WO2013063713A1

WO2013063713A1 - Method for inducing production of comprehensive immune cells

Info

Publication number: WO2013063713A1
Application number: PCT/CN2011/001811
Authority: WO
Inventors: 梁莹
Original assignee: 鑫品生医科技股份有限公司; 北京鑫谛合生医科技有限公司
Priority date: 2011-10-31
Filing date: 2011-10-31
Publication date: 2013-05-10

Abstract

Disclosed is a material and method for inducing production of comprehensive immune cells. The method comprises the following steps: an immunogenic composition is prepared, comprising at least two polypeptide libraries derived from the products of human herpes virus gene; the co-culture of the immunogenic composition with peripheral blood mononuclear cells (PBMC) is performed, and a mixed cell culture which contains immune cells is obtained; and the mixed cell culture is cultured in a suitable culture medium, and the comprehensive immune cells with specificity to human herpes virus are obtained when producing CD4T cells, CD8T cells and natural killer cells.

Description

Method for inducing the production of a comprehensive immune cell

Technical field

The main object of the present invention is to provide a method for inducing the production of a comprehensive immune cell, which can be used for inducing an immune response against herpesvirus in vitro, which is to use a medicine for treating a herpes virus infection disease, thereby applying to herpes Prevention and treatment of virus-related diseases. Background technique

The prevention and treatment of herpes virus-related diseases generally mainly use antiviral drugs, but in order to overcome the problems of drug toxicity, drug resistance, poor oral bioavailability, and low efficacy of using antiviral drugs, An immunotherapy with high specificity, low side effects, low resistance to drug resistance, and individualization has emerged. The existing immunotherapy for herpes virus-infected diseases includes: herpes virus antigen sources based on live virus, herpes virus-infected cells, and oozing virus genes. An expression vector (herpes virus gene expression vector) or a synthetic virulence virus protein or peptide fragment is used as an antigen to activate an ant i gen presenting cells that have a specific immune response to herpesvirus, and is expected to be activated by activation. The antigen presenting cells can stimulate the individual to produce EBV-specific toxic T cells (CTLs) and related cellular immune responses in vitro (e > o) or in vivo n 'ra). However, the use of viruses or virus-infected cells as a source of antigen has the potential for virus contamination and infection; and the use of full-length herpesvirus genes or proteins as antigen sources induces immune suppression or immunity. The problem of tolerance tolerance. Further, in the prior art, there is an immunotherapy for a herpes virus-infected disease, which is a method for producing activated immune cells in vitro, which mainly comprises the following steps: purified dendritic cells; activated tree The cells are mixed and cultured, and the activated dendritic cells are mixed with the peptide derived from the human herpesvirus gene product. The overall time is about 4 weeks, which is difficult to meet the need for activation of immune cells and immune function in a short period of time. Cellular needs. Summary of the invention

In view of the cumbersome preparation steps of the prior art immune cells, the high cost of preparation, and the long preparation time, it is difficult to meet the needs of the existing market. In order to solve the aforementioned problems, the present invention provides an induced production. A method for synthesizing immune cells, which can complete the production and production of immune cells and memory cells in a short period of time, and has confirmed that the herpesvirus-specific integrated immune cells produced have enhanced immune response ability, Helps improve the efficiency and quality of treatment for herpes virus-related diseases, thereby improving overall medical quality.

Accordingly, the present invention provides a method of inducing the production of a comprehensive immune function comprising the steps of: preparing an immunogenic composition comprising at least two peptide libraries derived from a human herpesvirus gene product

(peptide pool);

Co-culturing a peripheral blood mononuclear cell with the immunogenic composition to obtain a mixed cell culture containing immune cells and memory cells;

The mixed cell culture containing the immune cells is cultured in a specific culture condition, and the human herpesvirus-specific immune cells are obtained in the case of producing natural killer cells.

The "immune effector cell" as used herein refers to lymphocytes which exert an immune effect without further differentiation, including T cells having specific effects on antigens and natural killer cells, wherein T Cells, especially activated Th1, Th2 and CTL cells, have the ability to secrete immune molecules such as cytokines, cytotoxins and the like. Immune cells are different from naive cells (ie, unactivated lymphocytes) or resting cells (memory cells), which need to be proliferated, differentiated, or activated into immune cells to kill directly. Dead cell.

The "Natural killer cell" (NK cell), as is known, refers to a cell developed by bone marrow cells and having a non-specific cytotoxic effect, in general, peripheral blood. Cells that are sorted to express CD56 in monocytes but not to express CD3 are natural killer cells.

Preferably, the aforementioned derivative of human herpesvirus may be, but is not limited to: including (a) human herpes virus-1 (Li-1), also a simple trace virus-1 (herpes Simplex virus-1, HSV-1); (b) Human herpesvirus type 2 (HHV-2), also herpes simplex virus-2 (HSV-2); (c) Human herpesvirus type 3 (HHV- 3), that is, the herpes simplex virus (Varicella Zoster Virus, VZV), which causes varicella and herpes zoster, also known as varicella virus; (d) Human herpesvirus type 4 (HHV-4), ie Epstein-Barr Epstein-Barr Virus (EBV), a lymphoid virus that causes Birch's lymphoma and nasopharyngeal carcinoma; (e) Human herpesvirus type 5 (HHV-5), a giant cell virus (cytomegalovirus, CMV); (f) Human herpesvirus type 6 (HHV-6), the rose rash virus, causes the sixth disease, including infantile rose rash and childhood acute rash; (g) human herpesvirus type 7 (HHV-7), similar to HHV-6, causes the same symptoms; and (h) human herpesvirus type 8 HHV- 8), which is a monkey virus genus, referred to as kSHV, it can be found in Kaposi's sarcoma. More preferably, the aforementioned human herpesvirus is the fourth type of human herpesvirus.

Still more preferably, the aforementioned peptide library derived from the human herpesvirus type IV gene product is selected from the group consisting of EBV latent membrane protein-2, EBV nucleoprotein-1, EBV nucleus A peptide library derived from protein-3c and EBV-BZLF-1 polypeptide.

The term "peptide library" as used herein refers to a combination of complex peptide fragments having a partial amino acid overlap by spanning the entire sequence of the gene product. According to the present invention, each of the peptides in the aforementioned peptide library has a size between 10 and 15 amino acids.

For example, the aforementioned peptide library is composed of "pentadecap printed rice" J, which means a spanning - partial amino acid overlap of the entire sequence of gene products 15 The peptide pool formed by the peptide is the same. Similarly, the peptide library with other peptides is the same. Preferably, each of the fifteen peptide libraries derived from each polypeptide is adjacent to ten The pentapeptide has 11 consecutive amino acid residue overlaps.

Preferably, the aforementioned EBV latent membrane protein-2 has substantially the same sequence as SEQ ID NO: 1; EBV nucleoprotein-1 has substantially the same sequence as SEQ ID NO: 2; EBV nucleoprotein-3c has The sequence substantially identical to SEQ ID NO: 3; and the EBV-BZLF-1 polypeptide has substantially the same sequence as SEQ ID NO: 4. '

According to the present invention, the phrase "substantially the same" means that in the different species, the variation of the insertion, deletion or substitution of the amino acid sequence does not necessarily affect the configuration and activity of the constituent protein, so that the amino acid sequence has a certain The degree of similarity without affecting the configuration and activity of the protein is also within the scope of the present invention. The above degree of similarity is preferably 85% or more; more preferably 90% or more; and still more preferably 95% or more.

Preferably, the step of co-culturing a peripheral blood mononuclear cell with the immunogenic composition is performed with peripheral blood mononuclear cells at a concentration of between 1 g/mL and 5 g/mL of the immunogenic composition. to cultivate.

Preferably, in the method for inducing immune-producing cells, peripheral blood mononuclear cells are co-cultured with the immunogenic composition for 3 days.

Preferably, the specific culture conditions include culturing peripheral blood mononuclear cells and immunogenic composition in a specific medium. Preferably, the specific medium in the particular culture condition further comprises an immunostimulating agent.

In the method of inducing the production of immune cells, the immune cells are preferably lymphocytes. More preferably, the immune cells are from the same individual, that is, autologous immune cells (autologous immune cells) Cells). More preferably, the immune cell is a T cell.

The term "autologous" as used herein, as known to those skilled in the art, refers to originating from the same individual. By way of example, reference to an autologously derived immune cell refers to an immune cell derived from the self-speculating individual of the provided dendritic cell to avoid inducing an undesired immune response, such as heterogeneity. Heterologous immune response.

In the method for inducing the production of immune cells, the medium may be any medium suitable for culturing immune cells, such as, but not limited to, AIM-V medium having 2-5% human AB serum. . Preferably, the appropriate medium contains a cytokine selected from the group consisting of IL-2, IL-7, IL-15, and combinations thereof. DRAWINGS

Fig. 1 is a schematic view showing the composition of a fifteen-peptide library derived from EBV latent membrane protein-2 used in the present invention. Fig. 2A is a flow chart showing the amplification of antigen-specific immune cells by peripheral blood mononuclear cells in vitro (ex VI vo) activation according to the present invention.

Figure 2 is a flow chart showing the activation of amplifying antigen-specific immune cells by dendritic cells in vitro (a?). Fig. 3 is a graph showing the results of analyzing cell characteristics by flow cytometry in Example 1 of the present invention.

Fig. 4A is a flow chart showing the expression of IL2 and IFN-γ in the CD4 group in Example 2 of the present invention.

Fig. 4 is a flow chart showing the expression of IL2 and IFN-γ in the CD8 group in Experimental Example 2 of the present invention.

Main component symbol description

(1) EBV latent membrane protein - 2

(II) Fifteen peptide libraries derived from EBV latent membrane protein-2

(III) (112) (113) 15 member peptides

The present invention discloses a method for producing immune cells and inducing immune cells, which can obtain immune cells in a preparation time shorter than that required in the prior art to induce the production of immune cells. In the present invention, peripheral blood mononuclear cells are mixed with an immunogenic composition, wherein the immunogenic composition comprises four peptides derived from human herpesvirus, which have been confirmed in a preferred embodiment of the present invention. The immune cells can be prepared in 18 days, that is, the preparation time required for the method of the present invention can be reduced at least compared to the prior art. In a preferred embodiment of the invention, the peptide library derived from the human herpesvirus is a fifteen-peptide library in which two adjacent fifteen-peptides have an overlap of eleven consecutive amino acid residues. For example, as shown in Figure 1, EBV latent membrane protein-2 (1) has the sequence set forth in SEQ ID NO: 1, contains 497 amino acids, and fifteen wins derived from EBV latent membrane protein-2 The peptide library (11) may comprise 122 peptides, wherein a 15-member peptide (111) is a continuous 15 amino acid sequence having positions 1 to 15 and another 15 member peptide adjacent thereto (112) For a sequence of 11 consecutive amino acid residues having positions 5 to 15 and 4 consecutive amino acid residue sequences at positions 16 to 19, an adjacent 15-member peptide (113) has Repeating 11 amino acid partial overlaps at positions 9 to 19 and subsequent 4 consecutive amino acid residue sequences at positions 20 to 23, and so on, and finally remaining less than 4 consecutive amino acid residues, by The amino acid sequence of the overlapping portion was increased to satisfy the conditions of each fifteen peptides, 15 members (15-mer).

According to the present invention, the foregoing principles are also applicable to a peptide library derived from other described gene products (proteins), such as EBV nuclear protein-1 (full length 249 amino acids, the derived peptide library containing 60 peptides) ), EBV nucleoprotein-3c (full length 641 amino acids, the peptide library derived from it contains 265 peptides) and EBV-BZLF-1 polypeptide (full length 245 amino acids, the peptide library derived from it contains 59) Kind of peptide).

According to the invention, the term "immunostimulant" means any substance on the actual shield that enhances or enhances an immune response, including antibody or cell regulation, of a foreign antigen. A preferred immunopromoting agent comprises an adjuvant.

The specific adjuvant may be a commercially available reagent, for example, cytokine such as GM-CSF, IL-2, -7, -12, and the like, and other similar growth hormones may also be used as an adjuvant.

The technical means adopted by the present invention for achieving the intended purpose of the invention are further described below in conjunction with the drawings and preferred embodiments of the invention.

Example

General experimental materials and methods _

(1) peptide and antigen

The 15-mer peptide pools used in the examples below were purchased from JPT.

Peptide Technologies, Inc. (JPT Peptide Technologies GmbH, Berl in, Germany), which includes a majority of pentadecapeptides, which are spanned by the following peptide sequence with 11 amino acids The fifteen peptides of the partially overlapping sequence are composed of: EBV latent membrane protein-2 (SEQ ID NO: 1) (497 amino acids, the peptide library derived therefrom contains 122 peptides), EBV nuclear protein-1 (SEQ ID NO: 2) (249 amino acids, the peptide library derived from it contains 60 peptides), EBV nuclear eggs White-3C (SEQ ID NO: 3) (641 amino acids, derived peptide library containing 265 peptides) and EBV-BZLF-1 (SEQ ID NO: 4) (245 amino acids, derived peptides) The library contains 59 peptides). Other antigens and peptides can be synthesized by different methods. Among them, the peptide library derived from WT-1 (Wilm's tumor 1) (Swiss prot: P19544) (WT33; JPT Peptide Technologies GmbH, Berlin, Germany) and the core protein of hepatitis C virus (core protein of Hepatitis C virus) (NCBI ACCESSION : ABV46234 (1-191 //product="core protein") derived peptide library, ordered by JPT Peptide Technologies (Berlin, Germany) in Peptide Scan 15/11 , etc., is used as a non-specific peptide library.

(2) Separation of peripheral blood mononuclear cells (PBMCs) The plasma apheresis blood and whole blood used in the following examples were obtained from voluntary health donors. Peripheral blood mononuclear cells used in the following examples were prepared by gradient density centrifugation using Ficol l-Hypaque (GE Healthcare Bio-Sciences AB, NJ, USA) (Han S. et al., 2008, Molecular Therapy, 16 : 269-279). The survival rate of the prepared peripheral blood mononuclear cells was determined by trypan blue staining, and only cells of the group having a cell survival rate of more than 80% were used in the following examples.

' (3) EBV antigen-specific immune cells that are directly activated by peripheral blood mononuclear cells and ex vivo expanded,

The EBV antigen-specific immune cells were activated by peripheral blood mononuclear cells and ex vivo prepared according to the method shown in Fig. 2A. On day 0, peripheral blood mononuclear cells were obtained as described above, and then stimulated in the peripheral blood mononuclear cells by adding the above-mentioned 15-member peptide library (1-5 4 g/mL), and cultured in vitro to have 2-5%. Human AB serum in AIM-V medium. On day 3, IL-2 (Gentaur, Aachen, Germany), IL-7 (Gentaur), and IL-15 (Gentaur) were added. Next, a fresh medium having the cytokines was added every other day, and after the culture was expanded to the 17th day, the T cells in the culture were subjected to characteristic function analysis, and the analysis method was through flow. The cytometer was used for intracellular cytokine staining analysis.

(4) Isolation and culture methods of immune cells activated by dendritic cells activated by purified dendritic cells and ex vivo expanded are mainly according to the method proposed by Han et al. Han S. et al., 2008, Molecular Therapy, 16: 269-279). As shown in Fig. 2B, mature dendritic cells were prepared and purified in the manner described above by obtaining purified dendritic cells (Day-2) from donor blood 4 days prior to co-cultivation (Day-4). The above 15-member peptide library (5 g/mL) or other non-specific The sexual antigen lasted for 3 hours. Day 0 (Day 0) began to co-culture dendritic cells with homologous non-adherent peripheral blood mononuclear cells (PBMC) in a 1:20 ratio in vitro with 2-5% human AB Serum in AIM-V medium. On day 3, IL-2 (Gentaur, Aachen, Germany), IL-7 (Gentaur) and IL-15 (Gentaur) were added. Then, fresh cytokines-containing medium was added every other day, and the culture was expanded until the 17th day, and the overall preparation time was 22 days. The T cells in the culture were subjected to characteristic function analysis and analysis. The method is to perform intracellular cytokine staining analysis by flow cytometry.

(5) intracellular cytokine staining

Analysis of cell staining was performed according to the method described in Han et al. (Han et al., supra). Briefly, 3χ10 ⁵ expanded EBV immune effector cells (expanded EBV IE) use the above antigen-loaded homologous dendritic cells or peripheral blood mononuclear cells (antigen-loaded) Autologous DCs or PBMC) were stimulated in AM-V medium. One hour after the stimulation reaction, Monensin (Sigma) was added. After 5 hours, the cells were stained, fixed, permeablized for CD4 and CD8 and utilized antibodies against IL-2 and IFN-γ (both from BD Bioscience) and used FIX/PERM and PERM/Wash The solution (BD) was stained and analyzed by a flow cytometer.

Example 1

Two voluntary health donors were selected to obtain peripheral blood mononuclear cells (PBMC) or to isolate their dendritic cells (DC), respectively, with the EBV latent membrane protein-2 peptide library (Lmp2), EBV nuclear protein- 3c peptide library (EBNA3c) and EBV latent membrane protein-2, EBV nuclear protein-1, EBV nuclear protein_3c and EBV-BZLF-1 four peptide library combinations (4 mix) were stimulated separately, and the above After the culture method described in the general experimental materials and methods, anti-specific immune cells expanded by activation of dendritic cells or antigen-specific immune cells amplified by activation of peripheral blood mononuclear cells can be obtained respectively. . Antigen-specific immune cells derived from PBMC stimulation or activation by dendritic cells were analyzed by flow cytometry. The experimental results are shown in Fig. 3. The immune cells produced by the combination of PBMC or DC and the peptide library were mainly killed by killer T cells (CD8+). The PBMC is directly stimulated by the peptide library to produce a natural killer cell (NK cell) (CD3-/CD56+) compared to the group stimulated by DC and the peptide library, PBMC. The group can produce more NK cells.

Example 2

According to the foregoing experimental method and the flow chart of FIG. 2A and FIG. 2B, four peptide libraries and four peptide libraries are used. The PBMCs or DCs were separately stimulated, and DCs were separately treated to stimulate T cells to co-culture for 17 days. The immune cells obtained by activation of the above-mentioned stimuli were each stimulated for "once" for 5 hours with dendritic cells with specific antigens. The above-mentioned re-stimulated immune cells were observed for the amount of IFN-γ and IL-2 produced in the cells, thereby evaluating the function of EBV-specific cells. The experimental results are shown in Fig. 4Α and Fig. 4Β. Compared with the CD4 group, it can be observed in the CD8 group. After stimulation of PBMC or DC with a combination of four peptide libraries, the immune cells obtained by PBMC stimulation showed High IFN-γ cytokines have the best immune effect. N is a non-specific group, the peptide library used is WT-1, S is a specific group, and the above four EBV peptide libraries and four wins are used. Peptide library combination.

The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Although the present invention has been disclosed in the above preferred embodiments, it is not intended to limit the present invention. The equivalents of the technical solutions disclosed above may be modified or modified to equivalent variations, without departing from the technical scope of the present invention, without departing from the technical scope of the present invention, in accordance with the technical essence of the present invention. Any simple modifications, equivalent changes and modifications made to the above embodiments are still within the scope of the technical solutions of the present invention. '

Claims

Rights request

A method for inducing the production of a comprehensive immune cell, comprising the steps of:

Preparing an immunogenic composition comprising at least two peptide libraries derived from a human herpesvirus gene product; co-culturing a peripheral blood mononuclear cell with the immunogenic composition to obtain a mixed cell culture comprising Immune cells;

The mixed cell culture containing the immune cells is cultured in a specific culture condition and produced

In the case of CD4 T cells, CD8 T cells and natural killer cells, human herpesvirus-specific integrated immune cells are obtained.

The method for inducing the production of a comprehensive immune cell according to claim 1, wherein the human herpesvirus is the fourth type of human herpesvirus.

The method for inducing the production of a comprehensive immune cell according to claim 1, wherein the peptide library derived from the human herpesvirus type IV gene product is selected from the group consisting of: EBV latent membrane protein - 2. A library of peptides derived from EBV. nucleoprotein-1, EBV nucleoprotein-3c and EBV-BZLF-1 polypeptides without an N-terminal peptide.

4. The method of inducing the production of a comprehensive immune cell according to claim 3, wherein:

The EBV latent membrane protein-2 has substantially the same sequence as SEQ ID NO: 1;

EBV nucleoprotein-1 having no N-terminal peptide has substantially the same sequence as SEQ ID NO: 2;

EBV nucleoprotein-3c has a sequence substantially identical to SEQ ID NO: 3;

The EBV-BZLF-1 polypeptide has substantially the same sequence as SEQ ID NO: 4.

The method for inducing the production of a comprehensive immune cell according to claim 3, wherein each peptide in the peptide library has a size of between 10 and 15 amino acids.

The method of inducing the production of a comprehensive immune cell according to claim 3, wherein each peptide in the peptide library is a fifteen peptide.

The method of inducing the production of a comprehensive immune cell according to claim 6, wherein two adjacent fifteen peptides in the peptide library have an overlap of eleven consecutive amino acid residues.

The method of inducing the production of a comprehensive immune cell according to claim 1, wherein the specific culture condition comprises an immunopotentiator.

The method of inducing the production of a comprehensive immune cell according to any one of claims 1 to 8, wherein the immune cell is a lymphocyte.

The method for inducing the production of a comprehensive immune cell according to any one of claims 1 to 8, wherein the specific culture condition comprises a suitable medium containing a cytokine selected from the group consisting of The following groups are composed: IL-2, IL-7, IL-15, and combinations thereof.

The method for inducing the production of a comprehensive immune cell according to claim 9, wherein the specific culture condition comprises a suitable medium containing a cytokine selected from the group consisting of the following: Group: IL-2, IL-7, IL-15 and combinations thereof.