendritic Cell): dendritic cell

CIK (Cytokine-Induced Killer): cytokine-induced killer cells

PBMC (Peripheral blood mononary cell): peripheral blood mononuclear cells

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. This will not be repeated.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly described below.

FIG. 1 shows the results of flow cytometry for the expression levels of the DC surface molecular markers CD80, CD83, CD86 and HLA-DR.

FIG. 2 is a graph showing the results of flow analysis of expanded cultured CD3+ CTL cells.

FIG. 3 shows the killing effect of CTL against tumor cells with different expression levels of G12V mutant KRAS protein.

Detailed Description

To make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of the drug may reduce the number of cancer cells; reducing the size of the tumor; inhibit (i.e., slow to some extent and preferably prevent) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably prevent) tumor metastasis; inhibit tumor growth to some extent; and/or alleviate one or more symptoms associated with cancer to some extent. To the extent that the drug can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. For cancer therapy, efficacy can be measured, for example, by assessing time to disease progression (TTP) and/or determining Response Rate (RR).

The term "prophylactically effective amount" refers to an amount of a drug effective to prevent a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of the drug may reduce the risk of acquiring cancer in a subject, leaving it free of cancer for the lifetime or delaying the onset of cancer.

The term "cancer" refers to or describes a physiological condition in mammals that is generally characterized by unregulated cell growth. Examples of cancers include, but are not limited to, squamous cell cancer expressing G12V mutant Kras protein, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, liver cancer, stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer, and the like.

During cellular immunity, antigens are usually presented to the cell surface along with MHC complexes. Thus, the invention also provides an isolated cell capable of presenting a G12V mutant KRAS protein or a G12V mutant KRAS protein-MHC complex to the surface thereof. Illustratively, the cell is an immune cell, e.g., an APC, such as a DC (dendritic cell), or a B cell, or a T2 cell. Preferably, the cell presenting the G12V mutant KRAS protein or the G12V mutant KRAS protein-MHC complex is isolated. Cells presenting the polypeptide-MHC complex of the invention can be used to isolate T cells activated by the polypeptide or complex and further sorted for in vitro proliferation for reinfusion into a subject and T cell receptors.

The invention also provides the use of the cell, for example, in the preparation of a medicament for preventing or treating cancer.

The invention also provides a pharmaceutical composition, which comprises a pharmaceutically acceptable carrier and the cell.

For the pharmaceutical composition of the present invention, the dosage form may be any suitable administration route, such as injection (including subcutaneous, intramuscular, intraperitoneal or intravenous injection), inhalation or oral, or nasal, or anal routes. The compositions may be prepared by any method known in the art of pharmacy, for example, by mixing the active ingredient with the carrier or excipient under sterile conditions.

The cells of the invention may be provided in the form of a vaccine composition. The vaccine composition may be used for the treatment or prevention of cancer, wherein the vaccine composition may further comprise an adjuvant.

In the present invention, the cell is preferably an Antigen Presenting Cell (APC), for example selected from the group consisting of a monocyte, a monocyte derived cell, a macrophage, a Dendritic Cell (DC), preferably a DC cell.

Major Histocompatibility Complex (MHC) is a gene complex encoding a Human Leukocyte Antigen (HLA) gene. HLA genes are expressed as protein heterodimers displayed on the surface of human cells to circulating T cells. HLA genes are highly polymorphic, allowing them to fine tune the adaptive immune system.

MHC molecules fall into three classes: MHC I, MHC II and MHC III. MHC class I molecules consist of an alpha heavy chain and beta-2-microglobulin; MHC class II molecules consist of an alpha and a beta chain. The structure of MHC molecule contains a binding groove for non-covalent interaction with antigen; MHC iii encodes primarily complement components such as Tumor Necrosis Factor (TNF) and heat shock protein 70 (HSP 70).

MHC class I molecules are expressed on most nucleated cells. They present mainly endogenous proteins, defective ribosomal products (DRIP) and peptides generated by cleavage of larger peptides. However, exogenously derived peptides are also often found on MHC class I molecules. MHC class II molecules are found predominantly on Antigen Presenting Cells (APCs) and present predominantly, for example, peptides of exogenous or transmembrane proteins that are occupied by APCs during endocytosis and subsequently processed.

Complexes of G12V mutant KRAS protein and MHC class I are recognized by CD8 positive T cells bearing the corresponding T Cell Receptor (TCR), while complexes of G12V mutant KRAS protein and MHC class II molecules are recognized by CD4 positive helper T cells bearing the corresponding TCR.

CD4 ⁺ T helper cells play an important role in inducing and maintaining an efficient response of CD8 positive cytotoxic T cells. At the tumor site, T helper cells maintain a cytokine milieu that is beneficial for cytotoxic T Cells (CTLs) and attract effector cells, such as CTLs, natural Killer (NK) cells, macrophages, and granulocytes.

In the absence of inflammation, MHC class II molecule expression is primarily restricted to immune system cells, especially Antigen Presenting Cells (APCs). Expression of MHC class II molecules is found in tumor cells of cancer patients.

Term(s)

The term "isolated" means that a substance is removed from its original environment (e.g., the natural environment if it occurs naturally). For example, a native nucleotide or polypeptide in a living animal is not isolated, but a nucleotide or polypeptide isolated from some or all of the coexisting materials in the native system is isolated. Such polynucleotides may be part of a vector and/or such polynucleotides and polypeptides may be part of a composition, and as the vector or composition is not part of its natural environment, it remains isolated.

CD3+ T lymphocytes

CD3 is a class of antigens on the surface of T lymphocytes and CD3+ is a mature T lymphocyte surface marker. CD3 has five peptide chains, namely, the gamma, delta, epsilon, zeta and eta chains, which are transmembrane proteins. The transmembrane region of the CD3 molecule is connected with the transmembrane regions of two peptide chains of TCR through a salt bridge to form a TCR-CD3 complex, and the most common TCR/CD3 complex is composed of TCR alpha-beta/CD 3 gamma delta epsilon zeta and is jointly involved in the recognition of antigens by T cells. The activation signal generated by the TCR recognition of the antigen is transduced into T cells by CD 3.

CD4+ cell is an important immune cell in human immune system, is mainly expressed by helper T cell (Th), is receptor of Th cell TCR recognition antigen, combines with non-polypeptide region of MHC class II molecule, participates in Th cell TCR recognition antigen process, CD4+ is the most important pivotal cell for regulating immune response.

The CD8 molecule is a leukocyte differentiation antigen, a glycoprotein on part of the surface of T cells, which is used to help T Cell Receptors (TCRs) recognize antigens and participate in the transduction of T cell activation signals, also known as co-receptors for TCRs. CD 8-expressing T cells (CD 8+ T cells) typically differentiate into cytotoxic T Cells (CTLs) upon activation, capable of specifically killing target cells. Cytotoxic T cells are direct killer cells in the immune response.

The ratio of CD4+/CD8+ can be used as a clinical diagnosis sensitive index for judging the human immunologic dysfunction.

DC surface molecular marker

CD80 is a membrane antigen essential for T lymphocyte activation. Upon T lymphocyte activation, TCR/CD3 of the T lymphocyte binds to the MHC of the APC. CD80 is expressed on activated B lymphocytes, activated T lymphocytes, human T lymphocyte leukemia virus-1 (human T cell leukemia virus-1, HTLV-1) positive T lymphocytes, IFN-gamma stimulated monocytes, dendritic cells, but not on non-activated B lymphocytes, erythrocytes, granulocytes and monocytes.

CD83 belongs to the immunoglobulin superfamily, is a specific marker of dendritic cells, and is involved in antigen presentation and lymphocyte activation.

CD86 belongs to the immunoglobulin superfamily, has the name B7.2, and is expressed on the cell surfaces of dendritic cells, monocytes, memory T lymphocytes and the like.

HLA-DR is an MHC class II molecule containing 2 subunits (alpha and beta) of molecular weight 36kD and 27kD, respectively. HLA-DR is expressed on B lymphocytes, monocytes, macrophages, activated T lymphocytes, activated NK lymphocytes and human progenitor cells. It is also expressed in thymic epithelial cells, B lymphocyte-dependent regions of the spleen and lymph nodes, and B lymphocyte lymphomas. HLA-DR and CD1a are co-expressed in epidermal Langerhans cells.

The activation state of the DC cells can be embodied by detecting CD80/CD83/CD86/HLA-DR through flow cytometry, the method can be used for detecting the antigen presentation function of dendritic cells, detecting the treatment effect of a border immune state machine of the content in the peripheral blood before and after CIK cell treatment, and can also be used for evaluating the cell culture state in a CIK treatment scheme.

Interleukin

IL-7 is a glycoprotein secreted by bone marrow stromal cells, has a molecular weight of 25KD, and its gene is located on chromosome 8 and consists of 152 amino acids. IL-7 is produced primarily by bone marrow stromal cells, and its primary biological function is to promote proliferation and differentiation of T and B cell precursors.

IL-15 has similar biological functions to IL-2, and IL-15 and IL-2 share a gamma c receptor subunit, and both cytokines can induce the differentiation and promote the proliferation of T cells, B cells and NK cells. At present, IL-15 is often used in combination with IL-2 for the in vitro expansion culture of T cells or NK cells.

Another property of IL-15 in immunotherapy is the maintenance of memory T cells, which plays an important role in long-term antitumor activity. IL-15, as a pleiotropic cytokine, plays an important role in immunity, tumorigenesis, allergic reactions and autoimmune diseases.

Dendritic cell vaccine

According to the invention, the dendritic cell for the G12V mutation KRAS protein epitope is prepared by loading the G12V mutation KRAS protein-related antigen into the dendritic cell. The obtained vaccine is subjected to expression level detection of surface molecules CD80, CD83, CD86 and HLA-DR and in vitro tumor cell killing effect detection.

The invention designs a gene modified lentivirus sensitized DC (activated DC), which can effectively identify, process and treat tumor antigens and present the antigens to T cells to stimulate stronger antigen-specific T cell immune response.

The dendritic cell vaccine prepared by the invention is an effective vaccine with no side effect and strong targeting property, has a wide application range, and can be used for treating G12V mutation KRAS protein positive tumor diseases (but not limited to) including colorectal cancer, pancreatic cancer, gastric cancer and the like. Can also be used for adjuvant treatment of tumor, and can be used for associated treatment of tumor diseases by combining chemotherapy and other tumor treatment means.

Cytotoxic T lymphocytes

Cytotoxic T Lymphocytes (CTLs), also known as killer T lymphocytes, are a subset of leukocytes, are specific T cells that secrete various cytokines exclusively for immune function. It has killing effect on some viruses, tumor cells and other antigen substances. Therefore, it is an important link of the anti-tumor mechanism of the organism and is also one of the main effector cells of the tumor immune adoptive therapy.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: conditions described in a Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Example 1 preparation of lentiviruses loaded with G12V mutant KRAS protein-associated antigens

A modified dendritic cell, wherein a lentivirus vector loads G12V mutation KRAS protein related antigen to infect the dendritic cell, so that an antigen sequence of interest is integrated into the genome of the dendritic cell.

Construction of G12V mutant KRAS protein-associated antigen lentivirus expression vector

The DNA sequence corresponding to the amino acid residue of the G12V mutant KRAS protein was constructed into the lentiviral vector pLVX-mCMV-ZsGreen1-Puro using the restriction enzyme sites EcoR I and Not I.

2. Lentivirus packaging and extraction

2.1 293FT cells were collected by Trypsin digestion in a 10cm dish at 5X 10 ⁶ The cells were plated and 9mL of the medium after resuspension of the cells was added per 10cm of the culture dish.

2.2 preparing two 5mL centrifuge tubes which are divided into a tube a and a tube b, respectively adding 1.5mL serum-free Opti-MEM culture medium into the tube a and the tube b, adding 8 mu g mixed package helper plasmid and 6 mu g package target plasmid into the tube a, and gently mixing the materials; add 36. Mu.L of Lipofectamine 2000 to tube b and mix gently.

2.3 the mixed solution in tube b is added into tube a gently, mixed gently and mixed evenly, and incubated for 10min at room temperature to form DNAPIFACTAMINE 2000 compound.

2.4 adding the incubated DNA-Lipofectamine 2000 compound drop by drop and whirling into a 10cm culture dish, and gently whirling and mixing uniformly. 37 ℃ and 5% of CO ₂ The incubator was incubated overnight.

2.5After overnight incubation, the old medium was removed and the cells were relatively easy to shed, 12mL of DMEM +2% FBS virus-prepared medium was carefully added along the wall, 37 ℃ and 10% CO ₂ The incubator was incubated overnight.

2.6 after transfection, cell culture supernatant was collected at 40h, centrifuged at 1500rpm for 5min, and cell debris was removed. The supernatant was filtered through a 0.45 μm PES filter. 10cm dish 12mL fresh DMEM +2% FBS preparation medium carefully added along the wall, 37 ℃, 10% CO ₂ The incubator was incubated overnight.

2.7 cell culture supernatants were collected 62h after transfection and centrifuged at 2000rpm for 5min to remove cell debris. The supernatant was filtered through a 0.45 μm PES filter.

3. Virus titer detection

3.1 Collection of HT1080 cells in logarithmic growth phase and plating in 96-well plate, 6X 10 ³ Cell-mass/well plates, 100 μ L DMEM +10% FBS complete medium per well.

3.2 the following day 10-fold gradient dilutions of fluorescent expressing virus fluid were performed in DMEM serum-free medium, each gradient repeated 3 times. The specific operation is as follows: preparing 9 sterile centrifuge tubes, preparing 8 gradient application gradients, adding 80 mu L of DMEM into the first centrifuge tube, taking 10 mu L of a sample to be detected, and mixing uniformly. Then, 360. Mu.L of DMEM is added into each gradient centrifuge tube, and the samples with the last dilution gradient of 40. Mu.L are sequentially added and mixed evenly. Each gradient is marked with the added virus stock solution in turn as 100, 10-1, 10-2, 10-3, 10-4, 10-5, 10-6 and 10-7.

3.3 sequentially sucking out 90 mu L of culture medium in each gradient hole, and adding 90 mu L of gradient virus liquid diluted in corresponding gradient. 37 ℃ and 5% of CO ₂ The incubator was used for overnight incubation.

3.4 DMEM +10% FBS complete medium 100. Mu.L was added after 24 h.

3.5 after 72h of infection, the fluorescent expression condition is observed, the number of fluorescent cells is less along with the increase of dilution gradient fold, and the number of cells between gradients is consistent with the difference of dilution fold. The fluorescence expression virus titer was calculated by counting the number of fluorescent cells of the last dilution gradient at the maximum dilution of visible fluorescence. And detecting the virus titer of the target sequence by taking the titer as a reference.

3.6 Collection of logarithmic growth phase HT1080 cellsSpreading 6-well plate with cell number of 2 × 10 ⁵ 2mLDMEM +10% per well FBS complete medium.

And 3.7 infecting with target virus liquid and fluorescence expression virus liquid the next day, wherein the fluorescence expression virus liquid uses two gradients of 5 mu L and 1 mu L, the target virus liquid uses 1 mu L, and the target virus liquid is respectively diluted by 1mL of serum-free DMEM medium and mixed evenly. Aspirating 1mL of the culture medium from each well, adding the diluted virus solution to the corresponding well, gently shaking and mixing, and adjusting the concentration of CO at 37 ℃ to 5% ₂ The incubator was incubated overnight.

3.8 infection after 48h, trypsinize each well of cells, collect the pellet, rinse with cold PBS, and collect the pellet.

3.9 collecting cell sediment and extracting genome DNA according to the steps of the animal cell genome DNA extraction kit.

3.10 using 5. Mu.L of fluorescent expression virus infected cell genomic DNA as standard, diluting 6 gradients by 5-fold gradient, and complementing the change of the template amount after dilution with uninfected cell genomic DNA. QPCR detects the integrated copy number change of lentivirus LTR sequence on the genome DNA, and calculates the titer of the virus solution of interest.

Example 2 transfection, maturation Induction of DCs and preparation of specific CTLs

1) DC transfection and maturation induction: collecting peripheral blood of a volunteer, separating autologous plasma (inactivated at 56 ℃ for 30min and stored at 4 ℃) from peripheral blood mononuclear cells PBMC by using a Ficoll-Hypaque (polysucrose-diatrizoate) density gradient separation method, paving the PBMC in a T75 culture bottle with a serum-free x-vivo15 in a cell amount of 5-7 x 107, collecting suspension cells after 1h for separating CD3+ T lymphocytes, and stimulating adherent cells to differentiate the mononuclear cells into DC by replacing 1640 culture medium containing IL-4 (100 ng/mL), GM-CSF (100 ng/mL) and autologous plasma (2%).

Immature DCs were suspended in 24-well plates on day 3 by infecting them with lentiviruses as prepared in example 1. After 24 hours of sensitization, the medium was replaced with fresh DC induction medium and IFN-. Gamma.was added (50 ng/mL) and LPS (50 EU/mL) to promote differentiation and maturation of the DC.

On day 7, the maturation state of the DC was observed by microscopy, and the expression levels of the DC surface molecular markers CD80, CD83, CD86, and HLA-DR were measured by flow cytometry.

As shown in FIG. 1, the expression levels of CD80, CD83, CD86 and HLA-DR on the DC surface were significantly increased at day 7 after transfection and induction culture of DC, indicating that the cultured DC was mature and the antigen presenting ability was enhanced.

For comparison, immature DCs were suspended in 24-well plates infected on day 3 with lentiviruses that had been prepared as in example 1. After 24 hours of sensitization, the culture medium is replaced by a fresh DC induction culture medium, IFN-gamma (50 ng/mL) and LPS (50 EU/mL) are added, then the culture medium is kept still for half an hour at the temperature of-4 ℃ or 2 ℃ for 2 hours, then the temperature is gradually raised to 37 ℃, and the culture medium is continuously cultured at normal temperature to promote the differentiation and maturation of the DC.

On the 7 th day, the mature state of the DC can be observed through a microscope, and the expression levels of the DC surface molecular markers CD83 and HLA-DR detected by a flow cytometer are obviously higher than those of dendritic cells matured without low-temperature standing, and meanwhile, the expression levels of CD80 and CD86 are equivalent and have no obvious difference.

2) Preparation of antigen-specific CTLs: PBMC is spread on a bottle for 1h, collected suspension cells are separated by a magnetic bead negative selection method, CD3+ T lymphocytes are counted and frozen to liquid nitrogen, the T lymphocytes are recovered when the DC is induced to mature on the 7 th day, the antigen-loaded mature DC and the CD3+ T cells are co-cultured according to the proportion of 1; after completion of 2 rounds of sensitization, the cells were collected by flow analysis after supplementing every two days with an amplification medium (KBM 581 containing 500U/mL of IL-2), and when the cells proliferated to a considerable amount on day 14.

As shown in FIG. 2, the CD3+ CTL cells cultured by expansion have higher purity.

Example 3 tumor cell killing experiment by antigen-specific CTL

In order to verify the specific killing effect of antigen-specific CTL on colorectal cancer cell lines, the target cells in this example were SW480 cell lines with K12V mutation to Kras gene, and in vitro killing verification experiments of antigen-specific CTL were performed using HT29 cell lines without Kras gene mutation as a control, and other mutated Kras as a targeting reference. Tumor target cells and CTLs (E: T) were added to a 96-well plate at 1,1, 5 and 1.

The results shown in FIG. 3 show that, compared to the killing effect of the cell line HT-29, the CTL targeting the G12V mutant Kras protein prepared in example 2 has better killing effect and stronger specificity to the cell line SW480, while the CTL targeting the G12D mutant Kras protein has a better killing effect to the cell lines SW480 and HT-29, but slightly better killing effect than the CTL without load antigen, probably because it has certain recognition to other sites of the Kras protein. The immunotherapy method has a great promise for entering clinical treatment of colorectal cancer.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (10)

1. A dendritic cell capable of expressing a G12V mutant Kras protein.

2. The dendritic cell of claim 1, prepared by infecting a dendritic cell with a vector (e.g., lentivirus) comprising a nucleotide encoding a G12V mutant KRAS protein such that the dendritic cell expresses the G12V mutant KRAS protein and thereby loads a G12V mutant KRAS protein epitope onto the dendritic cell.

3. The dendritic cell according to claim 2, which is a matured dendritic cell further prepared by maturation induction culture.

4. The dendritic cell of claim 3, wherein the maturation inducing culture is an inducing culture on a medium containing IFN- γ and LPS to promote differentiation and maturation of the DC.

5. A vaccine for preventing or treating cancer, comprising the dendritic cell of any one of claims 1 to 4.

6. The vaccine of claim 5, further comprising T cells (e.g., CD3+ T cells).

7. Use of a dendritic cell according to any one of claims 1 to 4 in the manufacture of a vaccine for the treatment or prevention of a cancer expressing a G12V mutant Kras protein.

8. The use of claim 7, wherein the cancer is breast cancer, colorectal cancer, ovarian cancer, gastric cancer, or prostate cancer.

9. Activated T cells or CTLs prepared by co-culturing with the dendritic cells of any one of claims 1-4.

10. A cellular composition comprising the dendritic cell of any one of claims 1-4 and a T cell.

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