CN104491857A

CN104491857A - Antigen composition for immunotherapy of epstein-barr virus (EBV)-related diseases, biological preparation and preparation method of antigen composition

Info

Publication number: CN104491857A
Application number: CN201410817936.3A
Authority: CN
Inventors: 王亚栋; 郎婧; 李晓祥
Original assignee: Zmks International Cancer Therapy Biotechnologies Co Ltd
Current assignee: Zmks International Cancer Therapy Biotechnologies Co Ltd
Priority date: 2014-12-24
Filing date: 2014-12-24
Publication date: 2015-04-08
Anticipated expiration: 2034-12-24
Also published as: CN104491857B

Abstract

The invention provides an antigen composition for immunotherapy of epstein-barr virus (EBV)-related diseases, a biological agent containing the antigen composition and a preparation method of the antigen composition. The biological agent can be a vaccine, a medicine or a living cell preparation for preventing and treating the diseases. The invention provides a novel method for immunity breeding EBV-CTLs for treating EBV-related diseases including nasopharyngeal carcinoma; through a bioinformatics method and based on allelic expression characteristics of Chinese population on HLA-A, B and DR sites, the immunity breeding method comprises the following steps: analyzing different EBV virus strains, especially including virus strains derived from Guangdong Province, China, as well as existing sequences of possible epitope in three proteins, namely EBNA-1, LMP1 and LMP2, and preparing a peptide fragment including the sequences as an antigen, so that on the basis of cytokine combinations, antigen specific T cells more abundant in quantity and type in the EBV-CTLs are cultivated, and the cultivated cells are especially suitable for Chinese population. The method disclosed by the invention is short in culture cycle, strongly targeted and strong in effect of killing tumor cells.

Description

A kind of antigen composition for immunization therapy EBV relevant disease, biological preparation and preparation method thereof

Technical field

The present invention relates to a kind of antigen composition for immunization therapy EBV relevant disease, biological preparation and preparation method thereof, particularly relate to a kind of antigen composition being suitable for Chinese population treatment of nasopharyngeal carcinoma.Also relate to the biological preparation for immunization therapy EBV relevant disease comprising described antigen composition, this biological preparation can be vaccine or the medicine of this kind of disease of prevention and therapy.

Background technology

Nasopharyngeal carcinoma is the peculiar disease in area, Chinese Guangdong and Guangxi Provinces, has dependency with heredity, environment and living habit etc., especially in close relations with EBV (Epstein-Barr virus, EBV).EBV is the member that herpetoviridae tropic virus, lymphoid belongs to, entrained by the adult of more than 95%, with nasopharyngeal carcinoma have substantial connection, can detect that EBV genome exists in 90% nasopharyngeal carcinoma sample, that Burkitt african children lymphoma cell was successfully cultivated by In vitro Suspension in 1964 and builds strain by Epstein and Barr first, building in strain cell smear with electron microscopic observation to simplex virus particles, so gain the name.

Southern china (Guangdong, Guangxi) and Southeast Asia are global high risky area of nasopharyngeal carcinomas, and sickness rate is low 100 times of sending out regional.Causing the reason of this greatest differences to be considered to may be relevant to following two aspects: ethnic group genetic diversity and Epstein-Barr virus strain Area distribution diversity.Human leukocyte antigen (Human leukocyteantigen, HLA), i.e. major histocompatibility complex (major histocompatiabilitycomplex, MHC), be one group determine transplanted tissue whether compatible, with closely related, the closely linked gene group of immunne response, be the genic system that human body polymorphism is the abundantest.The allelic expression frequency of different HLA also exists larger difference in Caucasia crowd and Chinese Guangdong crowd.As in the crowd of Caucasia, the allele that HLA-A the most often expresses in site is A2, and in the crowd of Guangdong, the expression frequency of A11 molecule is about 50%, has comparative advantage.This species diversity makes Caucasia crowd and Chinese Guangdong crowd in the least unit-epi-position identifying 23Kda VCA, there is sizable difference.In the EBV associated epitope reported at present, majority is that A2, A24 are restricted.Make these results directly cannot be widely used in Chinese population.On the other hand, find in Guangdong and south east asia in many EBV Strain, compared with the advantage Strain B95-8 of European and American areas, sudden change is there is in some Chinese's prevalent HLA molecules restricted epitope sequences, the immunoreation of immune system cannot be processed, offering virus antigen to cause for EBV, thus the infection that these viruses are caused and tumor cannot be eliminated.

EBV, after host cells infected, can copy new virion on a large scale, also can tranquillization, also can transformed host cell in memory B cells for a long time, because which form the so-called different concealment stage, expresses different antigen.In the nasopharyngeal carcinoma and hodgkin's lymphoma of the EBV positive, EBV is in the so-called II type concealment stage, only expresses three kinds of virus antigens, is respectively EBNA-1, LMP1 and LMP2.If can screen in vitro and increase that those can identify the T lymphocyte specific of these three kinds of antigens, i.e. EBV-CTLs, by these cell infusion to patient, recover or improve the original cell immune response for EBV of patient, just can killing tumor cell in vivo, reach the object for the treatment of nasopharyngeal carcinoma.

EBV-CTLs by tentative for transplanting rear lymphoproliferative disease, the treatment of lymphoma and nasopharyngeal carcinoma.But currently used cultural method, or need with EBV transform autologous leukocytes system as irritation cell, cultivation for up to the several months, the timely treatment to patient cannot be realized; Or adopt the epi-position reported to carry out Selective Separation, stimulate and cultivate, because epi-position known is at present defined in minority Caucasia crowd's high frequency, to express HLA molecule restricted, makes most Chinese to apply.

Summary of the invention

The present invention solves the aforementioned problems in the prior proposition.

The invention provides a kind of antigen composition for immunization therapy EBV relevant disease.

Present invention also offers and a kind ofly comprise cell class biological preparation for immunization therapy EBV relevant disease of described antigen composition and preparation method thereof.

For achieving the above object, the present invention is by the following technical solutions:

First aspect of the present invention is to provide a kind of antigen composition for immunization therapy EBV relevant disease, and described antigen composition is the peptide section combination of particular sequence composition in three kinds of albumen EBNA-1, LMP1 and LMP2 in different EBV Strain source.

Wherein, described peptide section combination comprises at least three peptide sections, be preferably 3-90 bar peptide section, be more preferably 89 peptide sections, described single peptide segment length is preferably 15-25 aminoacid, is more preferably 20-25 aminoacid, and adjacent two peptide sections have 10-15, be more preferably overlap between 10-12 continuous amino acid residue.

Second aspect of the present invention is to provide a kind of biological preparation for immunization therapy EBV relevant disease, and described biological preparation comprises above-mentioned antigen composition.This biological preparation can be the vaccine of this kind of disease of prevention and therapy, medicine or living cells preparation.

Wherein, described EBV relevant disease comprise that EBV after nasopharyngeal carcinoma, chronic active EBV infection, bloodthirsty cell syndrome, hematopoietic stem cell transplantation or organ transplantation infects, lymphoproliferative disease and with the lymphoma of EBV height correlation and gastric cancer etc.

3rd aspect of the present invention is to provide a kind of preparation method of the biological preparation for immunization therapy EBV relevant disease, and step comprises:

Step 1, the co-cultivation in liquid cellular incubation base by immunocyte and described antigen, thus acquisition has corresponding antigens specific immune cell culture;

Step 2, is separated in the culture obtained from step 1 and amplification cultivation has antigen-reactive sexual cell;

Wherein, in described liquid cellular incubation base, except immunocyte and antigen, clonal antibody, cytokine and immunological adjuvant is also comprised.

Preferably, described immunocyte is selected from: any one of variant cell or autogenous cell, or without any one of gene-modified immunocyte or gene-modified immunocyte, or antigen presenting cell or lymphocytic any one.

Preferably, described immunocyte is selected from: any one or multiple or their derived cell of peripheral blood mononuclear cell, medullary cell, hemopoietic precursor cell or stem cell, be more preferably peripheral blood mononuclear cell.

Preferably, described in, there is antigen-reactive sexual cell to be selected from: tumor infiltrating lymphocyte, cytotoxic T cell and/or dendritic cell, be more preferably CD3 ⁺t lymphocyte, comprises CD8 ⁺t and CD4 ⁺t lymphocyte.

CD8 ⁺t and CD4 ⁺the antigen of T lymphocyte identification has a great difference, CD8 ⁺the epi-position of 8-10 the amino acid length that T cell identification MHC I quasi-molecule is offered, and CD4 ⁺t lymphocyte then can identify 15 amino acid lengths that MHC II quasi-molecule is offered or longer epi-position.CD8 ⁺t cell is considered to antineoplastic main effects cell, by release perforin/granzyme direct killing tumor cell, or by death signaling pathways, and the apoptosis of inducing tumor cell.CD4 ⁺t cells with antigenic specificity, is considered to CD8 ⁺t cell antineoplastic important composition composition, it is mainly through the form of paracrine, autocrine, auxiliary CD8 ⁺t cell and other immunocyte, as antigen presenting cell, improve the object immunologic rejection effect of tumor being reached to treatment tumor.Adopt donor source EBV-CTL for transplanting in the clinical trial of rear lymphoproliferative disease, prompting CD4 ⁺t cell ratio height person, can obtain better clinical response.

Present invention employs the combination of different cytokines, described cytokine be selected from GM-CSF, IL-2, IL-4, IL-7, IL-12, IL-15 any one or multiple, the wherein more preferably combination of GM-CSF, IL-2, IL-7, makes to cultivate the antigen-reactive sexual cell containing greater number and kind in the EBV-CTLs obtained.

The present invention adopts technique scheme, compared with prior art, has following technique effect:

A kind for the treatment of nasopharyngeal carcinoma newly provided by the invention is in the method for the immunity breeding EBV-CTLs of interior EBV relevant disease, the method adopts bioinformatics method, based on Chinese population at HLA-A, B and DR site allele expression characteristic, analyze different EBV Strain, especially the Strain in Chinese Guangdong source is comprised, three kinds of albumen EBNA-1, in LMP1 and LMP2 there is sequence in possibility epi-position, and preparation comprises the peptide section combination of these sequences as antigen, in conjunction with specific cells combinations of factors, make to cultivate in the EBV-CTLs obtained containing the more T cells with antigenic specificity of value volume and range of product, be particularly useful for Chinese population, the method cultivation cycle is short, with strong points, strong to tumor cytotoxicity effect.

Accompanying drawing explanation

Fig. 1 is the position that section of synthesized peptide is arranged in LMP1, LMP2 and EBNA1 sequence, and wherein Lycoperdon polymorphum Vitt is original protein sequence, and black is B98-8 sequence source peptide section, and clear band is GD1 and GD2 sequence source peptide section.

Fig. 2 is under different cytokines combination condition, and EBV-CTLs is to the reactivity of antigen, and wherein attached cell group is not gone in black block representative, and attached cell group is gone in grey block representative.

Fig. 3 is that the EBV-CTLs in different donor source is to the reactivity of antigenic peptides section.

Fig. 4 is that A010 originates EBV-CTLs to the lethal effect of autologous LCL.

Fig. 5 is the scattergram that EBV-CTLs prepared by the method for the invention comprises antigen reactivity CD4 and CD8 positive cell simultaneously.

Detailed description of the invention

Carry out detailed and concrete introduction below by specific embodiment to the present invention, to make better to understand the present invention, but following embodiment does not limit the scope of the invention.

First aspect of the present invention is to provide a kind of for the preparation method of immunization therapy nasopharyngeal carcinoma at interior EBV relevant disease medicine, and step comprises:

Step 1, the co-cultivation in liquid cellular incubation base by immunocyte and antigen, thus acquisition has corresponding antigens specific immune cell culture; Wherein, described antigen for different EBV Strain source be in expressed by the type III concealment stage three kinds of albumen EBNA-1, LMP1 and LMP2 in the peptide section combination of particular sequence composition;

Wherein, in described liquid cellular incubation base, except immunocyte and antigen, cytokine and immunological adjuvant is also comprised.

Preferably, described immunocyte is selected from peripheral blood mononuclear cell.

Wherein, described peptide section combination comprises at least three peptide sections, be preferably 3-90 bar peptide section, described single peptide segment length is preferably 15-25 aminoacid, is more preferably 20-25 aminoacid, adjacent two peptide sections have 10-15 individual, be more preferably overlap between 10-12 continuous amino acid residue.

Described have antigen-reactive sexual cell and be preferably CD3 ⁺t lymphocyte, comprises CD8 ⁺t and CD4 ⁺t lymphocyte.

Described cytokine be selected from GM-CSF, IL-2, IL-4, IL-7, IL-12, IL-15 any one or multiple, the wherein more preferably combination of GM-CSF, IL-2, IL-7.

1, the Design and synthesis of antigen:

The present invention with Chinese and Caucasian HLA developed by molecule difference for starting point, find out 5-7 allele of Chinese's predominant expression in HLA-A, B and DR tri-locus respectively, these allelic expression frequency sums, have exceeded more than 80% (asking for an interview following table 1) of this site all allele expression frequencies summation.These allele represent the expression of most people.Based on this subsequently, adopt bioinformatics method, under predicting these allele restrictive conditions, derive from the epi-position that may exist in EBNA-1, LMP1 and LMP2 tri-kinds of antigens in B95-8, GD1 and GD2 tri-kinds of Strain.Wherein, GD1 with GD2 comes from In Guangdong Province to be separated the Strain checking order and obtain in recent years, can reflect local popular Strain gene expression characteristics.Finally, synthesize a series of overlapping peptide section, these peptide segment length are about 20 aminoacid, the lap of adjacent two peptide sections is 10-12 aminoacid, cover all prediction epi-position distributed areas, need section of synthesized peptide LMP1 27 sections, LMP2 41 sections and EBNA1 21 sections (asking for an interview Fig. 1) altogether.

Table 1 Chinese HLA-A, B, DR site advantage allele

HLA-A	HLA-B	HLA-DRB1
			1101	4001	0901
2402	4601	1501
			0201	5801	0701
3303	1301	1201
			0207	1302	0803
	1502	0301

2, the stimulation of EBV-CTLs and cultivation:

The present invention's peptide section of about 20 amino acid lengths cultivates the EBV-CTL obtained as antigen, can stimulate simultaneously obtain antigenic specificity CD8 ⁺t and CD4 ⁺t lymphocyte (asking for an interview Fig. 5).

Present invention employs the cytokine of GM-CSF, IL-2 and IL-7 combination, make to cultivate in the EBV-CTLs obtained containing more antigen-reactive sexual cell (asking for an interview Fig. 2).

The immunocyte that the present invention obtains is healthy human peripheral blood mononuclearcell, cultivation obtains antigen reactivity EBV-CTLs, and the reactivity of EBV-CTLs to different peptide section in every donor source is all different, indicates method provided by the invention and has very high repeatability and adaptability (asking for an interview Fig. 3).

The present invention cultivates the EBV-CTLs that obtains with the target cell of load antigen, as after autologous LCL meets, effectively can kill and wound target cell, ask for an interview Fig. 4.

3, experimental technique:

1) experiment material and instrument:

EX VIVO 15 culture medium;

IL-7, is configured to 10 μ g/ml ,-20 DEG C of preservations with containing 0.1HAS PBS;

Injection employment recombinant il-2, is configured to 100000U/ml with PBS ,-20 DEG C of preservations;

Injection rhGM-CSF, is configured to 100000U/ml with PBS ,-20 DEG C of preservations;

PBS, not calcic, magnesium ion, cell culture level;

EBNA-1, LMP1 and LMP2 originate, 20aa overlapping peptide section, dissolve be configured to 1mM ,-20 DEG C of preservations with DMSO;

Ficoll plus；

DMSO；

Human IFN-γELISA KIT 96T；

BD Cytofix/Cytoperm ^tMplus Fixation/Permeabilization Kit is (with the Protein transport inhibitor B D GolgStop containing monensin ^tM);

Normal saline;

PMA, 1mg/vial, be configured to 10 μ g/ml with DMSO ,-20 DEG C of preservations;

Ionomycin, 1mg/vial, is configured to 250 μ g/ml with DMSO ,-20 DEG C of preservations;

CD3-pc7 labeled monoclonal antibody;

CD4-pc5 labeled monoclonal antibody;

CD8-FITC labeled monoclonal antibody;

IFN-γ-PE labelling monoclonal antibody body;

100 μm and 40 μm of aperture filter screens;

15ml and 50ml centrifuge tube;

5ml pipet;

96 hole U-shaped floor cells culture plates;

24 porocyte culture plates;

5-50 μ l and 50-300 μ l multichannel pipettor;

1.5 or 0.5ml centrifuge tube;

X-12R centrifuge;

Flow cytometer;

Counting chamber;

Microscope.

2) experimental procedure and method:

A. be separated from peripheral blood and obtain mononuclearcell:

A) by patient whole blood's anticoagulant heparin, be loaded into Ficoll surface, usually adopt 15ml to bore end centrifuge tube, add Ficoll 5ml in advance wherein, by 10ml whole blood Slow loading to its surface;

B) the centrifugal 20min of 900g, slowly slows down;

C) with pipette, extract tunica albuginea cellular layer, it is washed twice with PBS, and with cell screen cloth removal cell mass wherein, count.

B. antigenic stimulus is cultivated:

A) different by peptide source from its water-soluble character, all peptide sections are divided into 16 groups, and partial peptide section can be completely water-soluble, and partial peptide section is only soluble in 100%DMSO, all the other peptide sections are dissolved in the water of different ratio respectively: in DMSO solution, and the concentration of often kind of peptide section is 2mM; The object of adopting in this way is the content reducing DMSO in cultivating system, and the DMSO of more than 1% is considered to cell growth and has adverse effect;

B) by culture medium, cell density is adjusted to 1*10 ⁷/ ml, adds overlapping peptide mixture with 1:1000 volume ratio, fully after piping and druming mixing, hatches 30min at 37 DEG C;

C) wash twice with PBS, by culture medium, cell density is adjusted to 1*10 ⁷/ ml, adding GM-CSF to final concentration is 100ng/ml, inoculates in 96 hole U base plates, every hole 200 μ l, cultivate 48 hours after mixing;

D) every hole is inhaled and is abandoned 90 μ l supernatants, adds the culture medium 100 μ l containing IL-2100U/ml, IL-720ng/ml, continues cultivation 48 hours;

E) every hole is inhaled and is abandoned 90 μ l supernatants, adds the culture medium 100 μ l containing IL-2100U/ml, IL-720ng/ml, continues cultivation 48 hours.

C.EBV-CTLs specific detection:

A) difference obtained cell suspension 200 μ l, add in three cell culture tubes, supplementing culture medium is to 1ml, wherein a pipe adds EBV peptide mixer, one pipe adds PMA 1 μ l, and ionomycin 1 μ l is as positive control, and another pipe adds DMSO as negative control, Golgstop (monensin) 0.7 μ l is added again, at 37 DEG C, 5%CO at every pipe ₂cultivate 4 hours under condition;

B) centrifugal, abandon supernatant, with PBS washed cell once, after abandoning supernatant, with residual a small amount of liquid suspension cell, add PC5-CD4, PC7-CD3, FITC-CD8, at 2-8 DEG C, hatch 30min;

C) use PBS washed cell once, inhale abandon supernatant completely, vibration suspension cell, often pipe adds 250 μ l rupture of membranes fixatives, and at 2-8 DEG C, lucifuge hatches 30min;

D) often pipe adds 1ml rupture of membranes cleaning mixture washed cell once, centrifugal, and after abandoning supernatant, often pipe adds the PE-IFN-γ of respective amount, and lucifuge hatches 30min at 2-8 DEG C again;

E) after 1* rupture of membranes cleaning mixture washed cell twice, with 250 μ l rupture of membranes cleaning mixture re-suspended cells, upper machine testing CD3 ⁺iFN-γ ⁺cell, and CD4 ⁺iFN-γ ⁺and CD8 ⁺iFN-γ ⁺account for total cell proportion.

D. amplification cultivation:

A) as CD3 detected ⁺iFN-γ ⁺cell, starts rapid amplifying program;

B) cell density is adjusted to 2*10 by EBV-CTLs culture medium ⁶/ ml, proceeds to 25/75cm ²in Tissue Culture Flask;

C) adding IL-2 in the medium to final concentration is 500U/ml, adds hybrid peptide to 0.1 μM, and cultivate 7 days, every two to three days supplementary IL-2, until cell quantity reaches necessary requirement.

E. quality testing:

A) get part amplifying cells, centrifugal, abandon supernatant, washing is once, with the culture medium culturing 24 hours of not factor-containing, centrifugal, abandon supernatant, gets appropriate cell by step C method, detectable antigens specific cell ratio;

B) get appropriate cell and cell density is adjusted to 1*10 ⁶/ ml, inoculate in 96 hole flat-bottomed plates, every hole 100 μ l, dilute single peptide section respectively with culture medium 1:500, correspondence is inoculated in cell, every hole 100 μ l, blank adds 100 μ l culture medium, cultivates 18-24 hour, by the explanation of corresponding IFN-γ ELISA detection kit, detect the content of IFN-γ in sample, analyze the position at EBV-CTLs possibility epi-position place;

C) by state-promulgated pharmacopoeia correlation technique testing product microbial limit;

D) by state-promulgated pharmacopoeia correlation technique testing product endomycin content;

E) cell quantity and the Cell viability of final products is determined.

Be described in detail specific embodiments of the invention above, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and substituting also all among category of the present invention.Therefore, equalization conversion done without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.

Claims

1. for an antigen composition for immunization therapy EBV relevant disease, it is characterized in that, described antigen composition is the peptide section combination of particular sequence composition in three kinds of albumen EBNA-1, LMP1 and LMP2 in different EBV Strain source.

2. antigen composition according to claim 1, is characterized in that, in described antigen composition, single peptide segment length is 15-25 aminoacid.

3. antigen composition according to claim 1, is characterized in that, described peptide section is combined as 89 peptide sections, and adjacent two peptide sections have the overlap between 10 to 15 continuous amino acid residues.

4. for a biological preparation for immunization therapy EBV relevant disease, it is characterized in that, described biological preparation comprises antigen composition according to claim 1.

5. biological preparation according to claim 4, is characterized in that, described biological preparation is vaccine, medicine or living cells preparation.

6. biological preparation according to claim 4, it is characterized in that, described EBV relevant disease comprises that EBV after nasopharyngeal carcinoma, chronic active EBV infection, bloodthirsty cell syndrome, hematopoietic stem cell transplantation or organ transplantation infects, lymphoproliferative disease and with the lymphoma of EBV height correlation and gastric cancer etc.

7., for a preparation method for the biological preparation of immunization therapy EBV relevant disease, it is characterized in that, step comprises:

Step 1, by the co-cultivation in liquid cellular incubation base of antigen described in immunocyte and claim 1, thus acquisition has corresponding antigens specific immune cell culture;

8. preparation method according to claim 7, it is characterized in that, described immunocyte is selected from: any one of variant cell or autogenous cell, or without any one of gene-modified immunocyte or gene-modified immunocyte, or antigen presenting cell or lymphocytic any one.

9. preparation method according to claim 7, is characterized in that, described immunocyte is selected from: any one or multiple or their derived cell of peripheral blood mononuclear cell, medullary cell, hemopoietic forebody cell or stem cell.

10. preparation method according to claim 7, is characterized in that, described in have antigen-reactive sexual cell be CD3 ⁺t lymphocyte, comprises CD8 ⁺t and CD4 ⁺t lymphocyte.

11. preparation methoies according to claim 7, is characterized in that, described cytokine be selected from GM-CSF, IL-2, IL-4, IL-7, IL-12, IL-15 any one or multiple.