CN107904259A - A kind of AIDS for the treatment of merges CAR T cells of lymthoma and its preparation method and application - Google Patents
A kind of AIDS for the treatment of merges CAR T cells of lymthoma and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of preparation method treated AIDS and merge the CAR T cells of lymthoma, and, using CD8+T cells production CAR T cells, the source of the CD8+T cells is cord blood T cells, is included the following steps for it：Cord blood mononuclear cells are made from Cord blood, tumour and other cells are removed using human T-cell's purification kit, obtain T cell, and CD8+T cells are obtained using Beads enrichment；Utilize suitable culture medium and incentive condition activation CD8+T cells；CAR is transferred to CD8+T cells using slow virus, prepares CAR T cells；Using cell factor and activation stimulating factor, amplification in vitro is carried out to CAR T cells, reaches required effective dose.The CAR T cells prepared the invention further relates to a kind of above method and its application.CAR T cells prepared by the present invention, its cytoactive is good, growth rate is fast, reduces the risk that GVHD occurs；To the cord blood T cells for aids patient, CAR T cells only are prepared using CD8+T cells, reduce the CAR T of input by the risk of internal HIV infection.
Description
Technical field
The present invention relates to cellular immunotherapy technical field, more particularly to a kind of CAR- for treating AIDS and merging lymthoma
T cell and its preparation method and application, it is specially that a kind of Chimeric antigen receptor CAR is transferred to cord blood T cells and is built into inosculating antibody
The preparation method of original receptor T cell (CAR-T) and its cell preparation.
Background technology
Tumour is captured using immunological therapy means, is always the important side that immunology is applied in terms of translational medicine
To.With the development of various groups (genomics, protein science etc.), tumour cell is obtained due to the immunogenicity that mutation produces
Extensive accreditation, this has established the basis of theory for immunotherapy of tumors.Meanwhile as tumor immunology studies the product of itself
Tired, immunotherapy of tumors achieves huge progress in the recent period, and a series of new immunotherapeutics step into clinic.Current
Tumor immunology research, has established T cell and has killed the Central Position in immunotherapy of tumors, and CAR-T cells are exactly to combine
The targets identification of antibody and the tumor-killing function of T cell, the tumor-killing cell of manually modified generation.
The embedding and concept of antigen receptor T cell most proposed that they will know early in 1989 by Gross, Waks and Eshhar
The antibody expression of other TNP realizes antigentic specificity, non-MHC limitation the activation of T cell and the increasing of effect in T cell
By force, and the concept of application of the CAR-T technologies in oncotherapy is proposed.According to this principle, by with tumour-specific
Antibody is embedded in T cell, by assign T cell it is new kill knurl ability.Afterwards, CAR-T technologies are introduced in antitumor clinical test,
But the CAR-T cells of early stage only contain the first signal since its intracellular signal transmits domain, and the tumor type selected is entity
Knurl, final clinical effectiveness are not ideal.In 2008, the mechanism such as Fred Hutchison institutes of oncology used CAR-T
B cell lymphoma is treated, although treatment results are not ideal, the key of this clinical trial be to confirm with
The B cell of expression CD20 is comparatively safe as the CAR-T treatments of target spot.Then, to report an example B in NCI in 2010 thin
The successful case of born of the same parents' lymphoma treating, using the CAR-T for CD19, the lymthoma of patient is controlled, normal B cells also by
Remove, serum I g is significantly reduced, and the validity that the lymthoma in B cell source is treated for CAR-T provides theoretical and actual branch
Hold.In 2011, the team of the Carl doctors June leader of Univ Pennsylvania USA was by specific recognition CD19's
CAR-T is used for the treatment of the chronic lymphocytic leukemia in B cell source, it is shown that the effect of " healings ", then in recurrence hardly possible
Clinical trial is unfolded in the property controlled acute lymphoblastic leukemia, also achieves the effect of good.Due to this breakthrough progress with
And immunotherapy of tumors has been chosen as the progress of technological breakthrough in 2013 by the development of other immunoregulation means, Science magazines
First place.This success causes wide influence in countries in the world, and various countries start largely to carry out the scientific research based on CAR-T
With the clinical test of oncotherapy.
The structure of CAR is by extracellular antigen recognizing domain, extracellular hinge area, the signal transduction domain of transmembrane region and intracellular
Composition.Extracellular antigen recognition domain is typically to form (such as anti-CD19, CD20, EGFR, HER2, EGFRVIII by single-chain antibody
Deng), the ligand or acceptor (such as NKG2D of specific recognition tumour cell membrane surface molecule or some tumour specific antigens
Deng) etc..Extracellular hinge area be one section be used for separate antigen recognition domain and the space structure of transmembrane region, its purpose is to provide conjunction
Suitable locus so that extracellular antigen recognition domain can maintain correct structure before and after antigen is identified and conduct intracellular letter
Number, generally select CD8a or IgG one section is used as hinge area.Transmembrane region is to ensure knot that CAR molecules are positioned in film surface
Structure domain, generally selects the equimolecular transmembrane region of CD8a, CD28, CD137 or CD3.Intracellular signal transduction domain is mediation CAR letters
Number conduction key component, be typically one or several first signals (such as CD3zeta), (costimulatory signal is such as secondary signal
CD28, CD137, ICOS, OX40 etc.) combination.First generation CAR comprises only the first signal CD3zeta, and second generation CAR has one
First signal CD3zeta and a secondary signal, third generation CAR have a first signal CD3zeta and two secondary signal knots
Structure domain.Except third generation CAR-T, there are other new CAR-T layout strategies at present, i.e., draw on the basis of second generation CAR-T
The new adjusting molecule independently of CAR is entered, such as cell factor IL-12, other costimulating factors 41BBL etc., further to increase
The function of strong CAR-T.
The CD4+T cells of inhibition of HIV main infection people, cause T cell quantity drastically to decline, and then cause patient's immunity
Decline, that is, AIDS.In aids patient colony, the incidence of Partial tumors is higher than non-aids patient, especially right and wrong suddenly
Strange gold lymthoma (NHL), Kaposi sarcoma and cervical carcinoma.Compared with non-aids patient, the non-Hodgkin lymphoma of aids patient
Incidence higher, the death rate is also high, and show in symptom it is more pernicious, usually when diagnosis finds tumour, in swollen
The knurl illness weight phase, this is also likely to be one of the reason for its death rate is high.The most common relevant lymthoma of AIDS is the big B of diffusivity
Cell lymphoma (DLBCL), mainly primary central nervous system lymphoma (PCNSL) and Burkitt lymphoma (BL), and
Lymphoma primary effusion (PEL), Hodgkin lymphoma (HL), plasmablastic lymphoma incidence are lower.Other types
As t cell lymphoma, follicular lymphoma are more rare.In non-Hodgkin lymphoma, most importantly diffusivity large B cell leaching
Bar knurl, much higher than non-aids patient 60 times of aids patient incidence.
The specific mechanism of the high incidence of HIV infection and these tumours is still not clear, and may have with patient's immunity degradation
Close.It is difficult using the immune system killing tumour of conventional immunotherapy regulation and control patient itself due to the immunity degradation of aids patient
Spend larger.Due to for B cell surface targeted molecular CD19, the non-Hodgkin lymphoma of the CAR-T of CD20 in non-aids patient
Relative maturity in treatment, still plan CAR-T and apply in the Case treatment that AIDS merges lymthoma.
All it is to utilize CAR weights although the CAR-T treatments of aids patient and non-aids patient do not have essential distinction in principle
The new T cell that is oriented to makes its killing tumor cell, but due to the special circumstances of patient's HIV hypoimmunity, is treated in CAR-T real
There may be following aspect to pay attention in applying:1) can aids patient utilize CD8+T cells or NK cells production CAR-T or CAR-
NK, because CD4+CAR-T has by the risk of HIV infection, or removes CD4 or HIV co-receptors CCR5 using the method for gene editing
And the expression of CXCR4；2) itself hypoimmunity of aids patient, for the lymthoma in B cell source, it is thin further to delete B
Whether born of the same parents can cause the increase of opportunistic infections；3) physical condition of aids patient is weaker, for cell caused by CAR-T treatments
Whether factor storm and tumor lysis syndrome are resistant to lower and corresponding processing mode.
But on CAR-T cells are applied in the treatment that aids patient merges lymthoma, still suffer from problems with：
(1) blood of aids patient requires harshness, company is existing due to carrying inhibition of HIV to operating environment and condition
Condition is unable to reach requirement, although the qualification for having only a few mechanism that there is processing to carry virus spread blood, can not meet to face
The preparation of bed demand, therefore be badly in need of seeking the blood source beyond aids patient autoblood as T cell source；
(2) physical condition of aids patient is weaker, for cytokine storm and tumor lysis caused by CAR-T treatments
The tolerance of syndrome is lower, and Autologous T cells can not use, and the T cell of allosome donations needs that distribution type reaches half-matched and the above just may be used
Use, and it is possible to occur graft-versus-host reaction (GVHD) risk, therefore be badly in need of seek the low T cell of immunogenicity
As the T cell source for preparing CAR-T, the risk that GVHD occurs is reduced as far as possible.
(3) CAR-T prepared by patient's autogenous cell can only be used to me, due to individual difference, the cell of some patients
State is poor, prepares dosage and is unable to reach effective dose, causes feedback effect bad, if Healthy People is contributed, needs looking for qualified
Distribution type, process is very long easily to miss therapic opportunity.Therefore it is contemplated that the T cell source for seeking low immunogenicity.
(4) since CD4+CAR-T has by the risk of HIV infection, and cannot determine whether that following scheme can be used：For
Whether aids patient is inputted using CD8+T cells production CAR-T, or using the method removal CD4+T of gene editing with reducing
CAR-T also caused the risk of aggravation by internal HIV infection.
The content of the invention
The defects of it is an object of the invention to overcome in the prior art, by using cord blood T cells as preparing CAR-T's
T cell source, prepares CAR-T cells, the immunogenicity of bleeding of the umbilicus is low, is resistant to a greater extent especially with CD8+T cells
HLA distribution type be not inconsistent, the CAR-T of input is reduced by the risk of internal HIV infection using CD8+T cells, it is using external
Conversion expands corresponding CAR-T cells, and CAR-T should in the relevant malignant tumour of aids patient, the particularly tumour in B cell source
This has the prospect of light, also provides new thinking and direction for the relevant treating malignant tumor of aids patient.
The CAR-T for B cell surface targeted molecular CD19, CD20 prepared using patient's autoblood cell is thin in B
Application in born of the same parents' treatment of hematologic malignancies relative maturity, and special as being in a bad way for some, cell quality it is very poor or
AIDS merges the patient of lymthoma, autoimmune cell is not easy to as the T cell source for preparing CAR-T, and bleeding of the umbilicus is exempted from
Epidemic disease source property is low, and caused graft-versus-host reaction (GVHD) is small, and growth rate is fast, therefore uses cord blood T cells to prepare CAR-T and answer
In treatment for B cell source malignant hematologic disease.
To achieve the above object, the present invention adopts the following technical scheme that：
First purpose of the present invention is to provide a kind of CAR-T cells, and the CAR-T cells are thin as T using cord blood T cells
Born of the same parents source, the CAR-T cells prepare CAR-T cells using the extremely low cord blood T cells of immunogenicity, and cytoactive is good, propagation is fast
Degree is fast, for the treatment of B cell source malignant hematologic disease, reduces the risk that GVHD occurs, and can prepare on a large scale, should
For different patients, cost is saved.
Second object of the present invention is to provide a kind of preparation side treated AIDS and merge the CAR-T cells of lymthoma
Method, for the preparation method using CD8+T cells production CAR-T cells, the source of the CD8+T cells is cord blood T cells.
In order to further optimize above-mentioned preparation method, technical measures of the invention further include：
Further, the cord blood T cells reject CD4+T cells
Further, which includes following preparation process：
1) immunophenotyping is carried out to patient's tumour cell, confirms its surface expression；
2) Cord blood mononuclear cells are made from Cord blood, removes tumour using human T-cell's purification kit and other is thin
Born of the same parents, obtain T cell；CD4+T cells are rejected using T cell screening reagent, and CD8+T cells are obtained using Beads enrichment；
3) suitable culture medium and incentive condition activation step 2 are utilized) the CD8+T cells that obtain；
4) CAR is transferred to using slow virus by CD8+T cells according to the surface expression of step 1) tumour cell, prepares CAR-
T cell；
5) using cell factor and activation stimulating factor, amplification in vitro is carried out to CAR-T cells prepared by step 4), is reached
Required effective dose.
Further, the preparation method of the Cord blood mononuclear cells in the step 1) is as follows：
A) gather, screen volunteer's Cord blood；
B) Cord blood is centrifuged, obtains blood plasma, and by volume 1:0.9~1.1 adds DPBS or physiological saline
It is diluted；
C) lymphocyte separation medium is added into the diluted blood plasma, centrifuges, take mononuclear cell layer to be cultivated, will train
Foster thing centrifuges again, and precipitation is the Cord blood mononuclear cells.
Further, the inboard wall of test tube of collection Cord blood sets anti-coagulants in the step a), prevents umbilical cord blood clotting.
Further, the lymphocyte separation medium is commercial product, the blood plasma and the separation of lymphocytes
The volume ratio of liquid is 1~1.5:1；The condition of the step b) centrifugations is 1000~1500rmp, 10~30min；The step c)
The condition of centrifugation is 1000~1500rmp, 10~30min, then the condition centrifuged is 1000~1500rmp, 5~15min, is used
The purity of the Cord blood mononuclear cells of this method extraction is higher.
Further, the surface expression of tumour cell includes at least one of CD19, CD20 in the step 1).
Further, the incentive condition in the step 2) includes at least one of anti-CD3, anti-CD28.
Further, amplification is to be based on anti-CD3, anti-CD28 and IL-2 or special using antigen in the step 5)
The aAPC and IL-7, IL-15 of the opposite sex.
Third object of the present invention is to provide a kind of CAR-T cells prepared according to the above method.
Fourth object of the present invention is to provide a kind of preparation of above-mentioned CAR-T cells, and further, the preparation also wraps
Include medicinal diluent or excipient.
The 5th purpose of the present invention is to provide a kind of above-mentioned CAR-T cells and is preparing treatment or prevention AIDS merging leaching
Application in bar tumor medicine.
The 6th purpose of the present invention is to provide a kind of therapeutic process of the clinical tumor using above-mentioned CAR-T cells, its
It is broadly divided into the following steps：Cord blood mononuclear cells are extracted from Cord blood；T cell is obtained by purifying, screening；T is thin
Born of the same parents' Activated in Vitro；T cell CAR channel genes；CAR-T cells expand；The lymphocyte of patient is removed, and is conducive to CAR- to create
The environment of T cell amplification；CAR-T cells are fed back in patient body.
Compared with prior art, the present invention is had the advantages that using above-mentioned technical proposal：
(1) using cord blood T cells as the T cell source for preparing CAR-T.It can be carried out under existing experiment condition
Prepare.Compared with the T cell of allosome adult's donations, there is its uniqueness using cord blood T cells as the T cell source for preparing CAR-T
Advantage：1st, bleeding of the umbilicus derives from a wealth of sources, and unbilical blood bank is being established in many cities in the whole nation at present, and gatherer process is simple, and donor is without any
No pain is damaged, can for a long time preserve, take at any time；2nd, bleeding of the umbilicus is protected by placental barrier, by virus, the probability of germ contamination
It is low；3rd, bleeding of the umbilicus includes abundant candidate stem cell, and more original, has stronger amplification and differentiation capability, is Adult Human Bone Marrow
Or 10-20 times of peripheral hematopoietic stem cells；Although the 4th, there are the problem of heteroplastic transplantation, the immunogenicity of bleeding of the umbilicus is low, is resistant to more
HLA distribution type in big degree is not inconsistent, and is more easy to find the donor that HLA matches, therefore GVHD is small, and cytokine storm risk is smaller,
It is good to feed back effect.CAR-T is prepared using the extremely low cord blood T cells of immunogenicity, cytoactive is good, growth rate is fast, thin for B
The treatment of born of the same parents source malignant hematologic disease, reduces the risk that GVHD occurs, and can prepare on a large scale, applied to different trouble
Person, saves cost.
(2) to the cord blood T cells for aids patient, CD4+T cells are rejected with T cell screening reagent, and are only utilized
CD8+T cells prepare CAR-T, reduce the CAR-T of input by the risk of internal HIV infection.The present invention first applies CAR-T
Merge the treatment of lymthoma in AIDS, since HIV infection causes the decline of patient's immunity, the non-Hodgkin's leaching of aids patient
Bar knurl incidence is higher than non-aids patient, and due to the CD4+T cells of inhibition of HIV main infection people, causes T cell quantity anxious
Play declines, and the immune system killing tumour difficulty using conventional immunotherapy regulation and control patient itself is larger, therefore it is thin to filter out bleeding of the umbilicus T
CD8+T cells in born of the same parents prepare CAR-T target killing B cell nausea neoplastic hematologic disorders, while reduce CD4+CAR-T and felt by HIV
The risk of dye.
Brief description of the drawings
Fig. 1 and Fig. 2 is the preparation of CAR-T cells and the schematic diagram of therapeutic progresses in one embodiment of the invention.
Embodiment
The present invention provides a kind of preparation method treated AIDS and merge the CAR-T cells of lymthoma, it is thin using CD8+T
Born of the same parents produce CAR-T cells, and the source of the CD8+T cells is cord blood T cells；It is thin that wherein described cord blood T cells reject CD4+T
Born of the same parents.The application of the preparation, above-mentioned CAR-T cells that are prepared the invention further relates to above-mentioned CAR-T cells.
With reference to the accompanying drawings and examples, the embodiment of the present invention is further described.Following embodiments are only
For clearly illustrating technical scheme, and it is not intended to limit the protection scope of the present invention and limits the scope of the invention.
Embodiment 1
The present embodiment is the preparation process of the CAR-T cells for being directed to CD19, and as depicted in figs. 1 and 2, its specific steps is such as
Under：
1) immunophenotyping is carried out to patient's tumour cell, confirms the expression of its surface molecular CD19, CD19- can be passed through
CAR-T is treated；
2) Cord blood mononuclear cells are obtained by leucocyte list harvest, it is comprised the following steps that：
A) gather, screen volunteer's Cord blood, wherein the inboard wall of test tube of collection Cord blood sets anti-coagulants, prevent Cord blood
Solidification, wherein selective mechanisms project include carrying virus or hereditary family history；
B) 25min is centrifuged in 1200rmp to the Cord blood of collection, obtains blood plasma, and by volume 1：1 adds DPBS or physiology
Brine is diluted；
C) commercially available lymphocyte separation medium (such as Ficoll separating liquids), wherein blood plasma are added into the diluted blood plasma
Volume ratio with lymphocyte is 1：1.5, after mixing 30min, 30min is centrifuged with 1200rmp, takes mononuclear cell layer to be trained
Support, culture is centrifuged into 15min again with 1000rpm, precipitation is the Cord blood mononuclear cells.
3) tumour and other cells are removed using commercially available human T-cell's purification kit, obtains T cell；Using commercially available T
Cell screening reagent rejects CD4+T cells, and obtains CD8+T cells using the separation of commercially available magnetic bead separation kit；
4) using suitable culture medium (such as 15 culture mediums of Lonzax-vivo) and incentive condition (be typically anti-CD3 with
Anti-CD28) activating T cell, makes it be more advantageous to the transfection of CAR genes and amplification；
5) CD19-CAR-T cell transfectings, using conventional technical means, carry out the preparation and titre detection of CAR carriers, profit
CAR is transferred to T cell with slow virus, CAR-T cells are made；Gene of the CAR gene integrations of lentivirus mediated to T cell
Group, is stable long-term expression, has potential tumorigenesis possibility in theory, but current clinical data does not observe that tumorigenesis shows also
As；
6) using cell factor and activation stimulating factor, amplification in vitro is carried out to CAR-T cells, reaches required effective
Dosage, current main amplification method is the method for being based on anti-CD3, anti-CD28 and IL-2, it is possible to use antigen-specific
Property aAPC (artificial antigen is in delivery cell) and other cell factor such as IL-7, IL-15 etc., and carry out CAR positive rates and work
Rate detects；
When the personnel that the cell is used for AIDS Complicated lymthoma treat, as depicted in figs. 1 and 2, its therapeutic progresses
It is as follows：
7) patient removes the lymphocyte of itself using whole body irradiation or chemotherapeutics, creates and is conducive to CAR-T amplifications
Environment；
8) the effective dose CAR-T cells for preparing embodiment 1 are fed back, in vivo propagation, play effect, and periodic monitor body
The amplification situation of interior CAR-T cells and the killing situation of tumour cell.
Wherein, Chlamydia, mycoplasma, endotoxin and microorganism in the above-mentioned CAR-T cells being made are detected,
It is feminine gender that Chlamydia, mycoplasma, endotoxin and microorganism, which are detected result, in obtained CAR-T cells；By made from
CAR-T cells carry out the detection of CAR expression rates, and the expression rate of CAR is more than 55% in obtained CAR-T cells.
Embodiment 2
The CAR-T cells made of Cord blood in embodiment 1 are killed in vitro with the CAR-T cells made of peripheral blood
Wound contrast, specific experiment step are as follows：
The first step：Calcein-AM marks target cell；
Calcein-AM is diluted to l mg/mL with DMS0；The full culture medium of target cell is resuspended into 1 × 106/ mL's is close
Degree；Add 15 μM of Calcein-AM, 37 DEG C, 5%CO230min is cultivated, is gently mixed per 10min；1500rpm is centrifuged, and is gone
Clearly, it is resuspended, is repeated twice with full culture medium；
Second step：Killing；
The target cell marked is resuspended according to the density of 5000-50000/mL, takes 100 μ L to be added in 96 orifice plates,
According to appropriate ET than adding 100 μ L effector cells, every group 3 parallel；Meanwhile have that single A groups 6 are parallel, and only target is thin
Born of the same parents (spontaneous release)；Have that single B groups 6 are parallel, only target cell+2%Triton X-100 (maximum
release)；
3rd step：
37 DEG C, 5%CO2Cultivate 4 it is small when after, centrifugation, take 75 μ L of supernatant, be transferred on a new culture plate；Sample profit
(excitation is detected with pectramax Gemini dual-scanning microplate spectrofluorimeter
fiIter：485±9nm；band-pass filter：530 ± 9nm), data are shown in the form of AFU；Calculate cell cracking
Percentage：[(test release-spontaneous release)/(maximum release-spontaneous
release)]×100。
Cord blood CAR-T killing abilities and general peripheral blood CAR-T killing abilities are concluded that according to above-mentioned experiment
It is basically identical.
Experimental example 3
The present embodiment is to verifying its security and validity in obtained CAR-T cell bodies.
First with the validity of model of AIDS mouse verification CAR-T cells, comprise the following steps that：
1) humanization mouse model of AIDS is built：After humanization mouse Scid-hu-Thy/Liv mouse anesthesias, exposure
Kidney peplos position graft, inhibition of HIV, which is directly inoculated into graft, replicates HIV-1 infection models, then screens concurrent lymph
Knurl (volume 100mm3Left and right) mouse as subjects.
2) cell culture；
T cell culture：
Count and measure the motility rate of T cell, be resuspended after centrifugation with physiological saline, adjust its concentration as 5 × 105A/mL, always
Number reaches 6 × 105It is a.
CAR-T cell culture：
The CAR percentage compositions of CAR-T cells are measured, counts and measures motility rate, be resuspended after centrifugation with physiological saline, are adjusted
(living cells and CAR is expressed as the positive, the CAR-T cells being previously mentioned in this experimental example, refer both to effective CAR-T for effective CAR-T cells
Cell) concentration and prepare CAR-T cells.
3) humanization mouse model of AIDS is administered.The record administration same day is D0, calculates the time on the inoculation same day.Pass through
The mode of tail vein injection is administered, all equal single-doses of mouse, and specific administration is as shown in table 1.
Specifying information is administered in table 1
| Group | Cell is administered | Administered volume (μ L) | Dosage (a/only) |
| Blank group | Without (PBS) | 200 | 0 |
| Negative control group | Cord blood T cell | 200 | 1×106 |
| Experimental group | Cord blood CAR-T cells | 200 | 1×106 |
Carry out related evaluating drug effect：
1) mouse is observed
From being bought mouse, the survival state of 4 weeks continuous mouse of observation daily, the stringent physique for monitoring animal to after being administered
Situations such as feature, unfavorable condition such as weight loss more than 10%, depilation, diarrhea, conjunctivitis and paralysis that record may occur.
It is observed that learn, no significant difference between three groups.
2) body weight determination
It is grouped at random after measuring mouse weight during packet；Mouse weight is measured respectively before inoculation and before administration；It is every after administration
All 2 measure mouse weights.Weight can reflect mouse health status and tumor proliferation situation.Learn after measured, three groups
No significant difference between not.
3) measurement of gross tumor volume
The gross tumor volume of measure mouse, measures the tumour major diameter and minor axis of mouse, thus counts 2 times a week before inoculation after inoculation
Calculate gross tumor volume.Gross tumor volume can reflect the proliferative conditions of mouse interior tumor.With vernier caliper measurement tumour major diameter and short
Footpath, the difference between difference and each dosage group and negative control group between more each dosage group, as a result blank group and
Negative control group tumor size becomes larger, and experimental group tumor size is constant or is slightly reduced reduction.
4) measure of survival time of mice
The survival state of observation mouse daily, when mouse tumor volume reaches 3000mm3Death is denoted as, after record inoculation
The time of every dead mouse and group and counted in 4 weeks, gross tumor volume is not up to 3000mm after 4 weeks3Then it is denoted as survival mice.
Count the mean survival time of every group of mouse.Life cycle reflects the proliferative conditions of mouse interior tumor.Use vernier caliper measurement
Tumour major diameter and minor axis, learn after measured, and relative to other two groups, CAR-T cell therapies group can significantly extend life cycle.
5) tumor weight measures
Gross tumor volume is not up to 3000mm3Mouse, D28 to mouse carry out intraperitoneal anesthesia, dissect and peel off its tumour
Tissue, measures its weight.Tumor weight reflects the proliferative conditions of mouse interior tumor.With electronics balance measurement mouse weight,
Retain an effective digital after decimal point.Learn after measured, tumor weight does not increase substantially after CAR-T cell therapies, and T is thin
Tumor weight increase, PBS groups also dramatically increase after the treatment of born of the same parents treatment group, i.e., experimental group is treated with blank group and negative control group
Effect has significant difference.
6) graft-versus-host reaction (GVHD)
In D10~D30 days, blank group occurs without graft-versus-host reaction；Negative control group (T cell) there are about 20%
There is graft-versus-host reaction, experimental group (CAR-T cells) only has 2% and graft-versus-host reaction occurs, this shows umbilical cord
CAR-T cells prepared by blood T cell reduce the risk that GVHD occurs, the wherein performance of graft-versus-host reaction mainly such as
Under：Fever, loss of appetite, diarrhea, abdominal pain, vomiting and liver damage；Skin lesion：It is initially slight diffusivity red macula
Or papule, there is gargalesthesia；Mucosal injury：Erythroplakia, oedema, erosion, ulcer are showed, with pain；Viscera：Visceral lesion
Can at the same time occur with mucocutaneous lesions or successively be individually present.
The tumour that the above results can be seen that CAR-T cells provided by the invention and can significantly inhibit in animal pattern is given birth to
It is long, possess the function for the treatment of AIDS Complicated lymthoma.
The specific embodiment of the present invention is described in detail above, but it is only used as example, and the present invention is not intended to limit
In particular embodiments described above.To those skilled in the art, it is any to the equivalent modifications that carry out of the present invention and to replace
In generation, is also all among scope of the invention.Therefore, the impartial conversion made without departing from the spirit and scope of the invention and repair
Change, all should be contained within the scope of the invention.
Claims (10)
1. a kind of preparation method of CAR-T cells, it is characterised in that produce CAR-T cells, the CD8+T using CD8+T cells
The source of cell is cord blood T cells.
2. the preparation method of CAR-T cells according to claim 1, it is characterised in that the preparation method includes following step
Suddenly：
1) immunophenotyping is carried out to patient's tumour cell, confirms its surface expression；
2) Cord blood mononuclear cells are made from Cord blood, removes tumour and other cells using human T-cell's purification kit, obtains
T cell is obtained, and CD8+T cells are obtained using Beads enrichment；
3) suitable culture medium and incentive condition activation step 2 are utilized) the CD8+T cells that obtain；
4) CAR is transferred to using slow virus by CD8+T cells according to the surface expression of step 1) tumour cell, it is thin prepares CAR-T
Born of the same parents；
5) cell factor and activation stimulating factor are utilized, amplification in vitro is carried out to CAR-T cells prepared by step 4), reaches required
The effective dose wanted.
3. the preparation method of CAR-T cells according to claim 2, it is characterised in that the Cord blood in the step 1)
The preparation method of monocyte is as follows：
A) gather, screen volunteer's Cord blood；
B) institute's Cord blood is centrifuged, obtains blood plasma, and by volume 1:0.9~1.1 addition DPBS or physiological saline carry out dilute
Release；
C) lymphocyte separation medium is added into the diluted blood plasma, centrifuges, take mononuclear cell layer to be cultivated, by culture
Centrifuge again, precipitation is the Cord blood mononuclear cells.
4. the preparation method of CAR-T cells according to claim 2, it is characterised in that tumour cell in the step 1)
Surface expression include at least one of CD19, CD20.
5. the preparation method of CAR-T cells according to claim 2, it is characterised in that the stimulation bar in the step 2)
Part includes at least one of anti-CD3, anti-CD28.
6. the preparation method of CAR-T cells according to claim 2, it is characterised in that amplification is base in the step 5)
In anti-CD3, anti-CD28 and IL-2 or the aAPC and IL-7, IL-15 using antigentic specificity.
A kind of 7. CAR-T cells prepared according to method according to any one of claims 1 to 6.
A kind of 8. preparation of the CAR-T cells containing described in claim 7.
9. preparation according to claim 8, it is characterised in that the preparation further includes medicinal diluent or excipient.
10. a kind of CAR-T cells as claimed in claim 7 are in preparing treatment or prevention AIDS and merging lymphoid tumor medicament
Using.
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