CN113403275A

CN113403275A - Preparation method of new antigen of specific target cancer species

Info

Publication number: CN113403275A
Application number: CN202110742883.3A
Authority: CN
Inventors: 李力; 梁凯; 徐迎新
Original assignee: Beijing Baiyining Medical Technology Co ltd
Current assignee: Beijing Baiyining Medical Technology Co ltd
Priority date: 2021-07-01
Filing date: 2021-07-01
Publication date: 2021-09-17

Abstract

The invention discloses the technical field of antigen preparation methods, in particular to a preparation method of a novel antigen of a specific targeting cancer species, which comprises the following steps of 1) analyzing and preparing an accurate targeting tumor antigen; 2) collecting and separating and culturing DC cells; 3) preparing a sensitive activated DC cell by adopting a precise targeted tumor antigen and an activator; 4) blocking DC cell surface molecules, and harvesting a DC cell vaccine; the accurate target tumor antigen is prepared by using a small initial number of peripheral blood mononuclear cells, is effectively induced in vitro, amplifies accurate target tumor specific cytotoxic T cells, is used for clinical treatment of tumor patients, and has small interference on organisms. The preparation method effectively solves the problem of insufficient in-vitro amplification of DC cells and cytotoxic T cells; blocking of activated immunodetection sites in vitro culture of DC cells and cytotoxic T cells. The preparation method can effectively induce the accurate targeting tumor antigen specific cytotoxic T lymphocyte in vitro and in vivo, and has the functions of high efficiency and specific killing to the tumor cell.

Description

Preparation method of new antigen of specific target cancer species

Technical Field

The invention relates to the technical field of antigen preparation methods, in particular to a preparation method of a novel antigen of a specific targeting cancer species.

Background

Tumors are serious diseases threatening human life, at present, new cancer patients are increased every year in China and even all over the world, and the traditional treatment methods have great limitations. The existing operation only can remove visible tumor and surrounding visible lymph nodes, can not completely remove tumor cells and can not change the unbalance environment in the body of a patient; part of tumor cells can be killed by radiotherapy and chemotherapy, but part of solid tumors are not sensitive to radiotherapy and chemotherapy, and the side effects of the radiotherapy and chemotherapy bring negative effects to the immune system of a patient and cannot prevent the recurrence and metastasis of the tumors; the existing molecular targeted drugs have large application range limitations and are easy to generate drug resistance. Therefore, new precise therapeutic approaches and means need to be developed.

Studies have shown that reconstitution/restoration of patient self-fighting tumor immune system function is one of the strategies for treating tumors. In recent years, tumor immunity has become the fourth leading means of antitumor therapy, and the success of tumor immunotherapy needs to satisfy four key elements: 1) a good targeting tumor antigen; 2) a platform capable of correctly inducing and expanding immune cells; 3) cutting off the immunosuppressive mechanism; 4) promote tumor-specific Cytotoxic T (CTL) infiltration into tumor tissues. The optimal antigen is the key for ensuring the accurate targeting of the tumor specific immunotherapy.

DC cells, known as dendritic cells, are the professional antigen presenting cells that have attracted much attention in recent years, and can take up, process and present antigens to initiate T cell-mediated immune responses. DC cells have various sources, and the most convenient way is to separate peripheral blood to obtain mononuclear cells. Since expansion of DC cells and CTL cells is difficult, corresponding measures are usually taken on the number of cells at the beginning of culture in order to meet the number of cells required for therapy: 1) injecting GM-CSF for mobilizing bone marrow cells 3-5 days before collecting cells to increase the proportion of mononuclear cells in peripheral blood; 2) leukocyte separation, which collects a large amount of mononuclear cells from peripheral blood circulating blood. The above treatments in fact carry out a strong external intervention on the bone marrow and peripheral blood of the patient, further causing a great disturbance to the in vivo environment of the disordered immune system, especially after surgery, radiotherapy, chemotherapy of cancer patients. Therefore, the preparation method of the novel antigen of the specific targeting cancer species is provided, which has small interference on organisms, has less initial cell number, and can reach high standards in cell number and phenotype after culture and antigen activation.

Disclosure of Invention

The present invention is directed to a method for preparing a novel antigen specifically targeting cancer species, which solves the problems set forth in the background art.

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

a preparation method of a new antigen of a specific targeting cancer species comprises the following steps:

1) analyzing and preparing a precise target tumor antigen;

2) collecting and separating and culturing DC cells;

3) preparing a sensitive activated DC cell by adopting a precise targeted tumor antigen and an activator;

4) and (5) blocking DC cell surface molecules, and harvesting the DC cell vaccine.

Preferably, the method for collecting and separating the cultured DC cells in the step 2) is as follows:

C) isolation of mononuclear cells from peripheral blood: the anticoagulated blood is slowly added to the cell separation liquid from the vein, and the mononuclear cells are separated by centrifugation, 1-5X 10⁷Per ml;

B) isolation, induction and expansion of DC cells: after the cells were resuspended in DC cell culture medium, the cells were resuspended in 5% CO at 37 ℃₂Adhering the cells in the incubator for 2-5h, sucking out non-adherent suspension cells for cytotoxic T cells activated by tumor antigen sensitized DCs, wherein the adherent cells are DC cell precursors; adding a special DC cell culture medium, rh-IL-4 with a final concentration of 500-1000IU and rh-GM-CSF with a final concentration of 500-1000IU into the DC cell precursor, wherein the conditions of an incubator are 37 ℃ and 5 percent of CO₂And (5) incubating, inducing and amplifying for 7-10 days to obtain immature DC cells to be activated, and supplementing a DC cell culture medium and factors according to the cell state.

Preferably, the method for preparing the sensitized and activated DC cells by adopting the precisely targeted tumor antigen and the activator in the step 3) is as follows:

adopting an accurate targeting tumor antigen prepared by self-serum analysis, simultaneously adding an activator, and incubating to obtain sensitized and activated DC cells, wherein the activator is immunoglobulin, a monoclonal antibody, a molecular compound and a nucleic acid product;

the prepared DC cell surface sensitized and activated by the accurate target tumor antigen expresses CD80, CD83, CD86 and HLA-DR.

In vitro induction of targeted tumor-specific cytotoxic T cells: on the day of DC cell harvesting, extracting anticoagulated blood 40-60ml of the same tumor patient for the second time, separating the adherent culture of mononuclear cells, sucking nonadherent mononuclear cells, mixing with the DC cells harvested on the day with antigen sensitized and activated at a ratio of 4-8:1, placing into a culture flask, adding CTL special culture solution to start culture, and culturing under the conditions of 37 ℃ and 5% CO concentration in an incubator₂And (3) performing incubation culture, adding the CD3 monoclonal antibody, the CD28 monoclonal antibody and the rh-IL-2 for continuous culture the next day, and performing amplification culture on CTL cells by supplementing a proper amount of an individual culture solution containing the rh-IL-2CTL according to the growth amplification state of the cells.

Preferably, after step 3, the CTL cell culture reaches the peak period of logarithmic cell growth after 12-16 days, 1-10ug/ml of blocking monoclonal antibody is added before the CTL cell is harvested, molecules on the surface of the CTL cell are blocked, and the conditions of an incubator are 37 ℃ and 5% CO concentration₂Incubating for 1-3h, collecting cell suspension, centrifuging and washing for 2 times, collecting quality control sample, counting viable cells, resuspending the obtained CTL cells with 100ml of normal saline, adding human serum albumin, and preparing for intravenous drip.

Preferably, the analysis of step 1) produces a precisely targeted tumor antigen from the same individual as the peripheral blood of step 2).

Preferably, the type of the precise target tumor antigen in the step 1) is selected from tumor specific polypeptides, and the precise target tumor antigen is prepared by prediction based on gene sequencing and biological information analysis.

Preferably, the precise target tumor antigen in the step 1) is subjected to gene detection through serum separated from a patient to obtain a whole gene spectrum, a tumor high-expression gene segment is found out by utilizing biological information analysis, and the composite precise target tumor specific antigen is biosynthesized.

The invention has the beneficial effects that: the accurate target tumor antigen is prepared by using a small initial number of peripheral blood mononuclear cells, is effectively induced in vitro, amplifies accurate target tumor specific cytotoxic T Cells (CTL), is used for clinical treatment of tumor patients, and has small interference on organisms. The obtained DC cell vaccine effectively solves the problems of low immunogenicity of tumor antigens, incomplete and unclear target spots, difficult obtainment of tumor antigens of patients and the like in related preparation aspects; the preparation method effectively solves the problems of insufficient in-vitro amplification of DC cells and Cytotoxic T (CTL) and the problem of sealing of immunodetection points activated in-vitro culture of the DC cells and the Cytotoxic T (CTL); the preparation method can effectively induce accurate target tumor antigen specific Cytotoxic T (CTL) in vitro and in vivo, and has efficient specific killing function on tumors.

Drawings

FIG. 1 shows the ratio of DC cells in the examples of the present invention;

FIG. 2 shows the expression of CD80 on the surface of DC cells in an example of the present invention;

FIG. 3 shows the expression of CD83 on the surface of DC cells in an example of the present invention;

FIG. 4 shows the expression of CD86 on the surface of DC cells in an example of the present invention;

FIG. 5 shows the expression of HLA-DR on the surface of DC cells in an example of the present invention;

FIG. 6 shows the ratio of CTL cells in the present example;

FIG. 7 shows the expression of CD3 in CTL cells in the present example;

FIG. 8 shows the CD4 ratio of CTL cells CD3 according to the present invention;

FIG. 9 shows the CD8 ratio of CTL cells CD3 in the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example (b):

antigen preparation:

taking tumor tissues and normal tissues excised in the operation of a patient, respectively comparing the DNA sequences of the tumor tissues and the normal tissues, and identifying the specific somatic mutation of tumor cells;

predicting specific antigens possibly existing in the cells according to the mutation information, and meanwhile, predicting the HLA type of the patient by comparing results;

intercepting a peptide sequence containing mutant amino acid according to the length of 8-11 amino acid sequences of the peptide segment which can be combined by the HLA, and analyzing the combination force of the short peptide and the HLA of a patient by utilizing affinity prediction;

and (3) screening a batch of mutation points according to the affinity, the mutation reliability and the mutation frequency of the HLA, synthesizing the peptides by taking the mutation points as sequences for synthesizing the specific peptides, wherein the number of the peptides ranges from 5 to 20, and obtaining the specific targeted antigen.

Preparing a DC cell vaccine loading the targeted tumor antigen:

1) isolation of mononuclear cells from peripheral blood

Collecting sterile peripheral blood 45ml, slowly adding onto lymphocyte separation liquid, obtaining mononuclear cells by gradient density centrifugation, sucking tunica albuginea layer in a tube, transferring into a centrifuge tube containing normal saline, washing cells, discarding supernatant, adding special DC cell culture medium, and counting heavy suspension cells.

2) Directionally inducing and amplifying DC cells

Mononuclear cells at 37 ℃ with 5% CO concentration₂Adhering to wall h in incubator, sucking out non-adherent cells, adding rh-IL-4, rh _ GM-CSF500-1000IU (final concentration) incubator at 37 deg.C and 5% CO₂Incubating, directionally inducing and amplifying DC cells for 7-10 days, and supplementing a proper amount of special culture medium containing rh-IL-4, rh-GM-CSF500-1000IU (final concentration) according to the growth and amplification conditions of the cells.

3) Antigen-sensitized activated DC cells

Culturing DC cell for 7-9 days, and detecting to synthesize target tumor antigenAdding target tumor antigen proportionally, and incubating at 37 deg.C and 5% CO concentration₂Incubate overnight.

4) DC cell blocking surface molecules

Culturing DC cells for 8-10 days, adding blocking factor 7ug/ml (final concentration) before harvesting DC cells, and incubating at 37 deg.C and 5% CO concentration₂Incubate for 3 h.

5) Harvesting of DC cells

After the molecules on the surface of the DC cells are sealed, sucking cell suspension, washing the cells which are not exfoliated with precooled normal saline until the cells are exfoliated, collecting all cell suspension, washing and centrifuging for 2 times when the cells are completely exfoliated; the DC cells were resuspended in 1ml of physiological saline for treatment. Simultaneously, sampling and controlling the quality of the sample, and detecting microorganisms and endotoxin; the expression of CD80, CD83, CD86 and HLA-DR on the surface of the DC cells is detected by flow cytometry.

6) In vitro induction and amplification of tumor-specific Cytotoxic T (CTL) by DC cells sensitized and activated by targeting tumor antigen

On the day of DC cell vaccine harvest, extracting anticoagulated blood of 40-60ml for a patient twice, preparing DC cells by adherence, sucking fresh separated non-adherence mononuclear cells, mixing the cells with prepared mature DC cell vaccine in a ratio of 8:1, re-suspending the cells by using a starting culture medium special for CTL (cytotoxic T lymphocyte), transferring the cells to a corresponding cell culture bottle, and culturing the cells in an incubator at 37 ℃ and 5% CO concentration₂Incubating overnight, adding CD3 antibody, CD28 antibody, rh-IL-2 the next day, and incubating at 37 deg.C and 5% CO concentration₂And continuing the incubation. According to the cell growth amplification condition, a proper amount of special amplification culture solution containing rh-IL-2 for CTL is supplemented for CTL amplification culture.

Harvesting of Cytotoxic T (CTL)

When CTL is cultured for 15 days, 1-10ug/ml of blocking molecule antibody (final concentration) is added before CTL cells are harvested at the time of cell log growth peak, and the incubator is at 37 ℃ and 5% CO concentration₂Incubate for 3 h. Collecting cell suspension, centrifuging, washing for 2 times, collecting quality control sample, counting viable cells to obtain cell viability rate of over 95% and total cell number of 1 × 10¹⁰Left and right; the obtained CTL cells were resuspended in 100ml of physiological saline, and human serum albumin was added for intravenous dripThe application is as follows. And detecting the expression of cell phenotypes, namely CD3 and CD8 by flow cytometry.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. A method for preparing a novel antigen of a specific target cancer species is characterized in that: the preparation method comprises the following steps:

1) analyzing and preparing a precise target tumor antigen;

2) collecting and separating and culturing DC cells;

2. The method of claim 1, wherein the antigen is selected from the group consisting of: the method for collecting, separating and culturing the DC cells in the step 2) comprises the following steps:

A) isolation of mononuclear cells from peripheral blood: collecting anticoagulated blood from vein, slowly adding into lymphocyte separation solution, centrifuging to separate mononuclear cell 1-5 × 10⁷One per ml.

B) Isolation, induction and expansion of DC cells: after the cells were resuspended in DC cell culture medium, the cells were resuspended in 5% CO at 37 ℃₂Adhering the cells in the incubator for 2-5h, sucking out non-adherent suspension cells for cytotoxic T cells activated by tumor antigen sensitized DCs, wherein the adherent cells are DC cell precursors; adding a special DC cell culture medium, rh-IL-4 with a final concentration of 500-1000IU and rh-GM-CSF with a final concentration of 500-1000IU into the DC cell precursor, wherein the conditions of an incubator are 37 ℃ and 5 percent of CO₂And (5) incubating, inducing and amplifying for 7-10 days to obtain immature DC cells to be activated, and supplementing a DC cell culture solution and factors according to the cell state.

3. The method of claim 2, wherein the antigen is selected from the group consisting of: step 3) the method for preparing the sensitive activated DC cells by adopting the precise target tumor antigen and the activator comprises the following steps:

adopting an accurate targeting tumor antigen prepared by self-serum analysis, simultaneously adding an activator, and incubating to obtain sensitized and activated DC cells, wherein the activator is an immunoglobulin, a monoclonal antibody, a molecular compound and a bacterial product;

expressing CD80, CD83, CD86 and HLA-DR on the surface of the prepared DC cells sensitized and activated by the accurate targeting tumor antigen, inducing targeting tumor specific cytotoxic T cells in vitro, extracting anticoagulated blood 40-60ml of the same tumor patient for the second time on the day of harvesting the antigen sensitized DC cells prepared by first blood drawing, separating mononuclear cells for adherent culture, extracting nonadherent mononuclear cells, mixing the nonadherent mononuclear cells with the antigen sensitized and activated DC cells harvested on the day in a ratio of 4-8:1, placing a culture bottle, adding a CTL special culture medium for starting culture, and setting the conditions of an incubator at 37 ℃ and 5% CO concentration for 5 percent to start culture₂And (3) performing incubation culture, adding the CD3 monoclonal antibody, the CD28 monoclonal antibody and the rh-IL-2 for continuous culture the next day, and performing amplification culture on CTL cells by supplementing an appropriate amount of an individual rh-IL-2CTL culture medium according to the growth amplification state of the cells.

4. The method of claim 3, wherein the antigen is selected from the group consisting of: after the step 3), culturing the CTL cells to the 12 th to 16 th days until the logarithmic growth peak period of the cells is reached, adding 1-10ug/ml of closed monoclonal antibody before harvesting the CTL cells, closing molecules on the surfaces of the CTL cells, and incubating at 37 ℃ and 5% CO concentration₂Incubating for 1-3h, collecting cell suspension, centrifuging and washing for 2 times, collecting quality control sample, counting viable cells, resuspending the obtained CTL cells with 100ml of normal saline, adding human serum albumin, and performing intravenous drip.

5. The method of claim 2, wherein the antigen is selected from the group consisting of: the analysis and preparation of the accurate target tumor antigen in the step 1) and the peripheral blood in the step 2) are from the same individual.

6. The method of any one of claims 1 to 5, wherein the antigen is selected from the group consisting of: the type of the accurate target tumor antigen in the step 1) is selected from tumor specific polypeptide, and the accurate target tumor antigen is prepared by prediction based on gene sequencing and biological information analysis.

7. The method of claim 6, wherein the antigen is selected from the group consisting of: the precise target tumor antigen of the step 1) is subjected to gene detection through serum separated from a patient to obtain a whole gene spectrum, a tumor high-expression gene segment is found out by utilizing biological information analysis, and the compound precise target tumor specific antigen is biosynthesized.