CN101461942A - Dendritic cell vaccine carrying recombinant human HSP70 polypeptide complexes, preparation method and application - Google Patents
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- CN101461942A CN101461942A CNA2008100218966A CN200810021896A CN101461942A CN 101461942 A CN101461942 A CN 101461942A CN A2008100218966 A CNA2008100218966 A CN A2008100218966A CN 200810021896 A CN200810021896 A CN 200810021896A CN 101461942 A CN101461942 A CN 101461942A
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Abstract
The invention discloses a dendrite-shaped cell vaccine for loading a recombination human heat shock protein 70 polypeptide composite, a method for preparing the same and application thereof. The preparation method comprises: preparing a dendrite-shaped cell by designing and synthesizing an antigen polypeptide, forming the composition by the antigen polypeptide and the recombination human heat shock protein 70(rhHSP70), and loading the composite on the dendritic cell (DC) to obtain the vaccine. The vaccine has the advantages of multi-target points, double adjuvants and high efficacy, and can be used for resisting tumors and treating infectious diseases. The preparation process is simple and fast, and low in cost.
Description
Technical field:
The invention belongs to field of biological pharmacy, more specifically relate to antigen polypeptide and rhHSP70 (rhHSP70) are formed complex, again complex is carried on dendritic cell (DC) and prepares the dendritic cell polypeptide vaccine, be used for the treatment of tumor and infectious disease.
Background technology:
1. malignant tumor and infectious disease are fallen ill and the treatment present situation
(1) tumor invasion and therapeutic advance
Tumor and infectious disease are the serious threat human life and healthy major disease that generally acknowledge in the whole world.In the conference of the 18th world anticancer disease alliance, the research report that World Health Organization (WHO) delivers shows, whole world cancer condition will be serious day by day, new patient's number will be increased to 1,500 ten thousand by present every year 1000 ten thousand in 20 years from now on, and the number dead because of cancer also will increase to 1,000 ten thousand by annual 6000000.China's Epidemiological study shows that China urbanite malignant tumor in 2003 fatality rate is 94.71/10 ten thousand, and cancer becomes first deadly disease; Urban residents' malignant tumor patient case fatality rate is higher, is 104.01/10 ten thousand, occupies first of whole dead diseases.
Tumor has become human number one " killer ".The conventional method of existing treatment tumor (operation, radiotherapy and chemotherapy) though relief of symptoms to a certain extent, nearly 80% tumor patient stand to perform the operation, still dead after the misery of radiotherapy and chemotherapy because of the recurrence and the transfer of tumor.Therefore, development of new anti-tumor method and medicine are necessary and urgent.
Think that now generation, development and the host immune state of tumor have substantial connection.Therefore, so that tumor tissues suppresses the mode of tumor cell immunne response ability host's immunogenicity and enhancing body or the immunization therapy of eliminating tumor is the important method of antineoplaston by improving.Be acknowledged as " four-mode " of oncotherapy behind operation, radiation and chemotherapy based on the tumor biotherapy of immunization therapy, its curative effect is verified in the treatment of kinds of tumors.Become the research and development focus based on the antitumor cell immunization therapy of body cell immunologic mechanism and the research and development of therapeutic vaccine (as the DC vaccine).
(2) serious infectious diseases morbidity and treatment present situation
In recent years along with the use of preventative vaccine with popularize, common multiple infectious disease is effectively controlled, sickness rate descends significantly, but the harm of the great infectious disease of part is still very serious.
Tuberculosis (Tuberculosis; TB) be the global health problem that people pay close attention to always; although having started the effective cell immune response of protectiveness in infecting tubercule bacillus (MTB) back body, most of people (about 90%) stop it to develop into active tuberculosis; but follow the popular of drug resistance tubercule bacillus in recent years; and tubercule bacillus and HIV (human immunodeficiency virus) (Human Immunodeficiency Virus; HIVs) double infection; cause the incidence and mortality of TB to rise significantly, TB has become the No.1 killer in the infectious disease.
The WHO recent statistics shows that 1/3 (about 2,000,000,000) of global population infected MTB, annually surpasses 2,000,000 people and dies from tuberculosis.The annual de novo resistant tuberculosis patient's number of China accounts for global 1/4,2002 annual report New Development lunger more than 58 ten thousand examples, and 80% in the rural area.Owing to aspect reasons such as Economics and Managements, cause treatment not thoroughly or take medicine irregularly, cause the resistant tuberculosis patient to increase year by year.(Bacillus CalmetteGuerin is that unique getting permission is used to prevent vaccine lungy at present BCG) to bacillus calmette-guerin vaccine, is mainly used in the morbidity of prevention childhood tuberculosis, and adult's tuberculosis morbidity preventive effect is not waited from 0-80%.In view of the deficiency of BCG self (lost part and immunological memory and protectiveness associated protein etc. in BCG variation, the BCG preparation process) and drug resistance MTB's is popular, developing new generation vaccine is the important channel of controlling tuberculosis.
It then is the great public health problem in another whole world that hepatitis B virus (HBV) infects.Estimate that there are 300,000,000 Chronic HBV carriers in the whole world, China accounts for 1.3 hundred million.Wherein 50-75% is the Chronic HBV carrier that the activeness virus replication is arranged, and 30% may develop into liver cirrhosis among them, and 5-10% may develop into hepatocarcinoma.Estimate that die from global every year and HBV infects relevant about 1,000,000 people of chronic hepatopathy number.
Although new drugs such as a-interferon and lamivudine make the treatment of chronic hepatitis B improve, because the complexity of HBV reproduction process in liver does not find effective antiviral agents to be removed to the cccDNA that forms in the reproduction process so far as yet.Studies show that in a large number: the low or disappearance of chronic HBV infection person immunne response level, virus specific t cell quantity and function all reduce, the cell-mediated immunoreation of specific CTL of HBV and Th is faint, even detect less than the specific CTL reaction at HBV, this immune tolerance state is the root that causes the chronic persistent infection of HBV.Therefore, break immunologic tolerance, rebuilding chronic HBV infection patient-specific cellullar immunologic response may be the fresh approach that chronic HBV infection obtains effecting a radical cure.
2. the important function of specific T-cells immunoreation in antitumor, anti-infectious disease
(1) human immune system's mechanism of action
One of major function of human immune system is identification and removes dissident's (antigen), as intraindividual variation cell (as tumor cell), the host cell of pathogen infection (as the hepatocyte that infects HBV etc.), and the antibacterial (as TB) of exotic invasive, virus and parasite etc.
The human immune system can discern these dissident's antigen; activate the body natural immunity and specific immune response (humoral immunization of bone-marrow-derived lymphocyte mediation and the cellular immunization of T cell mediated); common these antigens of removing, the latter can produce immunological memory simultaneously, and human body is protected for a long time.Remove tumor, the interior infective virus (as HBV) of born of the same parents or intracellular bacteria (as TB) mainly by the specific T-cells immunoreation.
The specific T-cells immunoreation roughly is made up of four-stage: (1) antigen recognition: (Antigen Presenting Cell is APC) by surface receptor identification and engulf dissident's cell for human antigen's presenting cell; (2) antigen presentation: the cell of endocytosis is digested, is cracked into polypeptide by APC, and the latter combines with interior abundant MHC-I class of APC born of the same parents or II quasi-molecule and forms MHC-I-polypeptide or MHC-II-polypeptide complex respectively, and is transported to the APC surface; (3) t cell activation: the APC that carries the MHC-polypeptide complex migrates to lymph node and the T cell meets, the T cell is by surface receptor (T Cell Receptor, be activated after the MHC-polypeptide complex on TCRs and APC surface is discerned mutually, become the cytotoxic T lymphocyte (CTL) that antigenic specificity is arranged; (4) specific killing: the CTL that is activated is circulated to position, antigen place, kills and wounds behind the TCR specific recognition antigen presentation cell by the CTL surface; As seen, rely on the powerful immune defense system of human body self just, the cell of intraindividual variation, the antibacterial of outside invasion, virus etc. just are able to continuous removing, keep body homeostasis and balance.APC plays a significant role as the startup person of specific immune response.
(2) critical role of DCs in the specific T-cells immunoreation
Dendritic cell (Dendritic Cell, DC) be the most powerful sole duty antigen presenting cell (APC) of function in the body of finding up to now, and the antigen capture molecule of expressed in abundance (as MMR, DEC205, FcRs, TLRs), the antigen presentation molecule (as MHC-I, MHC-II, CD1) and immune costimulatory molecules (as CD40, CD54, CD80, CD83, CD86 etc.); The DCs of capture antigen migrates to lymph node, give T lymphocyte (first signal of T lymphocyte activator is provided) with the antigenic information submission after processing, the processing, the immune costimulatory molecules of its expression provides and activates the lymphocytic secondary signal of T, and the T cell is divided into the CTL with specific killing function through activation.Simultaneously, the enhancing body natural immunity is replied to produce a large amount of inflammatory factors (as IL-2, TNF α, IL-12, IFN γ etc.) through the DC of antigenic stimulus secretion, so DC has the laudatory title of " natural immunity adjuvant " again.
In view of the function of the powerful antigen capture of DC, processing, submission and activated T cell, the research of developing antitumor, anti-infectious disease therapeutic vaccine based on DC becomes global focus.External use antigen load DC feeds back the DC that carries antigen signals in the body, and excitating organism is the specific T-cells immunne response initiatively, removes the antigen presentation cell, lures different immunological memory simultaneously in vivo.This specific antigen signal that carries, the functional dendritic cell of startup/excitating organism specific cell immunoreaction is referred to as " DC vaccine " (DC Vaccine), and this immunotherapy method is considered to one of most advanced now, most promising cellular immunization treatment means.
3.DC vaccine is in the application progress in antitumor field
Research with the multi-form external load DC of antigen such as artificial synthetic known antigens polypeptide, recombinant antigen protein, coding for antigens RNA/DNA plasmid, cell pyrolysis liquid, cell extract, apoptotic cell all has a large amount of reports with application.U.S. Dendreon company uses prostate acid phosphatase (PAP) and GM-CSF fusion rotein as antigen load DC DC vaccine for treating product P rovenge
TM(APC8015) finished III phase clinical research in 2007, and by FDA experts' evaluation (Journal of Clinical Oncology, 2006,24 (19): 3089-3094).Barrou etc. from body DC, carry out adoptive therapy to 24 routine patients with prostate cancer with the external impact of recombined human PSA albumen (carcinoma of prostate related antigen), and the 6 male patients of routine circulation prostate gland cancer cell turn out cloudy after 6 months in treatment, detect still negative after 12 months; 11 routine patient PSA levels descend, 18 routine patients occur at PSA immunoreation (Barrou B, Benoit G.Cancer Immunol Immunother, 2004,53:453-460).
Nesselhut T etc. use patient with breast cancer's autologous tumor cell lysate or synthetic polypeptide (Her2-neu) load from body DC, feed back to the patient through Intradermal injection or vein.The result shows: 37% has produced clinical effect among the 143 routine patients.Wherein 7 people are alleviated fully, and 14 people are partly alleviated, 33 people's stable disease, and 3 people comprehensively reply, and prolong total life cycle (Nesselhut T, Matthes C, Marx D, et al.2005ASCO Annual Meeting).
4.DC the application progress of vaccine in anti-infectious disease
The desirable vaccine of infectivity resistant pathogen can not only produce lasting immunity, and can produce suitable immunne response to remove infection.Tubercule bacillus is born of the same parents' endophyte, desires to reach this purpose and mainly relies on cell-mediated immune response, rather than antibody response.Dillon SM etc. discovers that the DC of mice single inoculation tubercule bacillus purified protein derivative (PPD) sensitization can bring out intensive T cellullar immunologic response and produce IFN γ.Tubercule bacillus H37Rv strain Hsp65 has the antigenic characteristic of immunodominance, can induce the tuberculosis immunne response with enhancing body, and can activate gamma delta T cells.Simultaneously Hsp65 has the characteristic of carrier molecule effect and molecular chaperones, can assist and the protein fragments of its fusion, presents with the approach of MHC2I quasi-molecule in M Φ or DC, and can activate the effective stimulus agent that DC makes it to become primary tape CTL.Wuhan University Wei Ya is young to be waited with hsp65 and pEGFP2C1 fusion gene carrier transfection DC, and experiment in vitro shows that it can induce unsensitized splenocyte propagation, and the fusion gene of confirmation transfection can be expressed destination protein in DC, and can be presented effectively by DC.
Fazle Akbar etc. feed back the healthy volunteer who gives 5 HBsAg feminine genders with the recombinant HBsAg load behind body DC, 2 volunteers feed back and second week of back anti--HBsAg antibody occurs; 6 volunteers detect the non-responsiveness person and give to feed back for the second time after feeding back for the first time, occur in succession in one month resisting-HBsAg antibody (J Hepatology, 2007,47 (1): 60-66).Chen etc. with the ballistic DC of HBsAg through subcutaneous immune 19 routine CHB patients, observe 11/19 routine patient and clinical response occurs, wherein 10 routine patient HBeAg turn out cloudy, and HBeAg serology conversion (World J Gastroenterol appears in 5 examples, 2005,11 (12): 1806-1808).302 professors Wang Fusheng of hospital of PLA utilize HBsAg and two kinds of antigen combined load DC of HBcAg, and this vaccine can be induced (the patent of invention number: 200610078028.2) at the polyclone CTL of s181-193 epi-position and c18-27 epi-position;
5. the effect of heat shock protein in immunne response
(1) biologic activity of heat shock protein (HSPs)
(Heat Shock Proteins is one group of protein molecule family of guarding at biological evolution process camber HSPs) to heat shock protein, and this family member is divided into several big classes such as HSP100, HSP90, HSP70, HSP60, HSP28 according to the big I of molecular weight.HSPs plays a significant role in the processes such as the renaturation of denatured protein as folding, the transhipment of molecular chaperones (Chaperons) behind protein synthesis.
HSP70 is the heat shock protein of molecular weight about 70KD, is most important a member in the heat-shock protein family, is called as main heat shock protein.HSP70 albumen is made up of two parts, i.e. ATP enzyme district (ATPase domain) and substrate cog region or cry polypeptide land (Peptide-binding domain).The atpase activity district of HSP70 is positioned at 385 amino acid residues of HSP70 albumen aminoterminal; The n terminal fragment that separates this about 44KD can obtain a dependent ATP enzyme of non-polypeptide.Polypeptide combines with the HSP70 molecule and discharges is the process of a dependency ATP.The proteic polypeptide of HSP70 land is the amino acid fragment of about 27KD of c-terminus, can form the HSP-polypeptide complex in conjunction with the polypeptide (comprising tumor or virus-specific antigen polypeptide) of 5-25mer in the cell, participates in polypeptide in the intrasystem transhipment of endochylema nethike embrane.
Heat shock protein is known as and is connected the bridge of the natural immunity and specific immune reaction.Discover HSPs by with DC surface hsp receptor such as CD91, CD40, LOX1, TLR2/4 etc. are in conjunction with stimulating DC up-regulated expression panimmunity costimulatory molecules (CD83, CD80, MHC-I) and the immunocompetence factor (MIP, TNF α, IL-12 and IP-10 etc.), activate natural killer cell (NK) then, enhancing body nonspecific immune responses such as NKT cell are so HSPs has effect (the Srivastava PK.Curr Oncol Rep.2005Mar of " natural immunity adjuvant "; 7 (2): 104-8).
Srivastava finds: HSPs can mediate the submission of exogenous antigen polypeptide to MHC-I class and MHC-II quasi-molecule.Can cause T lymphocyte specific immunne response behind the HSP-antigen polypeptide complex immune animal of from tumor cell or virus infected cell, extracting at this tumor or virus, only immune peptide or only immune HSP then can not inducing specific immunne response (Binder RJ, Vatner R, Srivastava P.Tissue Antigens.2004Oct; 64 (4): 442-51.).
Based on this discovery, gp96 that U.S. Antigenics company (WWW.antigenics.com) will extract from patient tumors tissue or tumor cell and HSP70-polypeptide complex immunity tumor patient, corresponding product (
And AG-858) obtains the FDA approval and entered the I-II clinical trial phase.
The external impact dendritic cell of efflux body of tumor cell behind extraction such as China Cao Xue great waves academician heat treatment, experimental result shows can activate dendritic cell and T lymphocyte, effectively inducing antigen-specific and nonspecific immunoreation, the enhancing human body immunity function (number of patent application: 02145022.6, title: preparation of a kind of new and effective acellular vaccine and uses thereof).
6. the deficiency of existing DC vaccine
Antigenic antigenicity (Antigenicity), immunogenicity (Immunogenicity), antigen load system (Antigen Delivery System) and adjuvant (Adjuvents) are the important parameters that influences efficacy of vaccines in the DC vaccine design.The vaccine of above-mentioned simple antigen polypeptide load DC, or all there is deficiency in simple HSP70 antigen polypeptide compound vaccine.The stability of the former polypeptide self, polypeptide and DC surface bonded affinity of MHC molecule and stability etc. are the key factors that influences the DC polypeptide vaccine; Need the antigen polypeptide that is incorporated into HSP70 to be carried out submission again behind latter's immunity body by APC in the body (as DC), can start specific immune response, and tumor tissues is often secreted panimmunity inhibitive factor such as IL-10, TGF β etc. suppress the function of DC, thereby influence exciting of specific immune response.
Summary of the invention:
An object of the present invention is to improve the effect of polypeptide DC vaccine, provide a kind of flow process DC vaccine simple and direct, carrying recombinant human HSP 70 antigen polypeptide complex efficiently in order to overcome the defective of aforementioned polypeptides DC vaccine.
Another object of the present invention provides the preparation method of the DC vaccine of carrying recombinant human HSP 70 antigen polypeptide complex.
A further object of the invention provides the application of DC vaccine in the preparation antitumor drug of carrying recombinant human HSP 70 antigen polypeptide complex;
A further object of the invention provides the application of DC vaccine in preparation anti-infectious disease medicine of carrying recombinant human HSP 70 antigen polypeptide complex.
The theoretical basis of technical solution of the present invention:
(1) HSP70 has the natural biological characteristic (being referred to as molecular chaperones) in conjunction with polypeptide, makes polypeptide be difficult for degraded, thereby can keep the stability (antigenic stable) of polypeptide;
(2) HSP70 has the function of assisting polypeptide to transport at cell entoplasm net, has proved that HSP70 can be transported to the MHC-I quasi-molecule with its bonded polypeptide, forms MHC-I-polypeptide complex (first signal of t cell activation), excites the specific T-cells immunne response;
(3) DC is the strongest full-time antigen presenting cell of function of generally acknowledging, both as antigen load, the carrier of antigen presentation simultaneously because of the immune costimulatory molecules (secondary signal of t cell activation) of its expressed in abundance, has the effect of " natural immunity adjuvant " again;
(4) DC expresses the HSP70 receptor, and the HSP70 polypeptide complex can enter in the DC born of the same parents fast by the efficient endocytosis mechanism of hsp receptor mediation, with the antigen polypeptide submission to the MHC-I quasi-molecule, thereby improve antigen presentation efficient;
(5) HSP70 is in performance molecular chaperones effect, have the DC of promotion immunity costimulatory molecules (as CD80, CD83, CD86 etc.) up-regulated expression and cytokine (IL-12, TNF α etc.) excretory function, thus the ability that excites of DC vaccine improved, have " natural immunity adjuvant " effect; The objective of the invention is to realize by following measures:
A kind of dendritic cell vaccine of carrying recombinant human HSP 70 polypeptide complexes, comprising the following step:
1) recombinant human HSP 70 albumen (rhHSP70), as derive from Canadian Stressgen company, catalog number: ESP-555, purity〉90%;
2) design of antigen polypeptide and synthetic, purity〉90%, the biochemical Shanghai of gill company limited is synthetic;
3) preparation of recombinant human HSP 70 antigen polypeptide complex;
4) external evoked dendritic cell;
5) antigen load: the recombinant human HSP 70 antigen polypeptide complex that will prepare as stated above mixes with dendritic cell, cultivates certain hour jointly, is prepared into dendritic cell vaccine;
(6) quality testing of dendritic cell vaccine;
Described dendritic cell vaccine, wherein the recombinant human HSP 70 dietary protein origin is in Canadian Stressgen company, catalog number: ESP-555, purity〉90%;
Described dendritic cell vaccine, wherein the design considerations of antigen polypeptide is known has immunogenic tumour specific antigen (TSA), tumor associated antigen (TAA) or pathogen antigen (as HBV or TB) epi-position and carries out synthetic, the length of antigen polypeptide is 9 to 15 aminoacid sequences or longer as 9-25 aminoacid sequence, and purity is more than 90%.Wherein said tumor antigen is melanoma-associated antigen MART1, hepatocarcinoma related antigen AFP, carcinoma of prostate related antigen PSA, gastric cancer associated antigen CEA or breast cancer correlation antigen Her2-neu, including, but not limited to above-mentioned tumor antigen.Wherein said pathogen antigen is hepatitis b virus hbv, hepatitis C virus HCV, HIV (human immunodeficiency virus) HIV antigen or TB antigen, including, but not limited to above-mentioned pathogen.
Described dendritic cell vaccine, wherein recombinant human HSP 70 antigen polypeptide complex is with a certain amount of (10-100 μ M) HSP70 albumen and a certain amount of (50-300nM) antigen polypeptide, under 20 ℃ of-25 ℃ of temperature with contain ATP, KCl, MgCl
2The PBS buffer in common reaction 40min, add again after ADP reacts 40min jointly, dialysis is prepared from after removing free polypeptide;
Described dendritic cell vaccine, wherein external evoked dendritic cell is by after obtaining mononuclearcell cultivation certain hour, flush away is attached cell not, and the culture medium that adds the combination of cytokines of the combination of cytokines of combination of cytokines, GM-CSF and IFN α of combination of cytokines, GM-CSF and the IL-15 contain GM-CSF and IL-4 or GM-CSF and TNF α is induced the dendritic cell in monoblast source; Or get CD34+ precursor hematopoietic stem cell, and adding and contain GM-CSF, TNF α induces into CD34-DCs with the culture medium of Flt3-L cytokine.
Described dendritic cell vaccine, wherein HSP70 polypeptide complex and the dendritic cell consumption benchmark of educating altogether is 10 μ M HSP70 polypeptide complexes and 10
6Individual dendritic cell is educated altogether, and educating temperature altogether is 27 ℃-30 ℃; The time of educating is 4-6 hour altogether, and unconjugated HSP70 and HSP70 polypeptide complex are removed in results and washing, are prepared into dendritic cell vaccine.
Described dendritic cell vaccine, wherein cell purity (CD11c+HLADR+) greater than 80% and cell survival rate greater than 90%; The bacterin preparation endotoxin content is less than 5IU/ml; Gram-bacteria detects negative; The detection of mycoplasma feminine gender; Aseptic detection is qualified; Dendritic cell surface costimulatory molecules expression requires: CD80 is greater than 80%, and CD83 is greater than 50%, and CD86 is greater than 80%, and CD40 is greater than 90%, and CCR7 is greater than 50%;
Beneficial effect of the present invention
With utilize biochemical process from tissue, extract the HSP70 polypeptide complex as the immunogen method or with compare with HSP70 fusion protein expression plasmid transfection DC method by making up purpose antigen, utilize recombinant human HSP 70 albumen and antigen polypeptide at external formation complex load DC as immunogen, have that antigen is clear and definite, plurality of advantages such as flexible design, antigen convenient sources, safety and vaccine activity height:
1) can design and synthesize antigen polypeptide (9-25 aminoacid) according to the pathogen of known cancer related antigen or known infection targetedly, aminoacid sequence is clear and definite, and synthesis technique is simple and direct.As prostate related antigen polypeptide (PSA:CLAAGITYV); Hepatocarcinoma related antigen polypeptide (AFP:GVALQTMKQ); Melanoma-associated antigen polypeptide (MAGE3:FLWGPRALV); Breast cancer correlation antigen polypeptide (Her2-neu:KIFGSLAFL); HBV antigen polypeptide (HBsAg:FLLTRILTI; HBcAg:FLPSDFFPSV) etc.Biochemical process extracts the HSP70 polypeptide complex as immunogen from tissue, operating process relative complex, defective such as the bonded peptide sequence of HSP70 is indeterminate in the complex;
2) the HSP70 polypeptide complex that extracts with biochemical process is as immunogen, still need pass through complex processes such as human body APC (as DC) picked-up, antigen polypeptide submission after the immunity, can excite the specific T-cells immunne response, the quantity of APC and quality will be one of key factors that influences immune effect in this process.And with the dendritic cell of HSP70 polypeptide complex load as immunogen, then utilize dendritic cell full-time and powerful antigen uptake and submission function, in-vitro simulated and finish antigen load and submission process, dendritic cell has the function of " natural immunity adjuvant " again simultaneously, will obviously improve the effect of vaccine;
3) recombinant human HSP 70 albumen obtains (as Canadian Stressgen company, U.S. Sigma company etc.) easily, but the HSP70 polypeptide complex is in external prepared in batches.And that biochemical process is subject to is tissue-derived, the multifactor impact of quantity and quality, usually can not prepared in batches;
4) HSP70 has the biological nature in conjunction with polypeptide, can prepare the complex of multiple polypeptides and HSP70, and load DC simultaneously after the organic assembling as required excites the T cell clone of the multiple epitope of identification after the immunity, produces multiple-effect valency, firm immunne response.As melanoma express kinds of tumors antigen (GP100, MART1, MAGE3), available HSP70 respectively with these three kinds of antigenic peptides (GP100:IMDQVPFSV, MART1:AAGIGILTV; MAGE3:FLWGPRALV) form complex, loaded dendritic cell again;
5) recombiant plasmid transfection DC method is subject to behind transfection efficiency, destination protein expression efficiency, the expressing fusion protein influence at all too many levels such as submission efficient of DC endochylema endoantigen, and the safety of exogenous plasmid vector still is difficult to control so far.
2.HSP70-the DC immunologic competence of polypeptide complex load obviously improves
1) obviously raising is external with GM-CSF and IL-4 for the DC of HSP70-polypeptide complex load activation specific C D8T clone's ability, IFN α a, and IL-15, or the TNFa combinations of factors is induced HLA-A201
+Healthy volunteer's peripheral mononuclear cells is a dendritic cell.Use 10 μ g MART1 polypeptide (AAGIGILTV) respectively, the DC of 10 μ gMART1 polypeptide loads, 10 μ g HSP70-MART1 (complex that MART1 polypeptide and HSP70 form) repetitious stimulation is from body CD8T lymphocyte secondary, and is weekly.Collect the T lymphocyte (CTL) that stimulates, use the CD8T clone's of Flow cytometry specific recognition MART1 polypeptide frequency (Tetramer staining).Experimental result the results are shown in Figure shown in one from three independent healthy volunteers.
CTL1 is the cd8 t cell of independent MART1 polypeptide sensitization, and CTL2 is the cd8 t cell of MART1 polypeptide load DC sensitization, and CTL3 is the cd8 t cell of HSP70-MART1 polypeptide complex load DC sensitization;
The result shows: stimulate HLA-A201 with HSP70-polypeptide load DC
+MART1
+The ability of CD8T clonal expansion is the strongest, on average is respectively independent MART1 polypeptide, MART1 polypeptide load DC 107 times and 10 times.
2) CTL of the DC sensitization of HSP70 polypeptide complex load has the ability of killing and wounding target cell more by force
DC induces, antigen polypeptide load and cd8 t cell sensitization experimental procedure are the same, collects the T lymphocyte (CTL) that stimulates behind the secondary stimulus, with classical 4 hours
51The Cr release test detects the CTL killing activity, and target cell is the HLA-A201 of load MART1 polypeptide
+The T2 cell strain.CTL1 is the CD8T lymphocyte that the MART1 polypeptide directly stimulates, the CD8T lymphocyte of CTL2 for being stimulated with load MART1 polypeptide dendritic cell, the CD8T lymphocyte of CTL3 for being stimulated with load HSP70-MART1 complex dendritic cell.Experimental result is from three independent healthy volunteers, with average+/-standard deviation represents, the results are shown in Figure two.
The result shows: the ability the strongest (CTL3) of the CTL specific killing target cell that stimulates with HSP70-polypeptide load DC, compare with CTL1 with CTL2, and remarkable significant difference (the P value is respectively 0.05 and 0.07) is all arranged;
3.HSP70-contain dual " natural immunity adjuvant " in the DC vaccine design of polypeptide complex load, will obviously improve efficacy of vaccines.
The effect of HSP70 is to stablize polypeptide; Second effect is the efficient capture by the receptor-mediated HSP70 polypeptide complex of DC surface HSP70; The 3rd effect is that its bonded polypeptide is transported to the MHC-I molecule; The 4th effect is to promote the up-regulated expression of the immune costimulatory molecules in DC surface and the secretion of cytokine; HSP70 is the bridge that connects the natural immunity and specific immunity, and does not have individual polypeptide (Polymorphism), is the natural immunity adjuvant of generally acknowledging;
The immune costimulatory molecules of dendritic cell expressed in abundance is the indispensable stimulus signal of T cell activation, and the laudatory title of " natural immunity adjuvant " is also arranged.As antigen vectors, can keep the natural structure of its costimulatory molecules with living cells, be easy to TXi Baoshouti (TCR) identification and activate.The combined effect of two adjuvants both can increase the antigenicity of vaccine, can improve the immunogenicity of vaccine again, comprehensively improved the effect of DC vaccine.
4.HSP70 can raise the expression of dendritic cell vaccination costimulatory molecules
Induce in DC to add 0.1 μ g/mL on the 5th day respectively, 1.0 μ g/mL, 10 μ g/mL rhHSP70 or 100ng/mL LPS are common to be cultivated 48 hours, results DC, carry out surface marker detect (CD80, CD83, CD86); Figure three is a routine healthy volunteer DC fluidic cell testing result.
The result shows: HSP70 can raise the expression of DC CD83, CD80, CD86, thereby improves the immunocompetence of DC vaccine.
5. prepare easy, safe, nontoxic: in this vaccine production system, the mixing and pollute of no exogenous plasmid, carrier, chemical adjuvant; HSP70 albumen and DC cell all do not have antigenicity to intrasubject, and the probability that autoimmune disease takes place is minimum.
Description of drawings
Figure one is the MART1 antigen polypeptide stimulates specific C D8T clonal expansion with three kinds of different loads forms comparison
Figure two is MART1 antigen polypeptide CD8T lymphocyte fragmentation tests with three kinds of different loads form institute sensitization
Externally induce HLA-A201 with GM-CSF and IFN α combinations of factors+Healthy volunteer's peripheral mononuclear cells is tree Prominent shape cell; Be purified into from peripheral blood simultaneously that (kit is by German Miltenyi from body CD8T lymphocyte Biotech company provides, and the operating procedure by specification carries out). With Flow cytometry CD8T purity, reach 95% More than; Use respectively 10 μ g MART1 polypeptide (AAGIGILTV), 10 μ g MART1 polypeptide and HSP70 are compound Thing (HSP70-MART1) loaded dendritic cell is as sensibiligen; Use respectively 10 μ g MART1 polypeptide, 10 μ g The DC of MART1 polypeptide load, the DC repetitious stimulation of 10 μ g HSP70-MART1 polypeptide loads is from body CD8T lymph The cell secondary, weekly. Collect post-stimulatory T lymphocyte (CTL), with classical 4 hours51The Cr release test detects the CTL killing activity, and target cell is the HLA-A201 of load MART1 polypeptide+The T2 cell line. CTL1 Be the CD8T lymphocyte that the MART1 polypeptide directly stimulates, CTL2 is for using load MART1 polypeptide BMDC institute The CD8T lymphocyte that stimulates, the CD8T of CTL3 for stimulating with load HSP70-MART1 compound BMDC Lymphocyte. Experimental result is from three independent healthy volunteers, with average+/-standard deviation represents. Figure three variable concentrations rhHSP70 are to the impact of surface of dendritic cells costimulatory molecules expression
The specific embodiment
The present invention is further elaborated by the following examples:
Embodiment 1: the dendritic cell vaccine of four kinds of HSP70-melanoma-associated antigens of load polypeptide complex
1. the proteic preparation of melanoma-associated antigen polypeptide and rhHSP70
The restricted melanoma-associated antigen polypeptide fragment of the known HLA-A201 of synthetic, title and sequence are GP100:IMDQVPFSV; MART1:AAGIGILTV; MAGE3:FLWGPRALV and Try:YMDGTMSQV.Fully dissolve with DMSO, peptide concentration is 5mg/mL; RhHSP70 dissolves with PBS, and concentration is 0.1 μ g/mL.
2.rhHSP70-polypeptide complex preparation
Get 5 μ g (5 μ l) polypeptide and 10 μ g (100 μ l) rhHSP70 polypeptide respectively in 1.5mL EP pipe, add 400 μ l and contain 10nM ATP, 1mM KCl, 2mM MgCl
2The PBS buffer in, educated altogether 40 minutes in 20 ℃ behind the mixing, add and continue at 20 ℃ behind the 100nM ADP and hatched 40 minutes.Solution moved to (include 14kD molecular weight ultrafilter membrane) in the 0.5mL micro dialysis pipe and spend the night, remove, prepare rhHSP70-GP100 respectively, rhHSP70-MART1, rhHSP70-MAGE3 and rhHSP70-Tyr complex not in conjunction with polypeptide;
3. external evoked dendritic cell
Gather the melanoma patients (HLA-A201 of fresh anticoagulant heparin
+) peripheral blood or HLA-A201
+The normal health human peripheral obtains periphery mononuclearcell (PBMCs) behind the Ficoll density, PBMCs is suspended in the RPMI1640 culture medium that contains 2% (v/v) deactivation mixing people AB serum, and concentration is 2～3 * 10
6Individual cell/ml; Press every hole 3ml inoculating cell in 6 porocyte culture plates, in 5% (v/v) CO
2, place 90min in 37 ℃ of cell culture incubators and make adherent mononuclear cells, wash culture hole gently to remove not attached cell with the PBS buffer; Add 3ml in every hole and contain 5% (v/v) deactivation people AB serum, the RPMI1640 culture medium of 100ng/ml rhGM-CSF and 300ng/ml IFN α (DC inducing culture I).In inducing the 3rd day, the 1mL culture supernatant is inhaled in every hole gently, adds the fresh DC inducing culture I of 1ml and continues to induce; In the 5th day results DCs, to wash and be suspended in the PBS solution with PBS, concentration is 2 x 10
6Individual cell/ml;
4.HSP70-polypeptide complex load DCs
Get the above-mentioned four kinds of rhHSP70-polypeptide complexes that prepared of 250 μ l respectively, be added among the inductive DCs of 1mL, cultivated 4-6 hour in 27 ℃-30 ℃ behind the mixing.
5.DC vaccine results: collect above-mentioned cell, with being suspended in the normal saline that contains 2% (g/100ml) human albumin (HA) behind the PBS washing secondary, concentration is 2 x 10
6Individual cell/ml is melanoma dendritic cell polypeptide vaccine.
6. vaccine quality control criterion and detection method
(1) endotoxin detects: less than 5IU/ml; According to " three appendix XII of Chinese pharmacopoeia E bacterial endotoxins test detects;
(2) gram-bacteria detects: feminine gender; Gram staining method:
(3) aseptic detection: qualified; According to " three appendix XII of Chinese pharmacopoeia A sterility test method detects;
(4) detection of mycoplasma: feminine gender; The pcr amplification method;
(5) dendritic cell purity: CD11c+HLA-DR+ cell〉80%; Flow cytometry;
(6) dendritic cell survival rate:〉90%; Tongue is expected blue staining;
(7) the dendritic cell surface markers detects: CD80〉80%, CD83〉50%, CD86〉80%, CD40〉90%, CCR7〉50%; Flow cytometry;
Embodiment 2: the hepatocarcinoma dendritic cell vaccine of two kinds of HSP70-hepatocarcinoma of load antigen polypeptide complex
1. antigen polypeptide is synthetic: most of Patients with Primary high expressed alpha-fetoproteins (AFP), merge the chronic persistent infection of HBV, at this class liver cancer patient can AFP and HBV as target antigen such as AFP:GVALQTMKQ; HBsAg:FLLTRILTI; Polypeptide dissolving and concentration are with embodiment 1;
2.rhHSP70-the polypeptide complex preparation: operating procedure is prepared into two kinds of complex of HSP70-AFP and HSP70-HBsAg with implementing 1;
3. external evoked dendritic cell
Gather the Patients with Primary (HLA-A201 of fresh anticoagulant heparin
+) peripheral blood or HLA-A201
+Normal healthy people person's peripheral blood obtains periphery mononuclearcell (PBMCs) behind the Ficoll density, PBMCs is suspended in the RPMI1640 culture medium that contains 2% (v/v) deactivation mixing people AB serum, and concentration is 2-3 * 10
6Individual cell/ml; Press every hole 3ml inoculating cell in 6 porocyte culture plates, in 5% (v/v) CO
2, place in 37 ℃ of cell culture incubators and made adherent mononuclear cells in 90 minutes, wash culture hole gently to remove not attached cell with the PBS buffer; Add 3ml in every hole and contain 5% (v/v) deactivation people AB serum, the RPMI1640 culture medium of 100ng/ml rhGM-CSF and 50ng/ml IL-4 (DC inducing culture II).In inducing the 3rd day, the 1ml culture supernatant is inhaled in every hole gently, adds the fresh DC inducing culture II of 1ml and continues to induce; In the 5th day results DCs, wash and be suspended in the PBS solution with PBS, cell concentration is 2 x 10
6Individual cell/ml;
4.HSP70-polypeptide complex load DCs
Get the above-mentioned two kinds of rhHSP70-polypeptide complexes that prepared of 250 μ l respectively, be added among the inductive DCs of 1ml, cultivated 4-6 hour in 27 ℃-30 ℃ behind the mixing;
5.DC vaccine results: collect above-mentioned cell, with being suspended in the normal saline that contains 2% (g/100ml) human albumin (HA) behind the PBS washing secondary, concentration is 2 x 10
6Individual cell/ml is hepatocarcinoma dendritic cell polypeptide vaccine.
6. vaccine quality control criterion and detection method: with embodiment 1.
Embodiment 3: the dendritic cell vaccine of two kinds of HSP70-breast cancer antigens of load polypeptide complex
1. antigen polypeptide is synthetic: most of patient with breast cancer's high expressed MUC1 and Her2-neu related antigen; Can this synthesize polypeptide at this class patient with breast cancer, as MUC1:SLADPAHGV as target antigen; Her2-neu:KIFGSLAFL; Polypeptide dissolving and concentration are with embodiment 1;
2.rhHSP70-the polypeptide complex preparation: operating procedure is prepared into two kinds of complex of HSP70-MUC1 and HSP70-Her2-neu with implementing 1;
3. external evoked CD34-DCs
The HLA-201+ patient with breast cancer mobilizes through GM-CSF hemopoietic, gather mononuclearcell (PBMCs) with the cell collection instrument, add the separation and purification of CD34 monoclonal antibody magnetic bead and obtain CD34+ hemopoietic forebody cell (purity〉90%), it is suspended in contains in the X-VIVO culture medium that 5% (v/v) mix people AB inactivated serum.In culture medium, add GM-CSF (50ng/ml), TNF α (10ng/ml) and Flt
3-L (100ng/ml).Cultivate the 5th day additional fresh culture and cytokine, observe and in time adjust cell density.The 9th day CD34-DCs is induced in collection, washs and is suspended in the PBS solution with PBS, and concentration is 2 x 10
6Individual cell/ml;
4.HSP70-polypeptide complex load DCs
Get the above-mentioned two kinds of rhHSP70-polypeptide complexes that prepared of 250 μ l respectively, be added among the inductive DCs of 1ml, cultivated 4-6 hour in 27 ℃-30 ℃ behind the mixing;
5.DC vaccine results: collect above-mentioned cell, with being suspended in the normal saline that contains 2% (g/100ml) human albumin (HA) behind the PBS washing secondary, concentration is 2 x 10
6Individual cell/ml is breast carcinoma dendritic cell polypeptide vaccine.
6. vaccine quality detected parameters: with embodiment 1.
Embodiment 4: the hepatitis B dendritic cell polypeptide vaccine of two kinds of HSP70-HBV antigen polypeptides of load complex
1.HBV antigen polypeptide is synthetic: with HBV surface antigen polypeptide (HBsAg:FLLTRILTI) and HBV cAg polypeptide (HBcAg:FLPSDFFPSV) is target antigen; Polypeptide dissolving and concentration are with embodiment 1;
2.rhHSP70-the polypeptide complex preparation: operating procedure is prepared into two kinds of complex of HSP70-HBsAg and HSP70-HBcAg with implementing 1;
3. external evoked dendritic cell
Gather chronic hepatitis B patient (HLA-A201+) peripheral blood or the HLA-A201+ normal healthy people person peripheral blood of fresh anticoagulant heparin, behind the Ficoll density, obtain periphery mononuclearcell (PBMCs), PBMCs is suspended in the RPMI1640 culture medium that contains 2% (v/v) deactivation mixing people AB serum, and concentration is 2-3 * 10
6Individual cell/ml; Press every hole 3ml inoculating cell in 6 porocyte culture plates, in 5% (v/v) CO
2, place in 37 ℃ of cell culture incubators and made adherent mononuclear cells in 90 minutes, wash culture hole gently to remove not attached cell with the PBS buffer; Add 3ml in every hole and contain 5% (v/v) deactivation people AB serum, the RPMI1640 culture medium of 150ng/ml rhGM-CSF and 100ng/mlIL-15 (DC inducing culture III).In inducing the 3rd day, the 1ml culture supernatant is inhaled in every hole gently, adds the fresh DC inducing culture III of 1ml and continues to induce; In the 5th day results DCs, to wash and be suspended in the PBS solution with PBS, concentration is 2 x 10
6Individual cell/ml;
4.HSP70-polypeptide complex load DCs
Get the above-mentioned two kinds of rhHSP70-polypeptide complexes that prepared of 250 μ l respectively, be added among the inductive DCs of 1ml, cultivated 4-6 hour in 27 ℃-30 ℃ behind the mixing;
5.DC vaccine results: collect above-mentioned cell, with being suspended in the normal saline that contains 2% (g/100ml) human albumin (HA) behind the PBS washing secondary, concentration is 2 x 10
6Individual cell/ml is hepatitis B dendritic cell polypeptide vaccine.
6. vaccine quality detected parameters: with embodiment 1.
Claims (13)
1, a kind of dendritic cell polypeptide vaccine of carrying recombinant human heat shock protein 70 polypeptide complex, it is characterized in that: antigen polypeptide and rhHSP70 are formed complex, again complex is carried on dendritic cell and prepares the dendritic cell vaccine of carrying recombinant human heat shock protein 70 polypeptide complex.
2, the dendritic cell polypeptide vaccine of the described a kind of carrying recombinant human heat shock protein 70 polypeptide complex of claim 1, wherein said antigen polypeptide is to have immunogenic tumour specific antigen, tumor associated antigen or pathogen antigen epi-position and carry out synthetic according to known, the length of antigen polypeptide is 9-25 aminoacid sequence, and purity is more than 90%.
3, the dendritic cell polypeptide vaccine of the described a kind of carrying recombinant human heat shock protein 70 polypeptide complex of claim 2, wherein said tumor antigen is melanoma-associated antigen MART1, hepatocarcinoma related antigen AFP, carcinoma of prostate related antigen PSA, gastric cancer associated antigen CEA or breast cancer correlation antigen Her2-neu, including, but not limited to above-mentioned tumor antigen.
4, the dendritic cell polypeptide vaccine of the described a kind of carrying recombinant human heat shock protein 70 polypeptide complex of claim 2, wherein said pathogen antigen is hepatitis b virus hbv, hepatitis C virus HCV, HIV (human immunodeficiency virus) HIV antigen or TB antigen, including, but not limited to above-mentioned pathogen.
5, the dendritic cell polypeptide vaccine of the described a kind of carrying recombinant human heat shock protein 70 polypeptide complex of claim 2, the length of wherein said antigen polypeptide is 9-25 aminoacid sequence, purity is more than 90%.
6, the dendritic cell polypeptide vaccine of claim 1 or 2 described a kind of carrying recombinant human heat shock protein 70 polypeptide complexes, wherein said antigen polypeptide is GP100:IMDQVPFSV, MART1:AAGIGILTV, Try:YMDGTMSQV, MAGE3:FLWGPRALV, MUC1:SLADPAHGV, Her2-neu:KIFGSLAFL, AFP:GVALQTMKQ, HBsAg:FLLTRILTI or HBcAg:FLPSDFFPSV, including, but not limited to aforementioned polypeptides.
7, the preparation method of the dendritic cell polypeptide vaccine of any described a kind of carrying recombinant human heat shock protein 70 polypeptide complex of claim 1-6,
Comprising the following step:
1) recombinant human HSP 70 albumen (rhHSP70's) obtains;
2) design of antigen polypeptide is with synthetic;
3) preparation of recombinant human HSP 70 antigen polypeptide complex;
4) external evoked dendritic cell;
5) antigen load: the recombinant human HSP 70 antigen polypeptide complex that will prepare as stated above mixes with dendritic cell, cultivates certain hour jointly, is prepared into dendritic cell vaccine;
(6) quality testing of dendritic cell vaccine.
8, the described preparation method of claim 7 is characterized in that:
Wherein the design considerations of antigen polypeptide is known has immunogenic tumour specific antigen (TSA), tumor associated antigen (TAA) or pathogen antigen epi-position and carries out synthetic, and the length of antigen polypeptide is 9-25 aminoacid sequence, and purity is more than 90%;
Wherein recombinant human HSP 70 antigen polypeptide complex is HSP70 albumen and the 50-300nM antigen polypeptide with 10-100 μ M, is containing 10-20nM ATP, 1mM KCl, 2nM MgCl under 20 ℃ of-25 ℃ of temperature
2The PBS buffer in common reaction 20-60min, add again after 100-200nM ADP reacts 20-40min jointly, dialysis is prepared from after removing free polypeptide;
Wherein external evoked dendritic cell is that flush away is attached cell not, adds combination of cytokines, GM-CSF and the IFN of the combination of cytokines, GM-CSF and the IL-15 that contain GM-CSF and IL-4 by after obtaining mononuclearcell cultivation certain hour
The culture medium of the combination of cytokines of the combination of cytokines of α or GM-CSF and TNF α is induced the dendritic cell in monoblast source; Or get CD34+ precursor hematopoietic stem cell, adding contains GM-CSF, TNF α and induces into CD34-DCs with the culture medium of Flt3-L cytokine;
Wherein HSP70 polypeptide complex and dendritic cell altogether the ratio of educating be 10 μ g HSP70 polypeptide complexes and 10
6Individual dendritic cell is educated altogether, and educating temperature altogether is 27 ℃-30 ℃; The time of educating is 4-6 hour altogether, and unconjugated HSP70 and HSP70-polypeptide complex are removed in results and washing, are prepared into dendritic cell vaccine;
Described dendritic cell vaccine, wherein cell purity (CD11c+HLADR+) greater than 80% and cell survival rate greater than 90%; The bacterin preparation endotoxin content is less than 5IU/ml; Gram-bacteria detects negative; The detection of mycoplasma feminine gender; Aseptic detection is qualified; Dendritic cell surface costimulatory molecules expression requires: CD80 is greater than 80%, and CD83 is greater than 50%, and CD86 is greater than 80%, and CD40 is greater than 90%, and CCR7 is greater than 50%.
9, the described preparation method of claim 8, comprising the following step:
(a), fully dissolve antigen polypeptide, antigen polypeptide concentration is 5mg/ml with DMSO; The rhHSP70 is dissolved with PBS, and concentration is 0.1 μ g/ml;
(b), rhHSP70 polypeptide complex preparation, wherein step is:
Get 5 μ g antigen polypeptides and 10 μ g rhHSP70 polypeptide respectively in 1.5ml EP pipe, add 400 μ l and contain 10nM ATP, 1mM KCl, 2mMMgCl
2The PBS buffer in, educated altogether 40 minutes in 20 ℃ behind the mixing, add and continue at 20 ℃ behind the 100nM ADP and hatched 40 minutes, solution is moved in the micro dialysis pipe that 0.5ml includes 14kD molecular weight ultrafilter membrane spend the night, remove not in conjunction with polypeptide, prepare the rhHSP70-polypeptide complex;
(c), external evoked dendritic cell, wherein step is:
Gather the patient HLA-A201+ peripheral blood or the HLA-A201+ normal healthy people person peripheral blood of fresh anticoagulant heparin, behind the Ficoll density, obtain periphery mononuclearcell PBMCs, PBMCs is suspended in the RPMI1640 culture medium that contains 2% (v/v) deactivation mixing people AB serum, and concentration is 2-3 * 10
6Individual cell/ml; Press every hole 3ml inoculating cell in 6 porocyte culture plates, in 5% (v/v) CO
2, place 90min in 37 ℃ of cell culture incubators and make adherent mononuclear cells, wash culture hole gently to remove not attached cell with the PBS buffer; Add 3ml DC inducing culture in every hole, in inducing the 3rd day, the 1ml culture supernatant is inhaled in every hole gently, adds the fresh DC inducing culture of 1ml and continues to induce; In the 5th day results DC, to wash and be suspended in the PBS solution with PBS, concentration is 2 x 10
6Individual cell/ml;
(d), HSP70-polypeptide complex load DC, wherein step is:
Get the above-mentioned polypeptide complex that has prepared of 250 μ l respectively, be added among the inductive DC of 1ml, cultivated 4-6 hour in 27 ℃-30 ℃ behind the mixing;
(e), DC vaccine results: collect above-mentioned cell, with being suspended in the normal saline that contains 2% (g/100ml) human albumin HA behind the PBS washing secondary, concentration is 2 x 10
6Individual cell/ml is the dendritic cell polypeptide vaccine.
10, the described preparation method of claim 7, wherein said DC inducing culture be following any one: the RPMI1640 culture medium that contains 5% (v/v) deactivation people AB serum, 100ng/ml rhGM-CSF and 300ng/ml IFN α; Contain the RPMI1640 culture medium of 5% (v/v) deactivation people AB serum, 100ng/ml rhGM-CSF and 50ng/ml IL-4 or contain the RPMI1640 culture medium of 5% (v/v) deactivation people AB serum, 150ng/ml rhGM-CSF and 100ng/ml IL-15.
11, the described preparation method of claim 8, comprising the following step:
(a), fully dissolve antigen polypeptide, antigen polypeptide concentration is 5mg/ml with DMSO; The rhHSP70 is dissolved with PBS, and concentration is 0.1 μ g/ml;
(b), rhHSP70-polypeptide complex preparation, wherein step is:
Get 5 μ g antigen polypeptides and 10 μ g rhHSP70 polypeptide respectively in 1.5ml EP pipe, add 400 μ l and contain 10nM ATP, 1mM KCl, 2mM MgCl
2The PBS buffer in, educated altogether 40 minutes in 20 ℃ behind the mixing, add and continue at 20 ℃ behind the 100nM ADP and hatched 40 minutes, solution is moved in the micro dialysis pipe that 0.5ml includes 14kD molecular weight ultrafilter membrane spend the night, remove not in conjunction with polypeptide, prepare the rhHSP70-polypeptide complex;
(c), external evoked CD34-DCs, wherein step is:
HLA-201+ patient or normal person mobilize through GM-CSF hemopoietic, gather mononuclearcell PBMCs with the cell collection instrument, add the separation and purification of CD34 monoclonal antibody magnetic bead and obtain purity 90% CD34+ hemopoietic forebody cell, it is suspended in the X-VIVO culture medium that contains 5% (v/v) mixing people AB inactivated serum the TNF α of GM-CSF, the 10ng/ml of adding 50ng/mL and the Flt of 100ng/ml in culture medium
3-L cultivates the 5th day additional fresh culture and cytokine, observes and the timely cell density of adjusting, and collects the CD34-DCs that induced the 9th day, washs and is suspended in the PBS solution with PBS, and concentration is 2 x 10
6Individual cell/ml;
(d), HSP70-polypeptide complex load DCs, wherein step is:
Get the above-mentioned polypeptide complex that has prepared of 250 μ l, be added among the inductive DC of 1ml, cultivated 46 hours in 27 ℃-30 ℃ behind the mixing;
(e), DC vaccine results: collect above-mentioned cell, with being suspended in the normal saline that contains 2% (g/100ml) human albumin HA behind the PBS washing secondary, concentration is 2 x 10
6Individual cell/ml is the dendritic cell polypeptide vaccine.
12, the application of the dendritic cell polypeptide vaccine of any described carrying recombinant human heat shock protein 70 polypeptide complex of claim 1-6 in the preparation antitumor drug.
13, the application of the dendritic cell polypeptide vaccine of any described carrying recombinant human heat shock protein 70 polypeptide complex of claim 1-6 in preparation anti-infectious disease medicine.
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