CN102847143B - The cell class bio-pharmaceutical of allogene adoptive cellular immunotherapy disease in the blood system and preparation method - Google Patents
The cell class bio-pharmaceutical of allogene adoptive cellular immunotherapy disease in the blood system and preparation method Download PDFInfo
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Abstract
The invention provides one to prepare for allogene adoptive cellular immunotherapy disease in the blood system, such as MDS or leukemic cell class bio-pharmaceutical, biological preparation and preparation method.Immunocyte and antigen are placed in liquid cellular incubation base co-cultivation in culture vessel, thus obtain the specific immune cell culture with anti-corresponding antigens；The cell class bio-pharmaceutical with immune response is separated from the culture obtained.Immunocyte can be variant cell, does not haves allosome rejection during use, and safety is high；Bone marrow aspiration, bone marrow biopsy and bone marrow stem cell after treatment MDS are cultivated all has hemopoietic function to be obviously improved.Patient's well-tolerated in therapeutic process.The present invention treats MDS by improving this brand-new etiological treatment strategy of immunity of body, has effectively recovered hemopoietic function in the patient, and the treatment for other hematopoietic stem cell malignant clone diseases including leukemia has good prospect.
Description
Technical field
The present invention relates to a kind of medicine for allogene adoptive cellular immunotherapy disease in the blood system, particularly relate to a kind of adoptive cellular immunotherapy MDS or treat leukemic biological species medicine, and preparation method thereof.
Background technology
It is now recognized that multiple disease in the blood system, including erythrocyte disorder, such as aplastic anemia, paroxysmal nocturnal hemoglobinuria (PNH) etc.；Leukocyte disease, as various leukemia, malignant lymphoma, malignant lymphatic-Reticuloendotheliosis, plasma cell dyscrasia, histiocytosis disease, myelodysplastic syndrome, myeloproliferative disease (polycythemia vera, essential thrombocythemia, myelofibrosis) etc. all originate from clone's sexually transmitted disease (STD) of hematopoietic stem cell or pluripotent stem cell.
That representative is myelodysplastic syndrome (MyelodysplasticSyndromes, MDS).MDS is a kind of heterogeneous clone's sexually transmitted disease (STD) originating from hemopoietic medullary system committed stem cell or pluripotent stem cell, feature is that myelosis is enlivened, abnormal clone cell breaks up in bone marrow, dysmaturity, DH occurs, in situ or being destroyed soon after being released into blood at bone marrow, cause hemopoietic inefficiency, peripheral blood cells reduces, cell function is abnormal, and showing as intractable one is or the hypocellular hematopathy of polyphyly.
MDS is the Malignant hematologic diseases that a kind of height is Clonal.Sickness rate about 10/,100,000 ~ 12/,100,000 population, global range there are about 300,000 patients.Any age all can fall ill, and the ratio of men and women is 2:1.MDS involves middle-aged and elderly people more, and about 80% patient is more than 60 years old.MDS clinical manifestation is mainly anemia, and almost all of MDS patient has Anemia, as weak, tired.The MDS patient of about 60% has Neutrophilic granulocytopenia, low owing to there is functions of neutrophils simultaneously so that MDS patient is susceptible to infect, and the MDS that there are about 20% dies from infection.The MDS patient of about 20% has hemorrhage performance, is common in skin, respiratory tract, digestive tract etc., the person that also has intracranial hemorrhage.Some patients can have liver, spleen, lymph node silght enlargement, and small number of patients can have breastbone tenderness, rib or membra arthralgia.The MDS patient of 40% ~ 60% has thrombocytopenia, along with progression of disease may occur in which Progressive symmetric erythrokeratodermia thrombocytopenia.
According to hematology and the feature of bone marrow morphology, MDS is divided into 5 types by nineteen eighty-two FAB cooperative groups:
1) refractory anemia (RArefractoryanemia): clinical based on anemia, reticulocyte reduces, in peripheral blood, neutrophilic granulocyte and platelet are the most also to reduce.Original cell polar rare (< 1%) in peripheral blood.Myelosis is active or substantially enlivens, and how more apparent red system hypertrophy is, and < 15%, red cell morphology is abnormal common for ringed sideroblasts.Grain system and Megakaryocyte also have certain paramophia, but the lightest.Germinal cell < 5% in bone marrow.
2) refractory anemia companion's annular abrasive grit cytosis (RARSrefractoryanemiawithringsideroblasts): be that ringed sideroblasts occurs with the main distinction of RA, account for more than the 15% of bone marrow erythroblast.Peripheral blood leucocyte and platelet count are most normal, and serum ferritin increases.
3) refractory anemia with excess of blasts (RAEBrefractoryanemiawithexcessblasts): in peripheral blood, three is that cell great majority all have and reduce in various degree, and minority case only has two to be Leukopenia.Peripheral blood often has germinal cell to occur, but < 5%.In bone marrow, germinal cell is between 5%～19%.Grain system and red system juvenile cell quantity increase, and morphologic change is obvious.
4) chronic myelomonocytic leukemia (CMMLchronicmyelomonocyticleukemia): be more common in elderly.Liver, splenomegaly are common.There are anemia and thrombocytopenia.Topmost feature is to have more mononuclear cell in blood and bone marrow.Monocytic absolute number in peripheral blood > 1 × 109/ L, is often accompanied by neutrophilic leukocytosis and paramophia.Germinal cell < 5% in peripheral blood.Myelosis is substantially enlivened, grain: red ratio increases, and germinal cell is 5% ~ 20%.
5) refractory anemia with excess of blasts transformation type (RAEB-Trefractoryanemiawithexcessblastsintransformation): blood and bone marrow are in addition to some changes with RAEB, meet following characteristics: 1. germinal cell>=5% in peripheral blood, but<20%；2. germinal cell>=20% in bone marrow, but<30%；3. visible Auer body in germinal cell；The most about more than 50% patient develops into acute leukemia, and the survival of patients time is short, most less than 1 year.
WHO proposes new MDS typing standard, think that bone marrow blast reaches 20% and is acute leukemia, RAEB-t is classified as acute myeloid leukemia (AML), and CMML is classified as MDS/MPD(myelodysplastic syndrome/myeloproliferative diseases), remain RA, RAS, RAEB of FAB；And RA or RAS with 2 will be or 3 be that hypertrophy exception person is individually classified as the intractable Leukopenia companion abnormal (refractorycytopeniawithmultilineagedysplasia of polyphyly hypertrophy, RCMD), the RA of only No. 5 chromosome long arm disappearances is stood alone as 5q-syndrome；Also newly increase MDS to fail to classify (u-MDS).The most clinical MDS typing is used in parallel FAB and WHO standard.
RA and RAS patient is many based on anemia, and clinical progress is slow, median survival interval 3 ~ 6 years, leukemic transformation rate about 5% ~ 15%.RAEB and RAEB-t is many based on pancytopenia, anemia, hemorrhage and infect and be clear to, can be with splenomegaly, and disease progression is fast, and median survival time is respectively 12 months, 5 months, and leukemic transformation rate is up to 40%, 60%.CMML, based on anemia, can have infection with hemorrhage, and splenomegaly is common, median survival interval about 20 months, and about 30% is changed into AML.
The usual onset of MDS is slow, is gradually in progress, and small number of patients onset is drastically, there are about more than 50% within morbidity starts 1 year and be converted into leukemia, MDS has higher mortality rate, and its cause of death is in addition to leukemia, most owing to infecting, hemorrhage, especially intracranial hemorrhage.The cause of disease of constitutional MDS is still not clear, the factors such as biology, chemistry or physics that are presumably due to cause gene mutation, chromosomal abnormality makes certain cell clonal hypertrophy cancerated, already generally acknowledged, mutagenic agent such as virus, some drugs (such as chemotherapeutic), radiate (radiotherapy), industrial reaction agent (such as benzene, polyethylene) and environmental pollution etc. can carcinogenesis, mutagenic agent can cause rearrangement or the gene rearrangement of chromosome, it is also possible to only causes the change of gene expression to cause MDS.Secondary cases MDS patient often has obvious predisposing factors, benzene class arene compound, chemotherapeutics especially alkylating agent, lonizing radiation all can inducing cell gene mutation and cause MDS or other tumors to occur.
Being found by clonal analysis technical research such as G6PD isozyme, restriction fragment length polymorphism analysis, MDS is initiated by the Clonal disease of hematopoietic stem cell.Abnormal clone cell breaks up in bone marrow, dysmaturity, DH occurs, in situ or is destroyed soon after being released into blood at bone marrow, causes ineffective hematopoiesis.Part MDS patient can be found to have Oncogene Mutation (such as N-ras gene mutation) or chromosomal abnormality (such as+8 ,-7), and the exception of these genes may also assist in generation and the development of MDS.The function of MDS terminal cell is as low in neutrophilic granulocyte superoxide anion level, alkali phosphatase also compared with normal.But said gene changes many generations in MDS relatively late RAEB, RAEB-T type patient, and less in MDS in early days RA, RAS, this explanation gene mutation is still difficult to resolve releases whole MDS morbidity's reason.
The abnormal hematopoiesis stem cell clone of hyperplasia often has two kinds of evolution paths: one for evolving as hematopoietic potential decline due to hyperplasia, and bone marrow can transfer Hypolasia to, and clinical manifestation is hemopoietic function exhaustion, for MDS death reasons more than half.Another kind of approach develops into acute leukemia, and the patient that there are about 1/3rd in the MDS course of disease is converted into leukemia, mostly is AML.
The main purpose for the treatment of MDS is to reduce the complication caused because of anemia, leukopenia, thrombocytopenia, improves the quality of life of patient.MDS world prognostic scoring system (IPSS) is based on original cell proportion and karyotype in the quantity of patient's cytopenia, bone marrow and evaluates prognosis, is a basic tool of guiding treatment: low danger 0 point；Middle danger-1(Int-1) 0.5～1 point；Middle danger-2(Int-2) 1.5～2 points；High-risk >=2.5 points.Low danger or Int-1 level patient treatment are mainly made the life better quality; use Supporting Therapy, promote the treatments such as hemopoietic, induction differentiation and biological response modifier; and Int-2 level and high-risk group of MDS mainly improve survival, use Combination chemotherapy and the hematopoietic stem cell transplantation etc. of AML.
(1) Supporting Therapy
There is the cause of disease person of seeking should can stop contacting material or the environment of suspicious paathogenic factor as far as possible for Secondary cases MDS.Component blood transfusion is selected according to the state of an illness.The infected is had to select sensitive antibiotic to control in time to infect according to pathogen.MDS companion huge children anemia person can Supplement of folic acid, vitamin B12, RARS can give vitamin B6.
(2) androgen, such as stanozolol, 11-testosterone enanthate etc.
RA normal to caryogram is effective for stanozolol (stanozolol).Danazol may be effectively effective to thrombocytopenia to stanozolol nonresponder.After treatment effectively, hemoglobin, granulocyte and platelet counts increase.
(3) adrenocortical hormone
MDS is merged autoimmune disease, as effective in autoimmune hemolytic anemia, rheumatoid arthritis, big vasculitis or pure red cell aplasia.After medication 3 ~ 4 weeks, hemogram takes a turn for the better, to hematopoietic cell paramophia without effect.
(3) hemopoietic growth factor, such as G-CSF, erythropoietin (Epo) etc.
In being applicable to, low danger patient, most widely used general, the safest with EPO, curative effect can be increased with G-CSF, GM-CSF combination, but have the promotion cell worry to leukemic transformation.Interleukin Ⅲ (IL-3) and TPO can apply as one sees fit.They stimulate granulocyte and thrombocytopoiesis, have little effect on erythrocyte.
(1) GM-CSF and G-CSF: can improve the granulocyte in peripheral blood, mononuclear cell number, is primarily adapted for use in leukopenia the infected, especially antibiotic therapy invalid or be suspected to have fungal infections.
(2) EPO: to without blood transfusion history, the women of serum EPO ＜ 100 ~ 200MU/ml, caryogram are normal, and person curative effect low to transfusion dependent is preferable.EPO can also activate biologically active pdgf, useful to thrombocytopenia person.It is combined with retinoic acid or G-CSF and can strengthen EPO effect.
(3) interferon: have directly suppression tumor cell proliferation effect, leukemic clone can be suppressed, the most clinical seldom application.
(5) induction-differential therapy
All-trans retinoic acid and 1,1,25 (OH) can be used2VitD3, small part patient's hemogram improves.Hemopoietic growth factor (as G-CSF combines Epo) also has differentiation-inducing agents effect.
(1) retinoic acid: retinoic acid shows suppression malignant clone growth or promotes leukaemia's differentiation.ATRA+EPO is the one therapeutic scheme effective, tolerant treating danger MDS low, middle.
(2) 1,25 (OH)2VitD3: Rocaltrol 2 μ g/d, at least with 12 weeks, with retinoic acid share may effect more preferable.Propagation capable of inhibiting cell, promotion differentiation and regulation immunologic function, and have anti-myelofibrosis effect.
(3) arsenical (ATO): arsenic trioxide can suppress tumor cell proliferation, stops tumor-blood-vessel growth newborn, and Cell differentiation inducing activity is ripe, apoptosis, improves the mechanisms play antitumor actions such as body's immunity.Have Developing restraint and apoptosis-promoting effect to leukaemia, low dose of arsenical has induction of differentiation, effective to some patients.Arsenic trioxide in treatment MDS is effective, and safer, and untoward reaction is few, becomes a kind of new tool treating this disease, total effective percentage 50%.
(6) chemotherapy
Chemotherapy is to remove MDS malignant clone, recovers normal hematopoiesis, treats the main method of high-risk MDS.
(1) intensive chemotherapy: the scheme of anthracycline-containing, I(IDA) A scheme is better than F(fludarabine) A and T(topotecan) A scheme.And middle dosage Ara-C associating IDA and topotecan have obvious curative effects to intractable/recurrent AML and high-risk MDS, can be as salvage treatment measure.Scheme containing mitoxantrone is not suitable for the MDS patient in old age.In chemotherapy regimen, add G-CSF then to improve remission rate, the clinical setting improving patient, increase the case load that can accept AIIo-SCT.
(2) small dose chemotherapy: for old MDS patient, owing to the toxic and side effects of intensive chemotherapy is obvious, be not suitable for intense prior chemotherapy, then can be selected for small dose chemotherapy to extend life cycle, improve life quality.
A) Low Dose Cytosine Arabinoside: can improve curative effect with G-CSF use in conjunction, for high-risk MDS, then can improve cell number, reduce blood transfusion.Dosage is 5～25mg/d subcutaneous injections 10～within 20 days, is a course for the treatment of, and drug withdrawal repeats this course for the treatment of after 2 weeks, side effect is bone marrow depression.
B) low dose of L-Sarcolysinum: can try out in treating MDS patient old, high-risk, wherein myelosis reducing MDS is more preferable to the reaction of low dose of L-Sarcolysinum.Common dose is 2mg/d, oral, total amount 90～140mg.Non-evident effect.
C) CAG scheme: Ara-C10mg/m2, q12h × 14 day, aklavine 14mg/m2, × 4 days, G-CSF300 μ g/d, × 14 days, can try out in RAEB patient, achieve gratifying curative effect.
D) low dose of (high) harringtonine: low dose of homoharringtonine has induction differentiation and cell toxicant dual function.To oldaged physically weak, intentionally, liver, RAEB and the RAEB-T patient of renal damage, take the circumstances into consideration application.
(7) anti-apoptotic treatment
In MDS falls ill, play certain effect owing to apoptosis is abnormal, therefore anti-apoptotic treatment achieves certain curative effect.
(1) amifostine (amifostine, Amifostine) is phosphorylation Organic Alcohol wide spectrum cytoprotective, and its interior metabolism product has the effect that the normal ancestral cells of anti-oxidation protection is not killed by chemotherapeutics.Apoptosis can be reduced and stimulate the growth of Bone Morrow Hematopoietic Progenitor Cells.If adding with EPO and G-CSF, hemopoietic can be improved, and without obvious toxic-side effects.
(2) pentoxifylline (Pentoxifiline) is xanthine derivative, may interfere with the factors such as TNF-α, TNF-β and IL-1, reduces its apoptosis-promoting effect in MDS falls ill.
(8) anti-angiogenic medicaments
Thalidomide (reaction stops, thalidomide) has generation and the blood vessel formation against function of suppression TNF-α, it is possible to be effectively improved the Leukopenia of some MDS patient.Usage: 150～200mg/d.To improving anemia, reducing transfusion dependent has certain curative effect, original cell number less person good effect in bone marrow.Side effect mainly has weak, drowsiness, constipation, edema, myalgia, erythra etc..Recently Lenalidomide (lenalidomide Lenalidomide) is a kind of immunomodulator of new generation possessing antiangiogenic and anti-tumor activity, and clinical trial confirms that this medicine is to the low danger of transfusion dependent of companion's 5q-chromosomal abnormality and middle danger-1 myelodysplastic syndrome and multiple myeloma curative effect certainly.
(9) immunosuppressant
The low danger treated or middle danger patient need to be carried out, if not suitable for carrying out chemotherapy or stem cell transplantation (SCT), 1 antithymocyte globulin course for the treatment of (ATG) or cyclosporine A therapy should be accepted.ATG and CsA: being regulated the immunoreation of MDS by suppression Ts cell, promote MDS hematopoietic growth, the two is without crossing drug resistant.CsA has immunosuppressant, anti-apoptotic dual function, and reversible drug resistance simultaneously, is inverted CD4/CD8 ratio, and patient's curative effect of TCR-α or-β gene rearrangement is preferable.
(10) demethylation medicine
Reach Mactra sulcatria Deshayes (decitabine DACOGEN): Dacogen be applicable to IPSS marking system in danger-2 and high-risk first control, control myelodysplastic syndrome (MDS) patient again, including constitutional and insecondary MDS, according to all of hypotype of FAB typing.Decitabine is a demethylation medicine, belongs to cytidine acid-like substance, and its mechanism of action is unique.It can reverse aberrant DNA methylation, mix capture dna sequence DNA transferring enzyme, consume intracellular dnmt rna, it has ratio cytosine arabinoside and the higher in vivo cytotoxicity of azepine bag glycosides, it has more intensive Cell differentiation inducing activity ability than azepine bag glycosides and suppresses methylated ability, thus, the treatment of neoplastic disease has irreplaceable advantage and efficiency.
The operational version of decitabine is 20mg/m2, once a day, intravenous injection in 1 hour, continuous 5 days, every 4 courses for the treatment of Monday.Decitabine treatment overall security is higher, the seriously adverse reaction rate of (3 ~ 4 grades) low (＜ 5%), major side effects (untoward reaction) is Neutrophilic granulocytopenia, thrombocytopenia, the bone marrow depression performances such as anemia, but compared with Reinforcement chemotherapy, its treatment related mortality substantially reduces (P=0.001).Other toxic and side effects incidence rates are relatively low, can be alleviated by anti symptom treatment or decrement or delay medication.Reach Mactra sulcatria Deshayes to disable in the known patient to decitabine allergy.
(11) Allogeneic Hematopoietic Stem Cell Transplantation
Allogeneic stem cell transplantation is the unique hope cured by MDS at present.The indication of AIIo-SCT is age ＜ 55 years old, have HLA be harmonious donor high-risk and middle danger patient, to low danger patient should strictly grasp treatment indication.And Nonmyeloablative stem cell transplantation is applicable to older or that viscera function is the most complete patient.
Owing to MDS mostly is gerontal patient, transplant related mortality is higher, low danger patient's previously less transplanting.Recently as dropping, low intensive non-clear marrow hematopoietic stem cell transplantation technology is increasingly mature, and transplanting can be used for more low danger MDS patient.IPSS-Int-2 and high-risk person, whether especially young, germinal cell increases and is first considered as transplanting with prognosis mala karyotype person.
Lacking good medicine to for MDS at present, various Therapeutic Method effective percentage (improvement rate) are about about 30%, and effect is the most very good, and quality of life of patients is poor.Allogeneic Hematopoietic Stem Cell Transplantation is the currently the only therapy that can cure MDS, but has all Multiple Constraints, simultaneously because MDS mostly is gerontal patient, transplant related mortality is higher.Supporting Therapy is still current standard scheme.
Summary of the invention
For at present for multiple disease in the blood system, including erythrocyte disorder, such as aplastic anemia, paroxysmal nocturnal hemoglobinuria (PNH) etc.；Leukocyte disease, the problem all not having good medicine and method such as great majority such as various leukemia, malignant lymphoma, malignant lymphatic-Reticuloendotheliosis, plasma cell dyscrasia, histiocytosis disease, myelodysplastic syndrome, myeloproliferative disease (polycythemia vera, essential thrombocythemia, myelofibrosis), the invention provides one to prepare for allogene adoptive cellular immunotherapy disease in the blood system, the such as medicine of MDS or leukemia medicament and the preparation method of described medicine for one.
First purpose of the present invention is to provide a kind of method prepared for allogene adoptive cellular immunotherapy disease in the blood system medicine, and step includes:
Step 1, is placed in immunocyte and antigen co-cultivation in culture vessel in liquid cellular incubation base, thus obtains the specific immune cell culture with anti-corresponding antigens；
Step 2, separates the cell class bio-pharmaceutical with immune response from step 1 in the culture obtained；
Wherein, in described liquid cellular incubation base, in addition to immunocyte, component a) ~ e is also included):
A) cell mitogen is former: such as any one or a few in ConA, phytohaemagglutinin, phytolacca american, lipopolysaccharide, glucosan, anti-cd 3 antibodies.
B) antigen: described antigen is hematopoietic stem cell exception clone cell, can be derived from allogeneic, xenogenesis, the antigen of self.
C) superantigen: can be any one or the combination of two kinds, the exogenous superantigen: such as bacterial endotoxin, endogenous superantigen: such as any one or a few in retrovirus, heat shock protein in exogenous superantigen and endogenous superantigen.
D) cytokine: as derived from lymphocyte, lymphokine that mononuclear cell and other cells produce, monokine, the cytokine of activation inflammation and any one or a few in stimulating the cytokine of hemopoietic.
E) immunological adjuvant: such as any one or a few in inorganic adjuvant, organic adjuvant, synthetic adjuvant and oil preparation.
Wherein, described culture fluid can be basic culture solution increase serum or serum-free medium.
In method of the present invention, described immunocyte can be variant cell or autogenous cell, is preferably variant cell in the present invention.
In method of the present invention, described immunocyte can be wild-type immune cell, it is also possible to be gene-modified cell, and the most gene-modified cell.
In method of the present invention, the described cell with immune response, such as tumor infiltrating lymphocyte, Tumor-infiltrating lymphocytes, natural killer cell, tumor-associated macrophages, lethal mononuclear cell, cytotoxic T cell and/or the dendritic cell of activation.And preferably lymphocyte, such as T cell, B cell, NK cell etc., most preferably T cell.
Wherein, described immunocyte is preferably 5 ~ 100:1 with the cell quantity ratio of specific antigen.
Wherein, described immunocyte and specific antigen co-cultivation time are preferably 3 ~ 180 days.
According to a kind of preferred implementation of the method for the invention, wherein, step 1 can also add antigen presenting cell.
Wherein, described cell culture container is preferably three-dimensional large volume concentration cultivation container but it also may be other the most known culture vessel, including bioreactor.
A kind of preferred implementation according to the method for the invention, wherein, also include cell clone step: with the culture obtained in step 1, or the cell with immune response obtained in step 2 is raw material, described culture medium is cloned, obtains the cell strain with immune response.Described clone may is that 1) intermittent cyclic stimulus method；2) continued stimulus method.
Second object of the present invention is to provide the cell class bio-pharmaceutical for adoptive cellular immunotherapy disease in the blood system prepared by a kind of above-mentioned any means, and described medicine is prepared by method described in above-mentioned first purpose.Described disease in the blood system includes: 1) erythrocyte disorder, such as aplastic anemia, paroxysmal nocturnal hemoglobinuria (PNH) etc.；2) leukocyte disease, such as various leukemia, malignant lymphoma, malignant lymphatic-Reticuloendotheliosis, plasma cell dyscrasia, histiocytosis disease, myelodysplastic syndrome, myeloproliferative disease (polycythemia vera, essential thrombocythemia, myelofibrosis) etc..
Medicine of the present invention, can make the most available dosage form and/or by the most available approach input patient or demander body.
The 3rd purpose of the present invention is to provide a kind of biological preparation for allogene adoptive cellular immunotherapy disease in the blood system, including container, and is placed in the described medicine in described container, second mesh of the present invention.
Wherein, any one during described biological preparation can also include normal saline, albumin and other stabilizers.
In foregoing of the present invention, described " medicine " also includes for preventing MDS and leukemic vaccine.
In medicine containing T lymphocyte prepared by the method for the invention, T lymphocyte can be from the cell of allosome, does not haves allosome rejection during use, and safety is high；And pass through experimental verification.Such as using Drug therapy MDS provided by the present invention, the index such as hematochrome, leukocyte, erythrocyte substantially rises, and with being obviously improved of Clinical symptom and sign, antisecosis is normal；Bone marrow aspiration, bone marrow biopsy and bone marrow stem cell after treatment are cultivated all has hemopoietic function to be obviously improved.Patient's well-tolerated in therapeutic process.
Accompanying drawing explanation
Fig. 1 is the bone marrow biopsy photo in (on March 7th, 2011) before patient uses Drug therapy of the present invention；
Fig. 2 is (on JIUYUE 27th, 2011) bone marrow biopsy photo after patient begins to use Drug therapy of the present invention；
Fig. 3 is (on April 2nd, 2012) bone marrow biopsy photo after patient uses Drug therapy of the present invention.
Detailed description of the invention
The invention provides a kind of for medicine treating MDS and preparation method thereof, preparation process includes:
Step 1, is placed in immunocyte and antigen co-cultivation in culture vessel in liquid cellular incubation base, thus obtains the specific immune cell culture with anti-corresponding antigens；
Step 2, separates the cell class bio-pharmaceutical with immune response from step 1 in the culture obtained；
Wherein, in described liquid cellular incubation base, in addition to immunocyte, component a) ~ e is also included):
A) cell mitogen is former: such as any one or a few in ConA, phytohaemagglutinin, phytolacca american, lipopolysaccharide, glucosan, anti-cd 3 antibodies.
B) antigen: described antigen is hematopoietic stem cell exception clone cell, can be derived from allogeneic, xenogenesis, the antigen of self.
C) exogenous superantigen and/or endogenous superantigen: such as bacterial endotoxin, endogenous superantigen: such as any one or a few in retrovirus, heat shock protein；Can be any one or the combination of two kinds in exogenous superantigen and endogenous superantigen.
D) cytokine: as derived from lymphocyte, lymphokine that mononuclear cell and other cells produce, monokine, the cytokine of activation inflammation and any one or a few in stimulating the cytokine of hemopoietic.
E) immunological adjuvant: such as any one or a few in inorganic adjuvant, organic adjuvant, synthetic adjuvant and oil preparation.
Wherein, described culture fluid can be basic culture solution increase serum or serum-free medium.
Wherein, described immunocyte can be variant cell or autogenous cell, is preferably variant cell in the present invention.
Wherein, described immunocyte can be wild-type immune cell, it is also possible to be gene-modified cell, and the most gene-modified cell.
Wherein, the described cell with immune response, such as tumor infiltrating lymphocyte, Tumor-infiltrating lymphocytes, natural killer cell, tumor-associated macrophages, lethal mononuclear cell, cytotoxic T cell and/or the dendritic cell of activation.And preferably lymphocyte, such as T cell, B cell, NK cell etc., most preferably T cell.
Wherein, described immunocyte is preferably 5 ~ 100:1 with the part by weight of specific antigen.
Wherein, described immunocyte and specific antigen co-cultivation time are preferably 3 ~ 180 days.
Wherein, step 1 can also add antigen presenting cell.
Wherein, described cell culture container is preferably three-dimensional large volume concentration cultivation container but it also may be other the most known culture vessel, including bioreactor.
Wherein, it is also possible to include cell clone step: with the culture obtained in step 1, or the cell with immune response obtained in step 2 is raw material, clones in described culture medium, obtains the cell strain with immune response.Described clone may is that 1) intermittent cyclic stimulus method；2) continued stimulus method.
Medicine of the present invention, can make the most available dosage form and/or by the most available approach input patient or demander body.
The present invention also provides for a kind of biological preparation, including container, and is placed in the described medicine in described container, second mesh of the present invention.Wherein, described biological preparation can also include the components such as normal saline, albumin and other stabilizers.
Embodiment 1
Come from the mononuclearcell of allosome, with specific antigen (hematopoietic stem cell exception clone cell) co-cultivation in liquid culture medium, described culture medium also includes ConA, exogenous superantigen, lymphokine, immunological adjuvant, and wherein, ConA consumption in the medium is 1 μ g/L；Exogenous superantigen (pseudomonal toxin) consumption in the medium is 5 μ g/L；Lymphokine (interleukin II) consumption in the medium is 1000u/L；Immunological adjuvant (5% tween 80) consumption is 0.1mL/L.
T lymphocyte and specific antigen (Raji(Burkitt ＇ s lymphoma cell)) ratio be 5:1(cell quantity than).
By T lymphocyte and specific antigen co-cultivation 30 days, it is thus achieved that have the specific immune cell culture of anti-corresponding antigens.
The cell with immune response, predominantly cytotoxic T cell is isolated from culture.
Embodiment 2
Come from the mononuclearcell (gene-modified T lymphocyte) of allosome, with specific antigen (hematopoietic stem cell exception clone cell) co-cultivation in liquid culture medium, described culture medium also includes phytohaemagglutinin, endogenous superantigen, lymphokine, immunological adjuvant, wherein, phytohaemagglutinin (PHA) consumption in the medium is 5 μ g/L；Endogenous superantigen (pseudomonal toxin) consumption in the medium is 1 μ g/L；Lymphokine (interferon) consumption in the medium is 1000u/L；Immunological adjuvant (5% tween 80) consumption is 0.1mL/L.
T lymphocyte and specific antigen (MOLT-4(acute lymphoblastic leukemia cell)) ratio be 50:1(cell quantity than).
By T lymphocyte and specific antigen co-cultivation 90 days, it is thus achieved that have the specific immune cell culture of anti-corresponding antigens.
The cell with immune response, predominantly cytotoxic T cell is isolated from culture.
Embodiment 3
Coming from the mononuclearcell (gene-modified T lymphocyte) of allosome, with specific antigen (hematopoietic stem cell exception clone cell) co-cultivation in liquid culture medium, described culture medium includes phytohaemagglutinin, exogenous superantigen, lymphokine, immunological adjuvant.Wherein, phytohaemagglutinin consumption in the medium is 10 μ g/L；Exogenous superantigen (pseudomonal toxin) consumption in the medium is 5 μ g/L；Lymphokine (interleukin II) consumption in the medium is 500u/L；Immunological adjuvant (5% tween 80) consumption is 0.1mL/L.
T lymphocyte and specific antigen (the former leukaemia of the chronic marrow of K-562()) ratio be 100:1(cell quantity than).
By T lymphocyte and specific antigen co-cultivation 10 days, it is thus achieved that have the specific immune cell culture of anti-corresponding antigens.
The cell with immune response, predominantly cytotoxic T cell is isolated from culture.
Embodiment 4
Come from the single T lymphocyte of allosome, with specific antigen (hematopoietic stem cell exception clone cell) co-cultivation in liquid culture medium, described culture medium includes ConA, exogenous superantigen, lymphokine, immunological adjuvant, wherein, phytohaemagglutinin (PHA) consumption in the medium is 10 μ g/L；Exogenous superantigen (pseudomonal toxin) consumption in the medium is 1 μ g/L；Lymphokine (interleukin II) consumption in the medium is 800u/L；Immunological adjuvant (5% tween 80) consumption is 0.1mL/L.
T lymphocyte and Dami(megakaryoblastic leukemia cell) ratio be 10:1(cell quantity than).
By antigen with T lymphocyte (mononuclearcell) at 37 DEG C, 5%CO2Under the conditions of cultivate 7 days.
Resuspended, collecting interface cell by centrifugation, adjust cell concentration, add self MNC through irradiating, with non-irradiated own cells as feeder cells with antigen presenting cell, cultivate 7 days, repeat this step.Collect cell, after limiting dilution, continue under these conditions to cultivate, pass on and cultivate through intermittent stimulation.
The cell strain with immune response is isolated from above-mentioned culture.
Embodiment 5
Come from the single T lymphocyte of allosome, with specific antigen (hematopoietic stem cell exception clone cell) co-cultivation in liquid culture medium, described culture medium is serum-free medium, add and include ConA, exogenous superantigen, lymphokine, organic solvent, wherein, phytohaemagglutinin (PHA) consumption in the medium is 10 μ g/L；Exogenous superantigen (pseudomonal toxin) consumption in the medium is 1 μ g/L；Lymphokine (interleukin II) consumption in the medium is 800u/L.
T lymphocyte and HEL299(red white corpuscle leukaemia) ratio be 10:1(cell quantity than).
By antigen with T lymphocyte (mononuclearcell) at 37 DEG C, 5%CO2Under the conditions of cultivate 7 days.
Cultivated cell is added large volume biological reactor for cell culture, carries out concentration cultivation 30 days.
The cell with immune response, predominantly cytotoxic T cell is isolated from culture.
Effect example
Name: woods xx sex: men age: clinical diagnosis in 18 years old: MDS
Because of palor, weak dizziness, after fatigue, shortness of breath and palpitation is admitted to hospital in 2010-12-30 half a year.Check and find that three is minimizing, leukocyte 2.34 × 109/L, erythrocyte 2.14 × 109/L, hematochrome 81g/L, platelet 86 × 109/ L.Sqternal puncture shows that three is hyperplasia accompanied slight DH bone marrow smear.Disease progression is very fast, and five months after diagnosis i.e. need blood transfusion for the first time, maintain by blood transfusion thereafter, and transfuse blood during five wheat harvesting periods 4300mL altogether.
It is admitted to hospital for the first time to being admitted to hospital (on March 7,4 days to 2011 January in 2011, Tianjin blood grinds institute) for the second time
Clinical manifestation: palor, dizziness is weak to be increased the weight of, shortness of breath and palpitation after activity
Hemogram checking result: leukocyte, 2.34-5.25 × 109/ L, erythrocyte 1.9-2.42 × 109/ L, hemoglobin 78.2-96g/L, platelet 77-107 × 109/ L.
Bone marrow smear checks diagnostic comments: combining medical history, grain red two is hypoplasia bone marrow smear.
On March 7th, 2011, Fig. 1 was shown in by bone marrow biopsy (censorship tissue 1.0cm × 0.2cm × 0.2cm) photo, and myelosis is the lowest, and hematopoietic cell lacks, and has no megalokaryocyte.Reticular fiber staining is negative.
Second time is admitted to hospital (2011-03-08 to 2011-07-12 is in hospital) before making a definite diagnosis allogene adoptive cellular immunotherapy including third time
Clinical manifestation is: palor, and dizziness is weak substantially to be increased the weight of, and shortness of breath and palpitation after activity needs the 1200mL that transfuses blood.
The routine blood test leukocyte of patient, 2.60-4.66 × 109/ L, erythrocyte 1.3-2.83 × 109/ L, hemoglobin 48.2-92g/L, platelet 83-119 × 109/ L.
Bone marrow smear checks diagnostic comments: this position bone marrow grain huge two is hypertrophy, and red system ratio lowers, and grain red two is paramophia.
Start cellular immunotherapy to being admitted to hospital for the 4th time (13 days to 2011 July in 2011 on JIUYUE 28, Tianjin blood grinds institute)
Clinical manifestation: the state of an illness there is no and is clearly better.Blood transfusion: 2000ml.
July 15 to JIUYUE carries out 14 cellular immunotherapy on the 21st, every about 3 ~ 4 days once.
Hemogram checking result: leukocyte, 2.4-4.23 × 109/ L, erythrocyte 1.33-2.55 × 109/ L, hemoglobin 48-76g/L, platelet 51-115 × 109/ L.
Bone marrow smear inspection diagnostic comments: diagnostic comments: grain red huge three is hypoplasia bone marrow smear.
Fig. 2 is shown in by JIUYUE in 2011 bone marrow biopsy photo on the 27th, and myelosis is the lowest (＜ 10%), only a few inclined maturation period grain erythroid cells.Have no megalokaryocyte.Have no that germinal cell increases.
It is admitted to hospital for 4th time to being admitted to hospital for the 5th time (29 to 2011 years Decembers of JIUYUE in 2011 22 days, Tianjin blood grinds institute)
Blood transfusion: clinical manifestation: the state of an illness starts stable, transfusion requirement amount reduces (1100ml is 2011-11-25 for the last time)
Cell therapy: 11-29 to 12-7, five treatments, every 2-3 days once
Hemogram checking result: leukocyte, 3.0-5.55 × 109/ L, erythrocyte 2.04-2.97 × 109/ L, hemoglobin 61-93g/L, platelet 40-100 × 109/ L.
Bone marrow smear checks diagnostic comments: grain red huge three is hypoplasia bone marrow smear.
Bone marrow biopsy shows, myelosis is the lowest, adipose cell proliferation, and grain erythrocyte and megalokaryocyte lack, and reticular fiber staining is negative.
It is admitted to hospital for 5th time to being admitted to hospital for the 6th time (December in 2011 on April 2nd, 23 days 1, Tianjin blood grinds institute)
Clinical manifestation: the state of an illness starts to take a turn for the better, and symptom and sign alleviates, it is not necessary to transfuse blood again.
Cell therapy: on February 17,9 days to 2012 February in 2012, four treatments, every 2-3 days once.
Hemogram checking result: leukocyte, 3.03-7.16 × 109/ L, erythrocyte 2.28-3.39 × 109/ L, hemoglobin 87-123g/L, platelet 43-78 × 109/ L.
Bone marrow smear checks diagnostic comments: combine medical history, and compared with December in 2011 bone marrow on the 22nd, three is that hyperplasia degree increases, and germinal cell is clear to accompany the obvious atypia of mature erythrocyte.
On April 2nd, 2012, bone marrow biopsy showed, myelosis is relatively active (80%), grain red ratio reduce, grain is red is that each phase cell is visible, the most in young and following phase cell be main, the inmature phase cell of grain system slightly increases, megalokaryocyte is clear to, and leaflet core is main, it is seen that Dan Yuanhe megalokaryocyte and many circle core megalokaryocytes, reticular fiber staining (+), Fig. 3 is shown in by photo.
It is admitted to hospital so far for 6th time (on August 26,21 days to 2012 April in 2012)
Clinical manifestation: the state of an illness is clearly better further, symptom and sign substantially alleviates, and muscle power progressively recovers normal
Cell therapy: on May 18,12 days to 2012 May in 2012, four treatments, every 2 days once.
In on June 22,15 days to 2012 June in 2012, five treatments, every 1-2 days once.
Hemogram checking result: leukocyte, 4.18-7.46 × 109/ L, erythrocyte 2.61-4.45 × 109/ L, hemoglobin 99-162g/L, platelet 47-69 × 109/ L.
Patient from morbidity after within seven month, proceed by allogene adoptive cellular immunotherapy.Treatment 30 times the most altogether, using cell is the cytotoxic T cell (CD8+T cell, about 95%) of preparation in above-described embodiment.Cell quantity is 1X10 every time9, intravenous drip.It is admitted to hospital from second time and makes a definite diagnosis before allogene adoptive cellular immunotherapy (2011-03-08 to 2011-07-12), the routine blood test leukocyte 2.60-4.66 × 109/L of patient, erythrocyte 1.3-2.83 × 109/L, hemoglobin 48.2-92g/L, platelet 83-119 × 109/L.After treating two wheat harvesting periods, blood transfusion number of times and quantity reduce, maintenance of need not transfusing blood again after four months.When treating nearly 1 year (2012-04-21 to 2012-09-08), routine blood test leukocyte 4.18-7.46 × 109/L, erythrocyte 2.61-4.45 × 109/L, hemoglobin 99-162g/L, platelet, 47-69 × 109/L.Wherein hematochrome progressively rises to 16.2g/L from minimum 4.8g/L, increases highly significant 11.4g/L, and with being obviously improved of Clinical symptom and sign, antisecosis is normal.Bone marrow aspiration, bone marrow biopsy and bone marrow stem cell after treatment are cultivated all has hemopoietic function to be obviously improved.
In therapeutic process, patient tolerates very well, and wiping out and treating insect pests and plant diseases treatment had and be not required to outside the of short duration fear of cold of special handling, chills and fever the same day, did not had any other toxic and side effects.Do not observe diarrhoea, erythra, the performance of the graft versus host disease such as liver function injury.
Table 1, blood change before and after treatment
The cell class bio-pharmaceutical of adoptive cellular immunotherapy MDS provided by the present invention, has the advantage that
1) there is efficient recovery MDS hemopoietic function in the patient
Anemia is the modal clinical symptoms of MDS patient, and wherein the patient of 50% goes to a doctor because of anemia.When progression of disease, the patient of 90% has anemia, and the MDS patient of 80% needs to transfuse blood and maintains, and often leads to ferrum too much.The clinical manifestation of this case is based on anemia, and disease progression is very fast, and five months after diagnosis i.e. need blood transfusion for the first time, maintain by blood transfusion thereafter, and transfuse blood during five wheat harvesting periods 4300mL altogether.After application allogene adoptive cellular immunotherapy two wheat harvesting period, blood transfusion number of times and quantity reduce, maintenance of need not transfusing blood again after four months, and after treatment more than a year, hematochrome progressively rises to 16.2g/L from minimum 4.8g/L, increases highly significant 11.4g/L.
2) MDS is treated by this brand-new etiological treatment strategy of immunity of raising body
The traditional treatment of MDS includes immunosuppressant therapy.The evidence that MDS patient immune function is the most complete includes abnormal CD4/CD8 ratio, the cytotoxicity of activation-cell increases, and shows as compared with matched group, and the percentage ratio of AA and MDS patient CD8+CD28-and CD8+CD28-CD57+ cell increases.
CsA lowers the immunoreation of MDS by suppression Ts cell, promotes MDS hematopoietic growth.CsA has immunosuppressant, anti-apoptotic dual function, and reversible drug resistance simultaneously, is inverted CD4/CD8 ratio, and patient's curative effect of TCR-α or-β gene rearrangement is preferable.It addition, MDS is merged autoimmune disease, such as autoimmune hemolytic anemia, rheumatoid arthritis, big vasculitis, often use prednisone, to hematopoietic cell paramophia without effect.Traditional immunosuppressant substantially belongs to symptomatic treatment, offer limited effectiveness in the treatment of MDS, mostly is transient effect.
No. 8 chromosome trisomy positives (10%) of the Marrow chromosome inspection (Fish) of this example patient, before patient, CD3+CD4+T cell reduces, and CD3+CD8+T cell raises.Additionally, during this example patient's onset, liver function and thyroid function are the most normal, along with exception (before occurring in the treatment of allogene adoptive cellular immunotherapy) progressively occur in disease progression liver function and thyroid function, show as slight ALT and AST and raise and slight hypothyroidism.This may be adjoint with MDS immunologic dysfunction relevant, cause autoimmune to sexually revise.
Intractable cytopenia companion polyphyly abnormal development young to this example, clinical progress MDS patient faster, we use the strategy of a kind of brand-new immunity improving body, carry out adoptive cellular immunotherapy and obtain good curative effect, not only Clinical symptom and sign is obviously improved, and bone marrow aspiration result, bone marrow biopsy and bone marrow stem cell after treatment are cultivated and all had clear improvement.According to extensive consulting literatures, do not find and use this brand-new etiological treatment strategy of immunity improving body, by allogene adoptive cellular immunotherapy MDS, and obtain the similar report of good curative effect.This case belongs to reported first.
3) the alternative method of the another kind in addition to chemotherapy is provided for removing malignant clone
Chemotherapy is to remove MDS malignant clone, recovers normal hematopoiesis, treats the main method of high-risk MDS.Combined chemotherapy, the MDS patient good for organ function is it is contemplated that combined chemotherapy, and as anthracycline antibiotics combines cytosine arabinoside, the some patients accepting chemotherapy can obtain one period of catabasis.But stage of bone marrow is long after MDS chemotherapy, especially it is noted that strengthen Supporting Therapy and insulation blocking.
We treat with allogene adoptive cellular immunotherapy, it is thus achieved that continuable good curative effect.Meanwhile, bone marrow aspiration result, bone marrow biopsy and the bone marrow stem cell after treatment is cultivated and is all had clear improvement.Wherein, before treatment, bone marrow biopsy results display myelosis is the lowest (＜ 10%), only a few inclined maturation period grain erythroid cells.Have no megalokaryocyte.Have no that germinal cell increases.Reticular fiber staining is negative.After treatment, bone marrow biopsy is that myelosis is relatively active (80%), the red ratio of grain reduces, grain is red is that each phase cell is visible, all based on middle young and following phase cell, the inmature phase cell of grain system slightly increases, and megalokaryocyte is clear to, and leaflet core is main, visible Dan Yuanhe megalokaryocyte and many circle core megalokaryocytes, reticular fiber staining (+).Not only hemopoietic function of bone marrow after prompting allogene adoptive cellular immunotherapy treatment, and bone marrow microenvironment all has clear improvement.Speculate relevant by Graft versus leukemia (GVL) effect removing MDS malignant clone with immunocyte (cytotoxic T cell).This removes malignant clone for treating young high-risk MDS patient, changes natural history, recovers normal hematopoiesis, it is provided that the alternative method of another kind in addition to chemotherapy.
4) safety of allogene adoptive cellular immunotherapy is preferable, can be with drug combination, it is adaptable to middle-older patient
Therapeutic strategy Shi Yi world prognostic scoring system (IPSS) of MDS is foundation.Principle is: to low danger or MDS patient at advanced age, uses low-intensity treatment, and stimulating bone marrow hematogenesis, induction differentiation to add or be not added with small dose chemotherapy is main scheme, improves hemocyte and declines, improves the quality of living；To high-risk or young MDS patients, use high-intensity therapeutic, including chemotherapy and hematopoietic stem cell transplantation, eliminate MDS malignant clone, change natural history, recover normal hematopoiesis, but it is the highest to treat relevant case fatality rate.
Medicine shown in the present invention is significant for the current Therapeutic Principle of MDS.To low danger or MDS patient at advanced age; employing low-intensity is treated; stimulating bone marrow hematogenesis, induction differentiation to add or be not added with small dose chemotherapy is main scheme; although hemocyte can be improved to be declined; improve the quality of living; but owing to failing to treat in time the cause of disease, the control dynamics for disease is inadequate, and the state of an illness still has the possibility developing into secondary leukemia further.And allogene adoptive cellular immunotherapy myelodysplastic syndrome has that safety is good, toxic and side effects is low, can repeated multiple times use, this kind of low danger or the MDS patient at advanced age should be can use to carry out etiotropic treatment.It is also possible to individually or with additive method drug combination, change high-risk or young MDS patients's natural history, recover normal hematopoiesis, reduce the case fatality rate that treatment is relevant.
5) allogene adoptive cellular immunotherapy and the comparison of Allogeneic Hematopoietic Stem Cell Transplantation
Autologous and Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) all has been used for treating MDS.Autologous hematopoietic stem cell transplantation for treatment MDS is the most only feasible sub-fraction patient.These needs reach complete incidence graph at induction chemother, and can collect suitable autologous stem cell.Successfully autologous peripheral blood stem cells (PBSC) gathers and is limited by following factor: (1) graft pollutes, the implantation that (2) postpone, and (3) have the risk of recurrence (up to 72%) of height in the patient of 40 to 63 years old, and 2 years without disease life cycle only 25%.Therefore, the application of this method is very limited.
Although traditional clear marrow Allogeneic Hematopoietic Stem Cell Transplantation can significant reduction relapse rate (compared with the relapse rate of autoplastic 28% to 48%), transplant related mortality (TRM) is at a relatively high, up to 39% to 54%.Transplanting relevant complication, the occurrence frequency including graft versus host disease (GVHD) is higher and the order of severity increases.The indication of AIIo-SCT is age ＜ 55 years old, have HLA be harmonious donor high-risk and middle danger patient.Owing to MDS mostly is gerontal patient, transplant related mortality is higher, low danger patient's previously less transplanting.Recently as dropping, low intensive non-clear marrow hematopoietic stem cell transplantation technology is increasingly mature, and transplanting can be used for more low danger, the older or Insufficient MDS patient of main organs.For IPSS-Int-2 and high-risk person, whether especially young, germinal cell increases and is first considered as transplanting with prognosis mala karyotype person.
Allogeneic Hematopoietic Stem Cell Transplantation truly has higher success rate for the treatment of MDS.But allosome bone marrow hematopoietic stem cell transplantation needs HLA antigen distribution type, is difficult to find distribution type suitable allosome marrow hemopoietic stem cells.Distribution type process is very long, complicated, transplant after need to use expensive immunosuppressant (single unnecessary 400,000 RMB of expense transplanted), there is the problems such as higher nearly rejection at a specified future date (patient repels the allosome marrow hemopoietic stem cells of allosome marrow hemopoietic stem cells and transplanting and repels patient) probability.According to the result (including RIC and NST) of 2000 to 2008 years 30 MDS bone marrow transplantations reports, overall survival rate (OS) and be 22% and 20% during without disease survival rate (DFS) 2 years, is 79% and 79% when 4 years.In these are reported, the sickness rate of II-IV acute graft versus host disease is 9% and 63%, chronic graft versus host disease 18% and 80%, risk of recurrence 6% ~ 61%.It is 0% when transplanting relevant case fatality rate (TRM) 100 days, when 5 years 34%.
In this case therapeutic process, tolerance is very well, and wiping out and treating insect pests and plant diseases treatment had to be not required to outside the of short duration fear of cold of special handling, chills and fever the same day, did not had any other toxic and side effects.Do not observe diarrhoea, erythra, the performance of the graft versus host disease such as liver function injury.Medicine prepared by the present invention is in clinical practice, the nearly 500 example patients of chronotherapy more than 5 years, 18 months-88 years old age, at most use 110 times, up to 6 years time, through clinical multicenter application attestation: safety is good, without rejection and autoimmune disease, and patient about 50 ~ 60% generation GvHD that traditional uses DLI treats.
6) for the treatment of other hematopoietic stem cell malignant clone diseases including leukemia
MDS is the same with acute leukemia, is the Clonal disease grown up by the malignant clone that the hematopoietic stem cell of an exception is derivative.Mainly involving myeloid cell, make bone marrow grain, red and huge three be the invalid DH of cell, its apoptotic cell quantity substantially increases.Recently also finding in MDS and Secondary cases AMD sample, although not having germinal cell, MDS is similar with the tumor cell component in Secondary cases AML bone marrow, and basis MDS clone (containing 182 ~ 660 kinds of sudden changes) is continuously present in Secondary cases AML.This shows that MDS's is Clonal similar to Secondary cases AML.We carry out treatment with allogene adoptive cellular immunotherapy and obtain good effect.After treating two wheat harvesting periods, blood transfusion number of times and quantity reduce, maintenance of need not transfusing blood again after four months.Meanwhile, bone marrow aspiration result, bone marrow biopsy and the bone marrow stem cell after treatment is cultivated and is all had clear improvement.The relevant clinical meaning of the allogene adoptive cellular immunotherapy MDS merited attention has a following three points:
(1) allogene adoptive cellular immunotherapy can postpone or stop MDS to develop into leukemic process, prevents leukemic related complication.The usual onset of MDS is slow, is gradually in progress.The patient that there are about 1/3rd in the course of disease is converted into leukemia, mostly is AML.Small number of patients onset drastically, there are about more than 50% within morbidity starts 1 year and is converted into leukemia, have higher mortality rate, leukemic complication once occurs, and treats difficulty very greatly, expends the most surprising, if but set about from prevention, relatively easy, it is possible to alleviate the misery of patient.Research finds that the AML clone main in patient's AML bone marrow of MDS of Secondary cases derives from basis MDS clone, and it should be the maximally effective strategy of mutant clon cell proliferation eliminated and be easily caused progression of disease that this prompting carries out targeted therapy for these in early days sudden changes.
(2) allogene adoptive cellular immunotherapy can treat the leukemia that MDS is adjoint.MDS is once converted into AML, and treatment becomes extremely difficult.Because this disease has following feature: mostly 1. be old people.MDS involves middle-aged and elderly people more, and the case of more than 50 years old accounts for 50%-70%, except normal physiological function lowers in various degree, often merges the vitals diseases such as the heart, brain, kidney；The most common multiple chromosomal abnormality and complex chromosome abnormalities；3. high drug-resistance: Multidrug resistance gene MDR1 and P-glycoprotein are many in high expressed, and positive rate reaches 80%；The highest case fatality rate: MDS is once converted into AML natural history and is typically only 3 ~ 6 months.Compared with primary AML, the poor resistance of high intensity chemotherapy, chemotherapy related mortality is high in early days, and low-intensity induction chemother also is difficult to obtain complete incidence graph, occurs all kinds of complication after body is impaired, and recovery time is longer.Therefore, compared with primary AML, MDS the AML converted is bigger because of its treatment difficulty, and prognosis is poor, has the most belonged to intractable AML.
(3) the same with acute leukemia due to MDS, it it is all the Clonal disease grown up by the malignant clone that the hematopoietic stem cell of an exception is derivative, then the treatment of other hematopoietic stem cell malignant clone diseases that allogene adoptive cellular immunotherapy can also be applied to including leukemia.Including erythrocyte disorder, such as aplastic anemia, paroxysmal nocturnal hemoglobinuria (PNH) etc.；Leukocyte disease, such as various leukemia, malignant lymphoma, malignant lymphatic-Reticuloendotheliosis, plasma cell dyscrasia, histiocytosis disease, myelodysplastic syndrome, myeloproliferative disease (polycythemia vera, essential thrombocythemia, myelofibrosis) etc..
Being described in detail the specific embodiment of the present invention above, but it is intended only as example, the present invention is not restricted to particular embodiments described above.To those skilled in the art, any equivalent modifications carrying out the present invention and replacement are the most all among scope of the invention.Therefore, the impartial conversion made without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.
Claims (11)
1. the method prepared for allogene adoptive cellular immunotherapy disease in the blood system medicine, it is characterised in that step includes:
Step 1, is placed in immunocyte and antigen co-cultivation in culture vessel in liquid cellular incubation base, thus obtains the specific immune cell culture with anti-corresponding antigens；Wherein, described immunocyte is selected from variant cell；
Step 2, separates the cell class bio-pharmaceutical with immune response from step 1 in the culture obtained；
Wherein, in described liquid cellular incubation base, in addition to immunocyte, also include component a)～e):
A) cell mitogen is former, any one or a few in ConA, phytohaemagglutinin, phytolacca american, lipopolysaccharide, glucosan, anti-cd 3 antibodies；
B) antigen: described antigen is hematopoietic stem cell exception clone cell；
C) superantigen, any one or a combination thereof in exogenous superantigen and endogenous superantigen；Exogenous superantigen is bacterial endotoxin, and endogenous superantigen is selected from any one or a few in retrovirus, heat shock protein；
D) cytokine；
E) immunological adjuvant.
Method the most according to claim 1, it is characterized in that, cytokine is selected from: derive from lymphocyte, lymphokine that mononuclear cell and other cells produce, monokine, the cytokine of activation inflammation and any one or a few in stimulating the cytokine of hemopoietic.
Method the most according to claim 1, it is characterised in that culture fluid is selected from any one in basic culture solution increase serum or serum-free medium.
Method the most according to claim 1, it is characterised in that described immunocyte is selected from without any one of gene-modified immunocyte or gene-modified immunocyte.
5. according to the method described in claim 1 or 4, it is characterized in that, described in there is the cell of immune response be selected from: tumor infiltrating lymphocyte, Tumor-infiltrating lymphocytes, natural killer cell, tumor-associated macrophages, lethal mononuclear cell, cytotoxic T cell and/or the dendritic cell of activation.
Method the most according to claim 1, it is characterised in that be additionally added antigen presenting cell in step 1.
Method the most according to claim 1, it is characterised in that cell culture container is three-dimensional large volume concentration cultivation container.
Method the most according to claim 1, it is characterized in that, also include cell clone step: with the culture obtained in step 1, or the cell with immune response obtained in step 2 is raw material, described culture medium is cloned, obtains the cell strain with immune response.
9. the cell class bio-pharmaceutical for adoptive cellular immunotherapy disease in the blood system that prepared by a method as claimed in claim 1, it is characterised in that described disease in the blood system includes: erythrocyte disorder, leukocyte disease.
Bio-pharmaceutical the most according to claim 9, it is characterised in that described erythrocyte disorder includes: aplastic anemia, paroxysmal nocturnal hemoglobinuria；Described leukocyte disease includes: leukemia, malignant lymphoma, malignant lymphatic-Reticuloendotheliosis, plasma cell dyscrasia, histiocytosis disease, myeloproliferative disease.
11. 1 kinds of biological preparation for allogene adoptive cellular immunotherapy disease in the blood system, it is characterized in that, including container, and it is placed in the cell class bio-pharmaceutical for adoptive cellular immunotherapy disease in the blood system described in container, claim 9.
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