CN1806849A - Method of treating tumors - Google Patents
Method of treating tumors Download PDFInfo
- Publication number
- CN1806849A CN1806849A CNA2005100023767A CN200510002376A CN1806849A CN 1806849 A CN1806849 A CN 1806849A CN A2005100023767 A CNA2005100023767 A CN A2005100023767A CN 200510002376 A CN200510002376 A CN 200510002376A CN 1806849 A CN1806849 A CN 1806849A
- Authority
- CN
- China
- Prior art keywords
- tumor
- oncolytic
- therapeutic method
- cell
- antigen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a tumour treating method, which comprises the following steps: (1) increasing the tumor antigen burst size of tumour position which tumour patient needs to treat; (2) increasing protein content which sticks tumor antigen and was distinguished by antigen presenting cell in tumour position; (3) increasing participating immune cell content in tumour position; (4) overlapping at high limit with reaching maximum value time and location of (1) burst size, (2) protein content and (3) participating immune cell content.
Description
Technical field
The present invention relates to biomedical sector, especially relate to a kind of method that is used for the treatment of tumor.
Background technology
Be accompanied by improving constantly of medical science level, people are also more and more deep to the research of tumor therapeuticing method, and oneself not only is confined to conventional Therapeutic Method.The conventional treatments of tumor comprises operation, radiotherapy, chemotherapy, thermotherapy etc., and these methods can be used separately or be used in combination in the actual therapeutic process.But the conventional treatments toxicity, side effect of tumor is bigger, usually makes the part patient because of being difficult to bear the caused kickback therapy discontinued of toxic and side effects; And the effect of the conventional treatments of tumor is not good enough, and the patient is prone to the recurrence and the transfer of tumor after treatment.Therefore, people need seek that a kind of toxicity, side effect is less, therapeutic effect tumor therapeuticing method preferably.
Along with people to the going deep into of tumor molecular pathology research, and mutual relation clear between gene and the disease, the Biotherapeutics method of tumor is arisen at the historic moment.At present, the Biotherapeutics method of tumor mainly comprises the immunotherapy method and the gene therapy method of tumor.
The immunotherapy method of tumor refers to by the immune system that stimulates human body self and resists the treatment for cancer method.At present; there has been the technology can be with the tumor cell dissolution in vitro; and with this solute and the fusion of DC (dendritic cell) electricity; the DC that will carry tumor antigen again re-enters in the human body; anti-tumor function with startup or enhance immunity system; suppress tumor growth or make tumor regression, and obtain long protective immunological reaction, thereby reach the purpose of treatment tumor.According to the mechanism of immunotherapy, U.S. Antigenics biotech company develops the individuation knubble vaccine of a kind of HSP (heat shock protein)-oncopeptide complex.In the therapeutic process, at first tumor patient is implemented tumor resection; In cut tumor tissues, extract HSP-oncopeptide complex then; At last HSP-oncopeptide complex is injected in the body of this tumor patient, to reach the purpose of treatment tumor.But, because the tumor of Different Individual exists very big difference on the mutational spectrum of gene, in actual applications, just must will take from certain patient and re-enter accordingly in this patient's the body, and can not form a kind of tumor vaccine that is applicable to a plurality of patients with wide spectrum effect through external synthetic tumor vaccine.Therefore, above-mentioned immunotherapy method can not large-area applications in clinical; Moreover, because tumor vaccine need be in vitro extraction and synthetic, and operate strictly, thereby make this method be not easy to operation and successful implementation, this has limited the application of this method equally.
Research to tumor therapeuticing method is further found, can adopt various treatment meanss to come antineoplastic immunoreaction process in the analogue body in order consistently.Just have in the prior art and adopt chemotherapy binding immunoassay Therapeutic Method to treat the example of tumor.For example, adopt chemotherapy in conjunction with Thymopentin and IL-2 immune formulation Synergistic treatment colon cancer such as (interleukin II are called for short interleukin-22); Adopt chemotherapy in conjunction with GM-CSF (granulocyte/M-CSF), IL-2 and IFN α immune formulation Synergistic treatment melanomas such as (interferon-alpha).In actual applications, although adopting chemotherapy binding immunoassay Therapeutic Method to treat the tumor treatment effect improves, but owing to do not look after each key link of anti-tumor in vivo immunne response fully, perhaps fail to consider time facial difference (time that peak value occurs) and each peak-to-peak synergistic effect that each link of immunological effect produces, thereby make therapeutic effect not ideal enough.
Also provide in the prior art and adopted oncolytic virus to treat the method (abbreviating oncolytic therapy as) of tumor.This method mainly is to utilize oncolytic virus to duplicate and to cause the mechanism of cytolysis death to treat tumor in tumor cell.
The patent No. is US 5,677, and 178 United States Patent (USP) discloses a kind of oncolytic adenovirus ONYX-015.ONYX-015 is that a kind of E1B-55KD fragment of having deleted in the wild 5 type adenovirus DNA sequences of people makes up the specificity oncolytic adenovirus that forms afterwards.ONYX-015 does not have obvious influence to normal cell, but can optionally duplicate in tumor cell, breed, and causes oncolysis at last.People had begun to use ONYX-015 to carry out the clinical trial of tumor of head and neck, cerebral glioma, cancer of pancreas, former treating malignant tumor such as liver and gall tumor, colorectal cancer with liver metastases kitchen range, nonsmall-cell lung cancer and cervical cancer in 1996, and result of the test demonstrates this method and has sure curative effect and higher safety.
Granted publication number also discloses a kind of oncolytic adenovirus H101 (preserving number is CCTCC No.V98003) for the Chinese patent of CN1110553C.H101 be utilize technique for gene engineering that people's 5 type adenoviruss (Ad5) are carried out gene recombinaton and a kind of oncolytic adenovirus, it mainly is the E1B-55KD and the E3 district 19KD genetic fragment of having deleted people's 5 type adenoviruss.H101 can duplicate by specificity in tumor cell, finally causes cytolysis, thereby reaches the purpose of oncolytic.
At present, not only adopt oncolytic adenovirus H101 treatment tumor in the clinical trial separately, and attempted adopting the method for oncolytic adenovirus H101 combined chemotherapy to treat tumor.The chemotherapeutics that this method is used can be strongly select, nvelbine (NVB), cisplatin (CDDF), amycin, 5-FU (5-fluorouracil) etc., these chemotherapeutics in the DNA of tumor cell, cause the killing action to tumor by various machining functions.Wherein, the cisplatin hydrolysis forms [Pt (NH
3)
2(H
2O)
2]
2+, form a flat iron plate for making cakes compound with DNA, changed the template function of DNA; But the amycin intercalation of DNA also suppresses polymerase, thereby suppresses duplicating and transcribing of DNA; 5-FU is converted into 5-fluorodeoxyuridine acid (5F-dUMP) in human body, stop deoxyuridylic acid (dUMP) methyl to turn to deoxythymidylic acid (dTMP), thereby influences the synthetic of DNA.And oncolytic adenovirus H101 is by the massive duplication of H101 in tumor cell, disturbs the function of host cell to realize oncolytic, and does not have tangible cell cycle specific.Therefore, from anticancer mechanism, H101 and above-mentioned chemotherapeutics have certain synergism.And the experimental result of oncolytic adenovirus shows that chemotherapeutic such as cisplatin, 5-FU are to this viral replication capacity unrestraint effect.This shows that clinically oncolytic adenovirus H101 and embolic chemotherapy being united use is fully feasible in theory.And the actual clinical test also proves, the method that this oncolytic therapy and chemotherapy combined use, and its curative effect obviously is better than using separately the Therapeutic Method of chemotherapy.
In above-mentioned clinical trial, people also find to adopt oncolytic adenovirus treatment tumor, exist significant dependency between its curative effect and the heating.That is, no matter be simple oncolytic therapy, or the Therapeutic Method of oncolytic therapy combined chemotherapy, fever patient's curative effect is apparently higher than fever patient not.For example, when the shallow entry secondary disease kitchen range of treatment, the fever patient is respectively 81.3%, 78.1% with fever patient's curative effect not; And, presenting such rule equally for the distant place focus, the curative effect of the two is respectively 25.0%, 6.3%.
Similarly, it is found that and adopt oncolytic adenovirus treatment tumor that there are certain relation in its curative effect and heat shock protein, that is, when heat shock protein was able to great expression, curative effect was better.Therefore, in actual applications, having the tumor kitchen range of H101 to carry out local warming synchronously to injection expresses to induce local endogenous HSP, can improve the local curative effect of H101 equally greatly, and also obviously obtained better therapeutic effect in not injecting the focus of H101.Perhaps, insert the gene order of derivable heat shock protein 70 (HSP70) in the specificity oncolytic adenoviral gene group after changing structure, make virus duplicate, great expression HSP70 in the oncolytic.Like this, not only the tumor kitchen range at injection position place is dwindled, and can also act on far-end homology tumor kitchen range.
Although tumor therapeuticing method of the prior art be widely used in clinical in, but, because these tumor therapeuticing methods fail the whole bag of tricks such as radiotherapy, chemotherapy, oncolytic therapy, immunotherapy are effectively combined, therefore can not look after each link of anti-tumor in vivo immunity, the synergistic effect of the peak value (maximum) of perhaps not considering each link and being occurred so just makes that the curative effect of tumor therapeuticing method of the prior art is lower.
Summary of the invention
The technical problem that the present invention solves provides a kind of tumor treatment method, and this method has improved the curative effect of treatment tumor, has strengthened the multiple antigenic individual specificity's immunity of tumor.For addressing the above problem, the invention provides a kind of tumor treatment method, this method not may further comprise the steps according to the order of sequence:
(1) burst size of the tumor locus tumor antigen that tumor patient need treat is increased;
(2) enable to adhere to tumor antigen and increased at described tumor locus by the proteic content that antigen presenting cell is discerned;
(3) at described tumor locus the content of the cell that participates in immunity is increased;
(4) burst size of the described tumor antigen of step (1), the described proteic content that can adhere to tumor antigen and be discerned by antigen presenting cell of step (2), the described content that participates in the cell of immunity of step (3) reaches the peaked time and the place is overlapping to greatest extent.
In technique scheme of the present invention, the step that the release of tumor antigen is increased at described tumor locus can be accomplished in several ways, and for example described tumor locus is applied oncolytic reagent; To described tumor locus injection dehydrated alcohol, acetic acid, hot salt brine, hot distilled water; Described tumor locus is carried out radio-frequency (RF) ablation, microwave curing, high intensity focused ultrasound, laserthermia, cold therapy and other causes the method for tumor cell necrosis.
In optimized technical scheme, make the burst size of tumor antigen increase by described tumor locus being applied oncolytic reagent.Described oncolytic reagent comprises oncolytic microorganism, described oncolytic microorganism comprises oncolytic virus and oncolytic antibacterial, and described oncolytic virus comprises oncolytic adenovirus, oncolytic herpes simplex virus, oncolytic vesicular stomatitis virus, oncolytic Avian pneumo-encephalitis virus, oncolytic poliovirus, oncolytic Epstein-Barr virus and other the oncolytic virus that can optionally grow in tumor cell; Described oncolytic antibacterial comprises oncolytic salmonella typhi, oncolytic bifidus bacillus, oncolytic shigella, oncolytic Listerella, oncolytic bacillus pestis and other oncolytic antibacterials that can optionally grow in tumor cell.Described oncolytic reagent can also comprise that coding apoptogene, cytolysis gene, tumor necrosis factor gene, cysteine proteinase gene, y-globulin gene, HA-1 antitrypsin gene and other have the nucleotide sequence of the gene of oncolysis.
In optimized technical scheme, the described step that the burst size of tumor antigen is increased at tumor locus is to carry out before the step that tumor locus enables to adhere to tumor antigen and the proteic content discerned by antigen presenting cell increases.
In technique scheme of the present invention, the step that the content of the cell that participates in immunity is increased at described tumor locus realizes that by the patient being bestowed immune formulation the described cell that participates in immunity comprises antigen presenting cell, T cell.Described immune formulation preferably includes the molecule of interleukin-22, interleukin-13, interleukin 12, granulocyte/M-CSF, thymosin (Thymosin), tumor necrosis factor (TNF), interferon (INF), chemotactic factor, levamisole, immune costimulating factor and other energy enhance immunity effect.
In optimized technical scheme, Therapeutic Method provided by the invention also comprises described tumor patient is carried out chemotherapy, radiotherapy and/or applies the step of tumor molecular targeted agents to described tumor patient.Described chemotherapeutics comprises nvelbine, cisplatin, amycin, strong selecting and 5-fluorouracil.More preferably, the step of described chemotherapy or radiotherapy is to carry out before the step that the content that makes the cell that participates in immunity increases.The normal tumor molecular targeted agents that adopts comprises monoclonal antibody, micromolecular compound.Wherein, monoclonal antibody comprise He Saiting (other common name are: Hereeptin, Trastuzumab), Mabthera (Rituximab), IMC-C225 (cetuximab, Erbitux), Bevacizumab (Avastin) and other monoclonal antibody at the special target spot of tumor cell; Micromolecular compound comprises imatinib mesylate (other common name are: STI51, imatinib, Glivec), Iressa (other common name are: Iressa, ZD1839, Gefitinib), OSI774 (other common name are: Tarceva, erlotinib, R1415, CP358774, NSC718781) and other micromolecular compound at the special target spot of tumor cell.
In preferred technical scheme, when being treated, described tumor patient carries out omnidistance nutritional support, and the nutrition that described nutritional support replenishes comprises aminoacid, fat and trace element etc.
With respect to existing other technologies, the more remarkable treatment effect of tumor treatment method provided by the invention.This be because: at first, tumor therapeuticing method provided by the invention contained tumor antigen release, can adhere to tumor antigen and the protein expression level of being discerned by antigen presenting cell, tumor antigen present with immune effector cell to each link in the Chain of Immunology such as attack of tumor tissues, so not only can kill original position (injection site) tumor, and by starting the human immune system discerning and to kill and wound the tumor cell of transfer voluntarily, thereby curative effect is higher; Secondly, tumor therapeuticing method provided by the invention consider tumor antigen burst size after dissolving or the killing tumor cells peak value, can adhere to tumor antigen and the peak value of the expressing quantity discerned by antigen presenting cell and the when and where that peak value occurred that participates in the cell content of immunity, on when and where, as far as possible farthest realize " overlapping " by making above-mentioned peak value, so that each factor synergy and the bigger action effect of generation, thereby make curative effect increase.
Description of drawings
Describe the present invention in detail below in conjunction with the drawings and specific embodiments.
Fig. 1 is the flow chart of one embodiment of the present of invention;
Fig. 2 be embodiment illustrated in fig. 1 one the course of treatment administration time sketch map.
The specific embodiment
Tumor treatment method provided by the invention organically combines therapeutic modalities such as oncolytic therapy and immunization therapy, can not comprise the steps: according to the order of sequence
(1) content that tumor antigen is discharged at the tumor locus of the tumor patient of needs treatments increases;
In actual applications, can increase by the content that following several modes discharge the tumor locus tumor antigen of required treatment:
Mode one applies to described tumor locus that oncolytic reagent realizes.Wherein, oncolytic reagent can be oncolytic microorganism.Described oncolytic microorganism can be an oncolytic virus, for example oncolytic adenovirus, oncolytic herpes simplex virus, oncolytic vesicular stomatitis virus, oncolytic Avian pneumo-encephalitis virus, oncolytic poliovirus, oncolytic Epstein-Barr virus and other the oncolytic virus that can optionally grow in tumor cell; Also can be the oncolytic antibacterial, for example oncolytic salmonella typhi, oncolytic bifidus bacillus, oncolytic shigella, oncolytic Listerella, oncolytic bacillus pestis and other oncolytic antibacterials that can optionally in tumor cell, grow.Oncolytic reagent also can be that coding apoptogene, cytolysis gene, tumor necrosis factor gene, cysteine proteinase gene, y-globulin gene, HA-1 antitrypsin gene and other have the nucleotide sequence of the gene of oncolysis.
Mode two causes the reagent of tumor cell necrosis to tumor locus injection dehydrated alcohol, acetic acid, hot salt brine, hot distilled water and other.
Mode three is carried out radio-frequency (RF) ablation, microwave curing, high intensity focused ultrasound, laserthermia, cold therapy and other causes the method for tumor cell necrosis to tumor locus.
(2) enable to adhere to tumor antigen and increased at described tumor locus by the proteic content that antigen presenting cell is discerned; Wherein, tumor antigen be can adhere to and the albumen discerned by antigen presenting cell heat shock protein preferably, HSP70, HSP30, HSP60, HSP90, HSP94, HSP96, HSP110 and other heat shock protein specifically comprised.
In actual applications, can be implemented in the content increase that described tumor locus makes heat shock protein by tumor patient being applied stimulate; Also can by injection in above-mentioned steps (1) can the great expression heat shock protein oncolytic microorganism directly impel the increase of tumor locus heat shock protein content.
It is to be noted, although each included step of this method can be carried out not according to numeric order, but the preferred scheme of the present invention is: in the step that the content that tumor locus discharges tumor antigen increases is to carry out before the step that tumor locus enables to adhere to tumor antigen and the proteic content discerned by antigen presenting cell increases.
(3) at described tumor locus the content of the cell that participates in immunity is increased; Wherein, participate in immune cell and comprise antigen presenting cell, T cell etc.
(4) burst size of the described tumor antigen of step (1) (also can be the recruitment of burst size), the described proteic content that can adhere to tumor antigen and be discerned by antigen presenting cell of step (2) (also can be the amount that increases), the described content (also can be the amount that increases) that participates in the cell of immunity of step (3) reaches the peaked time and the place is overlapping to greatest extent.
In this article, the implication of used term " maximum " or " peak value " is identical in fact, refers to that all the described content of corresponding steps changes in time and forms summit value corresponding on the curve.
In actual applications, also oncolytic therapy, immunotherapy and nutritional support can be combined, form a kind of more preferred Therapeutic Method, it abbreviates the CHINA therapy as.Wherein, C represents " Cancer antigen release ", and promptly cancer antigen discharges; H represents " HSPincrease ", and promptly heat shock protein content increases; I represents " Immuno-streamformation ", and promptly flow immunoassay forms; N represents " Nutrition support ", i.e. nutritional support; A represents " Assembly ", i.e. each organic combination.
Below, be the mechanism that example is told about the CHINA therapy to use the stimulation of oncolytic reagent and local warming.Oncolytic reagent discharges individual specificity's kinds of tumors antigen in the dissolving tumor cell, by to applying the tumor kitchen range local warming of oncolytic reagent, induce generation " heat shock protein of inducibility ".The kinds of tumors antigen that these heat shock proteins are discharged during with the tumor cell cracking combines, and combines to give jejune dendritic cell with these antigen presentations with its specific receptor (CD91) that is present on the dendritic cell.The HSP70 that induces generation of heating can also promote the maturation of dendritic cell significantly.Dendritic cell is the strongest antigen presenting cell of function in the body, dendritic cell is carried out processed to antigen in maturation process, have transfer ability, migration is to the lymphatic organ of human body, and the tumor antigen submission after will being processed by the mhc class ii molecule on dendritic cell surface is given CD4
+The T cell, the exciting human immune system produces the specific immune response at tumor cell, by immune effector cell tumor cell is produced whole body, persistent fragmentation effect.
As seen from the above, the CHINA therapy combines oncolytic therapy and immunotherapy, optimize each links such as cell function of tumor antigen release, HSP expression, maturing dendritic cell and participation immunity in the anti tumor immune response, system, the when and where of reasonably arranging each factor to occur, it is farthest collaborative that they are realized, form the specific cell immunoreaction chain of body, thereby obtain optimum curative effect at self special tumor antigen.
Certainly, in actual applications, tumor treatment method provided by the invention can also comprise chemotherapy, radiotherapy or tumor molecular targeted agents therapy etc.Wherein, before increasing, the content that makes the cell that participates in immunity carries out having better effect of chemotherapy or radiotherapy.At present, the normal tumor molecular targeted agents that adopts comprises that monoclonal antibody, micromolecular compound are all in the method for the invention available.Wherein, monoclonal antibody comprise He Saiting (other common name are: Trastuzumab, Herceptin), Mabthera (Rituximab), IMC-C225 (cetuximab, Erbitux), Bevacizumab (Avastin) and other monoclonal antibody at the special target spot of tumor cell; Micromolecular compound comprises imatinib mesylate (ST151, imatinib, Glivec), Iressa (ZD1839, Gefitinib), OSI774 (Tarceva, erlotinib, R1415, CP358774, NSC718781) and other micromolecular compound at the special target spot of tumor cell.
Describe tumor treatment method provided by the invention in detail with the specific embodiment that adopts oncolytic adenovirus H101 below.
Present embodiment specifically comprises following each step:
1. need to determine the tumor locus of treatment, and it is implemented the chemotherapy and/or the radiotherapy of low dosage;
2. to the tumor locus injection oncolytic adenovirus H101 of needs treatments, with killing tumor cells and release tumor antigen specifically;
3. the tumor locus to the needs treatment heats, and the body temperature that makes the patient be heated the position by heating is higher than 1-7 degree centigrade of its normal body temperature, to induce expression of heat shock in the part; Described heat shock protein carries the tumor antigen of release, and submission gives antigen presenting cell, exciting in the body immunoreation at tumor cell, thereby local and far-end tumor is all had an effect;
4. in therapeutic process, carry out immunomodulating.Can impel immunomodulating by bestowing the immune formulation mode.Described immune formulation can adopt the molecule of IL-2, IL-3, IL-12, GM-CSF, thymosin, TNF, INF, chemotactic factor, levamisole, immune costimulating factor and other energy enhance immunity effect.Wherein, GM-CSF can be that the specific receptor on precursor surface combines with granulocyte and mononuclear phagocyte, promotes its proliferation and differentiation, produces neutrophilic granulocyte, eosinophilic granulocyte and mononuclear phagocyte); IL-2 can promote the killer cell propagation of cytotoxic T cell, natural killer cell and lymphokineactivation, and its killing activity is strengthened, and it can also promote lymphocytic emiocytosis antibody and interferon, participates in regulating the antineoplastic immunoreation; Thymosin is used for the inducing T cell differentiation and maturation, strengthens the generation of cytokine and the antibody response of enhancing B cell.
5. in treatment, the patient is carried out omnidistance nutritional support, comprise the additional balance of aminoacid, fat and trace element etc.;
6. the burst size of tumor antigen reaches peak value at the oncolytic adenovirus intratumor injection after 2-7 days, the expression of heat shock amount of heating induction reached peak value in 2-6 hour, acting on about 7 days of GM-CSF reaches peak value, acting on about 5 days of IL-2 and thymosin reaches peak value, and the effect of systemic immunity then is that curative effect is best when the effect peak value of each medicine and treatment measure is overlapping.
It is to be noted, oncolytic microorganism not only is confined to oncolytic adenovirus H101 in the above-described embodiment, can also adopt denomination of invention is " expressing the oncolytic microorganism and the application thereof of heat shock protein ", publication number is the disclosed oncolytic microorganism of the Chinese patent literature of CN1412295A, just need correspondingly adjust above-mentioned each step according to the characteristic of various oncolytic microorganisms.For example, when employing self can be expressed the oncolytic adenovirus H103 of heat shock protein, the heating operation of above-mentioned steps 3 be can omit, directly heat shock protein and body internal stimulus process expressed by H103.
Be that example further specifies the present invention with the treatment nonsmall-cell lung cancer below.
Please consult Fig. 1 and Fig. 2 simultaneously, at first definite 28 days is a course of treatment.In the present embodiment, tumor treatment method concrete steps provided by the invention are as follows:
1. the 1st of the course of treatment the day: at first, with nvelbine (NVB) 25mg/m
2Add normal saline 100ml, intravenous drip 15-30 minute; Then, get blood vessel express developed, cisplatin (CDDF) 40mg/m with the 250ml normal saline
2Cooperate aquation;
2. the 2nd of the course of treatment the day: cisplatin (CDDF) 40mg/m
2Cooperate aquation;
3. the 8th of the course of treatment the day: at first, with nvelbine (NVB) 25mg/m
2Add normal saline 100ml, intravenous drip 15-30 minute; Then, get blood vessel express developed with the 250ml normal saline;
4. the 10th, 12,14,16,18 of the course of treatment the day (next day once): subcutaneous injection GM-CSF 150ug, once a day; The the 10th, 12,14,16,18 day of the course of treatment (next day once): subcutaneous injection Thymosin alpha 1 1.6mg;
5. the 14th of the course of treatment the day: 5+rhIL-2 of intratumor injection H101,1,000,000 units;
6. 15-18 days of the course of treatment (continuous 4 days): intramuscular injection rhIL-2 1,000,000 units, once a day;
7. 15-18 days of the course of treatment (continuous 4 days): 42.5 ℃ of radio frequency heating of chest one hour, once a day.
Wherein, injection IL-2 is in order to improve IL-10 in the tumor in tumor, and TGF-β (transforming growth factor) preponderates and is beneficial to " microenvironment " of growth of tumour cell; Apply the maturation that GM-CSF can impel DC; Apply the effect that thymosin is used to strengthen the T cell.
Among Fig. 2, d1 represents first day, and d2 represents second day, by that analogy.
In the present embodiment, by patient's chest being carried out 42.5 ℃ of radio frequencies heating at 15-18 days continuous 4 days of the course of treatment, impel the HSP at this position to express, make the expression place unanimity of the release place of tumor antigen and the inductive endogenous HSP that heats, thereby make the spatiality that immunoreation takes place realize unification, make curative effect better.
Further specify the present invention below by the statistic experiment example of a plurality of patients in the clinical trial and the individuality test example of single patient.
Test example 1
Test example 1 is a plurality of patients' a statistic experiment example, and it mainly is the desk study clinical trial of carrying out at the nonsmall-cell lung cancer patients with terminal.Wherein, Ru Xuan patient all is can't perform the operation or perform the operation III, the IV phase patient of (or chemotherapy) back recurrence and the patient of partly be unwilling acceptance, radiotherapy in the treatment.
The therapeutic scheme that adopts in the test example 1 is: the CT guiding is percutaneous puncture intratumor injection H101 and IL-2 down, and in conjunction with local warming's (42.5 ℃) chemotherapy in addition again.
The result of the therapeutic scheme that test example 1 adopts is referring to table 1.
The result of the test statistical table of table 1. test example 1
| Sex | Age | Diagnosis | Diagnostic Time | H101 treatment time | Curative effect | ST | TTP |
| The man | 45 | Adenocarcinoma (IV) | 2002.10 | 2002.11 | PR | 13 months | 13 months |
| Man * | 75 | Adenocarcinoma (IIIb) | 2002.11 | 2002.11 | SD | May | May |
| Man * | 69 | Adenocarcinoma (IV) | 2002.12 | 2003.1 | PR | ＞23 months | 23 months |
| Man * | 75 | Adenocarcinoma (III) | 2002.5 | 2003.2 | MR | 14 months | 14 months |
| The woman | 47 | Adenocarcinoma (IV) | 2003.2 | 2003.2 | MR | ＞21 months | 20 months |
| The man | 71 | Scale cancer (IIb) | 2003.3 | 2003.3 | SD | December | December |
| The man | 75 | Non-small cell (IIIa) | 2003.3 | 2003.3 | SD | ＞20 months | 16 months |
| The man | 46 | Adenocarcinoma (IV) | 2003.3 | 2003.4 | PR | ＞19 months | JIUYUE |
| The man | 47 | Scale cancer (IIIa) | 2003.10 | 2003.11 | PD | ＞November | May |
| The woman | 72 | Non-small cell | 2004.8 | 2004.9 | SD | ＞March | ＞March |
| (IV) |
Wherein, * represents not accept chemotherapy, and the implication of other abbreviation is as follows in the table:
CR (Complete Response): expression is alleviated fully.
PR (Partial Response): the expression part is alleviated, and the minimizing that specifically refers to gross tumor volume is greater than 50%.
MR (Minor Response): expression takes a turn for the better, and specifically refers to dwindling greater than 25% but less than 50% of gross tumor volume.
SD (Stable Disease); Expression is stable, specifically refers to dwindling less than 25% or increasing less than 25% of gross tumor volume.
PD (Progressive Disease): expression progress, the increase that specifically refers to gross tumor volume is greater than 25% or new focus occurs.
TTP (Time to progression): the time of progress is treated in expression.
ST (survival time): represent life cycle.
Need to prove that in the terminological interpretation of above-mentioned his-and-hers watches 1, gross tumor volume refers to the product of two maximum perpendicular diameter of tumor focus.
As can be seen from Table 1, adopt comprehensive immunotherapy of oncolytic therapy and the chemotherapy of H101 to treat a line nonsmall-cell lung cancer patients with terminal 10 examples exploratoryly, (response rate is 30% RR) to its response rate, with the response rate size of employing chemotherapy is similar separately in the conventional treatments.Have the curative effect of 9 examples to reach more than the SD among the 10 routine patients, the TTP of 7 examples reached more than 9 months, and the ST of 7 examples reached more than 11 months, and these indexs obviously are better than adopting separately the situation of chemotherapy.
It is pointed out that the patient that major part is accepted chemotherapy in the test example 1 all carried out chemotherapy before injection H101.Certainly, also can or carry out chemotherapy afterwards in the injection H101 while in the actual therapeutic, just chemotherapeutics can cause damage to immune system, and it is remarkable to make that curative effect is not so good as present embodiment.
After the test, will test 5 large sample tests of example 1 and classical chemotherapy and compare, the result is referring to table 2.
The comparison of test results table of table 2. test example 1 and 5 large samples of classical chemotherapy
| 5 large sample tests of classical chemotherapy meansigma methods (minima-maximum) | Test example 1 | |
| Objective response rate (%) | 21(17-37) | 30(3/10) |
| The tumour progression time (moon) | 4.5(3.5-5.5) | ＞11.9 (more than the 5-21) |
| Median survival interval (moon) | 8.8(6.7-11.3) | ＞13 |
| The annual rate (%) of depositing | 36(27-46) | 70 |
Wherein, the scheme of test example 1 employing is: H101+ chemotherapy+local heat+IL-2.
5 large sample tests are: ECOG1594, SWOG, Italian Lung Cancer trial, EORTC08975 and Tax326.
As can be seen from Table 2, patient's tumour progression time average is about 12 months in the test example 1, and median survival interval is about 13 months, and the annual rate of depositing is about 70%.Correspondingly, tumour progression time, median survival interval, the annual rate of depositing of 5 large sample tests of internationally recognizable classical chemotherapy are respectively 4.5,8.8 and 36.This shows, adopt tumor treatment method provided by the invention, its curative effect obviously is better than adopting 5 large sample tests of classical chemotherapy.
Be understandable that, in oncolytic, heat in the test example 1 to promote expression of heat shock, make the tumor specific antigen in the tumor cell to be given the human immune system by holographic ground of heat shock protein submission, and be not only that certain tumour-specific of single submission or dependency antigen are given immune system, thereby, can be so that individual patient obtains curative effect preferably.
Further be understandable that, the whole process of test example 1 is finished in vivo, and selected patient can use identical medicine and step in treatment, obtain the therapeutic effect of individuation, therefore, this Therapeutic Method is not only easy and simple to handle, and has strengthened the multiple antigenic individual specificity's immunity of tumor.
Therefore, from test example 1 method provided by the invention as can be seen can be clinical large-area applications.
Be that example illustrates tumor treatment method of the present invention with the individuality test below.
Test example 2
The patient 1, is 45 years old male patient, do not have recurrent cough under the inducement in JIUYUE, 2002.
In October, 2002, CT showed: the middle lobe of right lung occupy-place, accompany two lungs in a large number being dispersed in property millet appearance send out kitchen range, mediastinal lymph node enlargement, right side moderate hydrothorax.In October, 2002, hydrothorax pathology showed: adenocarcinoma cell.Be diagnosed as: two lung alveolar cell carcinomas, right lung adenocarcinoma.Be: T4N3M1/IV (IV phase) by stages.KPS (scoring of Ka Shi muscle power situation): 80.
Begin to adopt in October, 2002 tumor treatment method of the present invention to treat.Concrete therapeutic scheme is: chemotherapy+H101+IL-2+ local heat.Wherein, chemotherapeutic is selected strong select 1.6mg, cisplatin 100mg for use; Percutaneous puncture injection H101, IL-2 in the thoracic cavity; 42.5 ℃ of thoracic cavity local heat.Treat and check chest films showed after 16 days: lung marking is clear, and rib diaphragm angle occurs, and hydrothorax disappears substantially.December in 2002 is after first course of treatment, and symptoms such as the patient is uncomfortable in chest, cough take a turn for the better; The thoracic cavity drawing liquid is 3～4ml (treatment prosocoel drawing liquid reaches 300ml) only.CT shows: send out kitchen range in the lung and obviously reduce, hydrothorax reduces, and lung marking is clear.After in January in 2003 second course of treatment, CT shows: hydrothorax disappears, and sends out kitchen range in the lung and obviously reduces more than 70% before than treatment, and lung marking is clear, and patient's symptom of coughing obviously alleviates.But because platelet low (1500) is suspended chemotherapy.Once used docetaxel Taxoterc+ cisplatin before beginning the 3rd course of treatment of March in 2003 separately.Began for the 4th course of treatment in April, 2003.Take tumor molecular targeted agents Iressa during the whole treatment.
Tumor treatment method of the present invention is PR to patient 1 curative effect; Patient 1 life span is 13 months.
Find out that from above-mentioned result of the test the curative effect of tumor treatment method of the present invention is comparatively remarkable.Infers former because tumor molecular targeted agents Iressa should have synergism with H101 according to the inventor, and infer that this synergistic mechanism is as follows, but the present invention is not subjected to the restriction of following supposition:
1.H101 entering tumor cell plays a dual role: promptly, (1) activation PI3K/ATK (phosphoinositide kinases/activation tyrosine kinase), RAS/RAF approach are to duplicate oncolytic virus; (2) cracking tumor cell.But two approach of PI3K/ATK, RAS/RAF have the protection tumor cell, promote division growth, resist effect of apoptosis, and like this, tumor will be difficult for being killed and wounded and cracking.And Iressa can suppress above two approach just, and therefore, the burst times after the H101 replicative phase is bestowed Iressa, can increase the splitting action of H101 to tumor.
2.H101 in tumor, duplicate, fight for intracellular resource with tumor cell, tumor cell is caused selection pressure, tumor cell with stronger multiplication capacity obtains selection advantage, but also may depend on the propagation activation signals approach of cell simultaneously, and PI3K/ATK, RAS/RAF are two main signal pathways wherein.Therefore, the use of H101 may make tumor cell more responsive to Iressa, thereby makes Iressa killing tumor cells more efficiently.In addition, E1A acts on common pathway by downward modulation EGFR inducing apoptosis of tumour cell with Iressa, has Synergistic killing effect.
3.T cell occupies key position in tumour immunity, mainly by apoptosis receptor/ligand (Fas-FasL), perforin/granzyme and TNF tumor is brought into play apoptosis and splitting action.And PI3K/ATK, RAS/RAF have anti-apoptosis activity, make tumor cell be difficult for being killed and wounded by lymphocyte.Therefore, Iressa may make tumor cell more responsive to the lethal effect of T cell to the inhibition of these two approach, thereby improves the whole body therapeutic effect of immunization therapy.
4.Iressa have the selectivity of height, to not obviously influence of immune system, can lower tumor load simultaneously, make the local oncolytic of H101, Iressa suppress tumor performance cooperative compensating.
Test example 3
The patient 2, are 47 years old women.Begin dry cough in February, 2003, increase the weight of March, but do not have expectorant, nothing spitting of blood and acomia heat symptom-complex shape.In March, 2003, CT showed: about 2 * 3 * 2cm lump of left hilus pulumonis, inferior lobe of left lung one about 4.2 * 4.1cm lump.Be diagnosed as: left adenocarcinoma of lung.Be: T4N2M1/IV (IV phase) by stages.KPS：80。
After first course of treatment, patient's 2 cough sxs.Second course of treatment of April in 2003.The 3rd course of treatment of June in 2003.The 4th course of treatment of August in 2003.
Through the treatment of four courses of treatment, patient 2 tumor size is obviously dwindled, and occurs downright bad, liquefaction kitchen range in lump.Patient cough, symptom such as uncomfortable in chest are clearly better.
Tumor treatment method of the present invention is PR to patient 1 curative effect; Patient 1 life span is greater than 20 months.
Therapeutic process and therapeutic outcome by patient 1 and patient 2 show that the curative effect of tumor treatment method of the present invention is better than the tumor therapeuticing method that prior art provides.
It is pointed out that the above only is a preferred implementation of the present invention.For the ordinary skill in the art, can also make some improvements and modifications under the prerequisite that does not break away from ultimate principle of the present invention, these improvements and modifications all are regarded as protection scope of the present invention.
Claims (29)
1. tumor treatment method, this method not may further comprise the steps according to the order of sequence:
(1) burst size of the tumor locus tumor antigen that tumor patient need treat is increased;
(2) enable to adhere to tumor antigen and increased at described tumor locus by the proteic content that antigen presenting cell is discerned;
(3) at described tumor locus the content of the cell that participates in immunity is increased;
(4) burst size of the described tumor antigen of step (1), the described proteic content that can adhere to tumor antigen and be discerned by antigen presenting cell of step (2), the described content that participates in the cell of immunity of step (3) reaches the peaked time and the place is overlapping to greatest extent.
2. Therapeutic Method according to claim 1, wherein, the step that the burst size of tumor antigen is increased at described tumor locus realizes by described tumor locus being applied oncolytic reagent.
3. Therapeutic Method according to claim 2, wherein, described oncolytic reagent comprises oncolytic microorganism.
4. Therapeutic Method according to claim 3, wherein, described oncolytic microorganism comprises the oncolytic virus that can optionally grow in tumor cell.
5. Therapeutic Method according to claim 4, wherein, described oncolytic virus comprises oncolytic adenovirus, oncolytic herpes simplex virus, oncolytic vesicular stomatitis virus, oncolytic Avian pneumo-encephalitis virus, oncolytic poliovirus or oncolytic Epstein-Barr virus.
6. Therapeutic Method according to claim 3, wherein, described oncolytic microorganism comprises the oncolytic antibacterial that can optionally grow in tumor cell.
7. Therapeutic Method according to claim 6, wherein, described oncolytic antibacterial comprises oncolytic salmonella typhi, oncolytic bifidus bacillus, oncolytic shigella, oncolytic Listerella or oncolytic bacillus pestis.
8. Therapeutic Method according to claim 2, wherein, described oncolytic reagent comprises that coding apoptogene, cytolysis gene, tumor necrosis factor gene, cysteine proteinase gene, y-globulin gene, HA-1 antitrypsin gene or other have the nucleotide sequence of the gene of oncolysis.
9. Therapeutic Method according to claim 1, wherein, the step that the burst size of tumor antigen is increased at described tumor locus is by causing the reagent of tumor cell necrosis to be realized to described tumor locus injection dehydrated alcohol, acetic acid, hot salt brine, hot distilled water or other.
10. Therapeutic Method according to claim 1, wherein, the step that the burst size of tumor antigen is increased at described tumor locus is by described tumor locus being carried out radio-frequency (RF) ablation, microwave curing, high intensity focused ultrasound, laserthermia, cold therapy or other causes the method for tumor cell necrosis to realize.
11. Therapeutic Method according to claim 1 wherein, describedly can adhere to tumor antigen and is heat shock protein by the albumen that antigen presenting cell is discerned.
12. Therapeutic Method according to claim 11, wherein, described heat shock protein comprises heat shock protein 70, heat shock protein 30, heat shock protein 60, heat shock protein 90, heat shock protein 94, heat shock protein 96, heat shock protein 110 or other heat shock protein.
13. Therapeutic Method according to claim 11, wherein, the step that heat shock protein content is increased at described tumor locus realizes by described tumor locus being carried out local excitation.
14. Therapeutic Method according to claim 13, wherein, described stimulation comprise heat, stimulation that anoxia, cold, infection, radiation, ethanol or other can inducing cell generate heat shock protein.
15. Therapeutic Method according to claim 14, wherein, described heating comprises and makes the heat body temperature at position of patient be higher than heating of 1-7 degree centigrade of its normal body temperature.
16. Therapeutic Method according to claim 1, wherein, the described step that the burst size of tumor antigen is increased at tumor locus is to carry out before the step that tumor locus enables to adhere to tumor antigen and the proteic content discerned by antigen presenting cell increases.
17. Therapeutic Method according to claim 1, wherein, the described cell that participates in immunity comprises antigen presenting cell, T cell.
18. Therapeutic Method according to claim 1, wherein, the step that the content of the cell that participates in immunity is increased at described tumor locus realizes by the patient being bestowed immune formulation.
19. Therapeutic Method according to claim 18, wherein, described immune formulation comprises the molecule of interleukin-22, interleukin-13, interleukin 12, granulocyte/M-CSF, thymosin, tumor necrosis factor, interferon, chemotactic factor, levamisole, immune costimulating factor or other energy enhance immunity effect.
20. Therapeutic Method according to claim 1 wherein, also comprises the step of described tumor patient being carried out chemotherapy.
21. Therapeutic Method according to claim 20, wherein, the medicine that described chemotherapy is used comprises nvelbine, cisplatin, amycin, strong selecting or 5-fluorouracil.
22. Therapeutic Method according to claim 1 wherein, also comprises the step of described tumor patient being carried out radiotherapy.
23. according to claim 20 or 22 described Therapeutic Method, wherein, the step of described chemotherapy or radiotherapy is to carry out before the step that the content that makes the cell that participates in immunity increases.
24. Therapeutic Method according to claim 1 wherein, also comprises the step that described tumor patient is applied the tumor molecular targeted agents.
25. Therapeutic Method according to claim 24, wherein, described tumor molecular targeted agents comprises monoclonal antibody.
26. Therapeutic Method according to claim 25, wherein, described monoclonal antibody comprises He Saiting, Mabthera, IMC-C225, Bevacizumab or other monoclonal antibody at the special target spot of tumor cell.
27. Therapeutic Method according to claim 24, wherein, described tumor molecular targeted agents comprises the micromolecular compound at the special target spot of tumor cell.
28. Therapeutic Method according to claim 27, wherein, described micromolecular compound comprises imatinib mesylate, Iressa or OSI774.
29. according to each described Therapeutic Method of claim 1-28, wherein, also protect when drawing together described tumor patient treatment and carry out omnidistance nutritional support, the nutrition that described nutritional support replenishes comprises aminoacid, fat and trace element.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2005100023767A CN1806849A (en) | 2005-01-19 | 2005-01-19 | Method of treating tumors |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2005100023767A CN1806849A (en) | 2005-01-19 | 2005-01-19 | Method of treating tumors |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1806849A true CN1806849A (en) | 2006-07-26 |
Family
ID=36839059
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2005100023767A Pending CN1806849A (en) | 2005-01-19 | 2005-01-19 | Method of treating tumors |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1806849A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104415335A (en) * | 2013-09-02 | 2015-03-18 | 北京中康万达医药科技有限公司 | Immunological therapy method and device for in-vivo individual system |
| CN105102613A (en) * | 2013-03-12 | 2015-11-25 | 免疫创新治疗有限公司 | ablative immunotherapy |
| CN111850041A (en) * | 2020-07-30 | 2020-10-30 | 药鼎(北京)国际细胞医学技术有限公司 | Viral construct containing IL12 bicistron for treating liver cancer and application and construction method thereof |
| CN114632157A (en) * | 2020-12-16 | 2022-06-17 | 上海三维生物技术有限公司 | Application of oncolytic virus and chemotherapeutic drug in synergistic inhibition of local advanced cervical cancer |
-
2005
- 2005-01-19 CN CNA2005100023767A patent/CN1806849A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105102613A (en) * | 2013-03-12 | 2015-11-25 | 免疫创新治疗有限公司 | ablative immunotherapy |
| CN104415335A (en) * | 2013-09-02 | 2015-03-18 | 北京中康万达医药科技有限公司 | Immunological therapy method and device for in-vivo individual system |
| US10980859B2 (en) | 2013-09-02 | 2021-04-20 | Hangzhou Converd Co., Ltd. | In vivo individualized systemic immunotherapeutic method and device |
| CN111850041A (en) * | 2020-07-30 | 2020-10-30 | 药鼎(北京)国际细胞医学技术有限公司 | Viral construct containing IL12 bicistron for treating liver cancer and application and construction method thereof |
| CN114632157A (en) * | 2020-12-16 | 2022-06-17 | 上海三维生物技术有限公司 | Application of oncolytic virus and chemotherapeutic drug in synergistic inhibition of local advanced cervical cancer |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104415335A (en) | Immunological therapy method and device for in-vivo individual system | |
| JP2022031331A (en) | Method for treating tumor by using recombinant interferon with changed spatial configuration | |
| CN108261426A (en) | Pharmaceutical composition and its application in the drug for the treatment of tumour and/or cancer | |
| CN1709409A (en) | Anti-tumor agent, beverages and foods using the same, and a process for manufacturing the anti-tumor agent | |
| CN1259418C (en) | Recombinant adenoviruses for sodium/iodide symporter (NIS) | |
| Yin et al. | Antitumor effects of oncolytic herpes simplex virus type 2 against colorectal cancer in vitro and in vivo | |
| CN1806849A (en) | Method of treating tumors | |
| CN1883286A (en) | A tea milk and method for preparing same | |
| CN106166294A (en) | A kind of compound for preoperative intervention radiotherapy in the treatment tumor | |
| Zhijun et al. | Clinical effects of shenqi fuzheng injection in the neoadjuvant chemotherapy for local advanced breast cancer and the effects on T-lymphocyte subsets | |
| CN1895337A (en) | Pharmaceutical use of Tangshen and Milkvetch root composition for improving late tumor patient life quality | |
| CN104666842A (en) | Hericium erinaceus health product and preparation method thereof | |
| CN1424057A (en) | Chinese medicine immunomodulator and its preparation method and use in pharmacy | |
| El-Khoueiry | AFM24 in Combination With Autologous NK Cells (SNK01) in Patients With Advanced/Metastatic Epidermal Growth Factor Receptor (EGFR) Expressing Solid Tumors: Initial Results From the Phase 1 Dose Escalation Study | |
| CN1294987C (en) | Protein having antitumor function, and high performance expression in vitro | |
| CN1211110C (en) | Chinese medicine composition for treating tumour | |
| CN1537564A (en) | Traditional chinese medicine prepns. for treating broad-spectrum cancers | |
| Lan et al. | Synergistic effect of a combination of granulocyte macrophage colony-stimulating factor and thymosin α1 on Lewis lung cancer transplanted tumor in mice | |
| CN1219054C (en) | Structure for recombinant adenovirus with double killer function and application in tumor treatment | |
| CN101628114B (en) | Broad spectrum anti-tumor composite vaccine | |
| CN1618978A (en) | Construction and application of cytokine IL-24 eukaryotic expression vector | |
| CN1850107A (en) | Fatty cream composition for maligant splanchnocoele hydrops and its preparing method | |
| TWI692361B (en) | Use of antrodia salmonea mycelium in alleviation of cancer cachexia | |
| CN1607003A (en) | Hepatitis C Adjuvant Therapy | |
| CN1834242A (en) | Use of artificial proliferative inhibiting gene-adenovirus expression carrier |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |