CN1761475A - Leukocyte cell banks - Google Patents

Abstract

The invention relates to a novel form of leukapheresis (isolated leukapheresis), to processes and apparatus for carrying out isolated leukapheresis, to leukocyte cell banks created thereby and to various forms of therapy based thereon.

Description

Leukocyte cell banks

Technical field

The present invention relates to new leukocyte list and adopt form (isolating leukocyte list is adopted), implement the method and apparatus that isolating leukocyte list is adopted, consequent leukocyte cell banks (leukocyte cell bank) and the various forms of treatments of carrying out based on them.

Background technology

Cell bank

The cell bank industry is a kind of service occupation, and it is preserved living cells for future use.This industry has many decades history, for example stores the spermatid of cattle so that cow is carried out the artificial fertilization.

Along with the technological progress of biomedical research and field of tissue engineering technology, people recognize that carrying out multiple alternative medicine with human stem cell has multiple probability.These progress cause more and more needing to separate the stem cell of individuality the facilitated method of also frozen standby (using from body).For example, increasing people has recognized that this class stem cell preservation thing increases fast in the privately owned cell bank of result to storing the demand of umbilical cord hemocytoblast.

Along with the demand of pair cell and tissue constantly increases, the cell bank industry is recognized practical problem gradually.Known at present, the cell bank that aims to provide long-term cell source is easy to be subjected to stochastic events, cause part or all to preserve thing and lose activity, and cell bank is big more, and period of storage is long more, and the risk relevant with this random event is just big more.The integrity of preserving thing is also most important: preserve the decisive action that integrity that preparation, preservation, operation and the occupation mode of thing might the pair cell storehouse plays key: this point, for the cross-contamination of preserving thing can cause transmission of disease cause improperly or dangerous physiology consequence (as, might be owing to when autotransplantation, given the allos cellular material) situation be even more important.For bigger cell bank, information storage, processing and preservation thing classification catalogue also are extremely important.

This class problem makes many countries put into effect increasing laws and regulations, manage cell bank generation, keep and use: at present in Britain, the cell bank industry is by an integrated management organizational controls.

Should acute autotransplantation (CAT) therapy

Recently document description has been arranged a kind of therapy (seeing WO 00/29551 and WO 01/88099), it takes out various tissues (comprising leukocyte) from healthy, they are stored in tissue or the cell bank, when in the future some day emergency taking place and to carry out autotransplantation, can carry out autotransplantation with them.This therapy is referred to herein as should acute autotransplantation (contingent autologous transplantation, CAT) therapy.

For any concrete tissue or cell type, only some healthy population may need the CAT therapy.Therefore, the CAT therapy whether effectively, key depends on whether can produce comprehensive type cell tissue storehouse, comprise the preservation thing from most of people in this colony in the described storehouse.

Therefore there is viewpoint to think, will helps the CAT therapy by making up comprehensive tissue bank.But the character of CAT therapy itself has determined it that any this class tissue bank is had its unique and strict requirement.Particularly, the CAT therapy require to have a large amount of blood donors to participate in (thereby a large amount of preservation things are arranged), relatively long storage life arranged, in long-time well maintained tissue function and in final treatment application, excellent adaptability is being arranged.

For leukocyte cell banks, these problems are especially urgent, because the absolute quantity of the cell that leukocyte cell banks can provide is fewer, the function that might key depends on a small set of cell is renderd a service in final treatment, and because different cells adapts to storage by different way, the leukocytic activity of storage might change as time passes.Up to now, still nobody constructs the leukocyte cell banks that is fit to CAT.

Adopt the leukocyte that separation can be used for the CAT therapy by the leukocyte list

The leukocyte list is adopted the blood sampling art (apheresis) that (Leukapheresis) is a kind of specific form, relates to from the blood that obtains Selective Separation and takes out leukocyte, and the remainder with blood feeds back to the blood donor then.Adopt in the process at the leukocyte list, the blood flow that makes taking-up is through cell separation apparatus, and this device will have nuclear leukocyte and erythrocyte and separating plasma external.Erythrocyte and blood plasma are fed back to the blood donor, and this also is the part of separation process.This process is carried out continuously, constantly has blood to be removed and almost is to turn back in the body after having carried out various extractions simultaneously.Therefore, the leukocyte list is adopted and is made and whole blood of blood donor might be taken out several times and feed back, and isolates and preserve a large amount of leukocyte under the situation that does not injure this person.This technology thereby depend on and can set up a kind of vein to the venous extracorporeal circulation of blood, and from this circulation blood, extract leukocyte.

It generally all is automatic that the leukocyte list is adopted, centrifugal or filtering technique carries out with fluid successive or that be interrupted, can be referring to " Leukapheresis and Granulocyte Transfusions ", by AmericanAssociation of Blood Banks, Washington DC (1975) publishes.

Implement device description to some extent in US 3489145 and US 3655123 that centrifugal leukocyte list is adopted, implement device description to some extent in US 3802432 and US 3892236 that filtering type leukocyte list is adopted.Gravity type leukocyte list is adopted (gravity leukapheresis) and is only utilized gravity to implement leukocyte separation and collection, and this method is described in US 4111199 to some extent.

The automatic leukocyte list device for picking that can be purchased at present has number of different types, comprise: FenwalCS-3000 (Baxter Healthcare, Chicago, IL), Cobe 2997 (Cobe BCT, Lakewood, CO), Cobe Spectra, Cobe 2991, and Haemonetics V50 (Haemonetics Corp., Braintree, MA).Wherein any may be used to the present invention for this, but preferred Cobe  system (CobeBCT, Lakewood, CO, USA), it can extract the 40%-50% of total white blood cells from the whole blood of its separator of flowing through, and the flow velocity of its per minute can reach 40-60ml or bigger.

The someone proposes in the recent period, adopts with the leukocyte list and creates the used B lymphocyte repertoire (seeing WO00/29551 and WO 01/88099) of CAT therapy.But adopt the restriction that the business-like comprehensive type B lymphocyte repertoire of generation is subjected to blood donor's convenience and comfortableness with the leukocyte list.For example, the blood donor is limited at time that blood collection mechanism carries out blood supply of can visiting usually.Therefore, in a single day the blood donor arrives blood sampling mechanism, and it is a key factor in Blood Sampling Room institute's time spent really.But the leukocyte list is adopted generally needs 2-4 hour (than the required time long number of the blood that a unit is provided doubly).Bring the problem of blood donor's comfortableness thus: a lot of people feel to fear very much when seeing real blood collection procedure, and the blood donor must insert (normally two at a syringe needle on one's body at least in whole process, be used for drain and feed back blood), bring potential danger thus.

Therefore, need a kind of method for preparing leukocyte cell banks, it can avoid the problem that occurred when using the leukocyte list to adopt, can also make blood donor more convenient (and sense of discomfort is less), thereby might create the comprehensive type leukocyte cell banks that is applicable to the CAT therapy.

Summary of the invention

The present invention be based on (to small part based on) below find: the blood sample of gathering from the blood donor with conventional method, the processing of the leukocyte list device for picking that process can be purchased can be built the leukocyte source of providing convenience in the storehouse for leukocyte: need not blood donor's " line " is used these devices.

By the leukocyte preparation of this " isolating leukocyte list is adopted " method preparation, can be used directly in the treatment of various diseases and carry out autotransplantation, perhaps after being carried out various processing, leukocyte is used further to described autotransplantation.

Therefore, first aspect present invention provides isolating leukocyte list to adopt, wherein leukocyte list device for picking and provide between the individuality of blood sample and do not have fluid communication and/or remaining blood sample not to be fed back to this individuality.

The preferably automatic leukocyte list device for picking of leukocyte list device for picking.Especially preferred fluid centrifugal leukocyte list device for picking or the filtering flow formula leukocyte list device for picking successive or that be interrupted that uses successive or interruption.

Leukocyte list device for picking can comprise: (a) segregation apparatus (for example, centrifugal rotor or filter); (b) the leukocyte list is adopted tubing (tubing set); And (c) one or more pump, described pump is used for the blood sample transportation is transported to collection container by described plumbing installation with isolating leukocyte.

Described method is preferably further comprising the steps of:

(a) make the leukocyte dormancy (for example, preserve (cryogenicpreservation) and make it dormancy) that collects by profound hypothermia; With, alternatively

(b) leukocyte of dormancy is recovered (for example, making it recovery) by thawing and/or diluting.

If use profound hypothermia to preserve, for simplicity, this profound hypothermia is preserved and is comprised and be refrigerated to approximately-160 ℃ or low temperature more, and this can realize with liquid nitrogen.Long-time if desired the preservation and/or reservation function better can be refrigerated to approximately-269 ℃ or low temperature more with liquid helium.

Extensively multiple suitable profound hypothermia is preserved in the medium, anyly may be used to the present invention, preferably comprises the medium of suitable penetrance cryoprotective agent.That be especially suitable for use as the penetrance cryoprotective agent is DMSO, its working concentration, such as, can be up to 10%.

Profound hypothermia is preserved medium and can also be comprised: anticoagulant (as acid acid citrate dextrose, EDTA, heparin, or their mixture), nuclease (for example DNA enzyme and/or RNA enzyme) and physiology acceptable medium (for example, phosphate buffer).Profound hypothermia is preserved medium can also comprise proteinaceous compositions, as serum or serum composition and/or sugar (sugar) and/or polysaccharide (polysaccharide) (they are particularly preferred in the embodiment that adopts submersible type freezing (plunge freezing)).

Preserve the particularly preferred proteinaceous compositions of using in the medium at profound hypothermia of the present invention and comprise blood albumin (for example, bovine serum albumin or human serum albumin).Especially easily be to use isolating human serum from blood donor's blood sample.It can be used as the isolating by-product of leukocyte and separates with leukocyte.

Second aspect present invention provides and has been used for from the blood sample Selective Separation and the purposes of taking out leukocytic leukocyte list device for picking, described blood sample and provide between the blood of individuality of this blood sample and do not have fluid communication (that is, being isolating blood sample as herein described).

Third aspect present invention provides preparation to be used for the method for autoplastic leukocyte compositions, may further comprise the steps: the isolating blood sample from blood donor's individuality (a) is provided; (b) with leukocyte list device for picking Selective Separation and collect leukocyte from this blood sample.

Fourth aspect present invention provides preparation to be used for the method for the leukocyte compositions of restorative autotransplantation (restorativeautotransplantation), may further comprise the steps: the isolating blood sample from blood donor's individuality (a) is provided; (b) with leukocyte list device for picking Selective Separation and collect leukocyte from this blood sample; (c) make the leukocyte dormancy (for example, preserve (cryogenicpreservation) and make it dormancy) of collecting by profound hypothermia; With, (d) makes the leukocyte recovery (for example, making it recovery by thawing and/or diluting) of dormancy alternatively.

Fifth aspect present invention provides the method for the leukocyte compositions of preparation being used for the treatment of property autotransplantation (remedialautotransplantation), may further comprise the steps: the isolating blood sample from blood donor's individuality (a) is provided; (b) with leukocyte list device for picking Selective Separation and collect leukocyte from this blood sample; (c) handle collected leukocyte; With, (d) makes treated leukocyte dormancy (for example, making it dormancy by the profound hypothermia preservation) alternatively.

Sixth aspect present invention provides the method for preparing leukocyte cell banks, may further comprise the steps: the isolating blood sample from blood donor's individuality (a) is provided; (b) with leukocyte list device for picking Selective Separation and collect leukocyte from this blood sample; (c) make the leukocyte dormancy of collecting (for example, make it dormancy by the profound hypothermia preservation), and with these step repeated application in a series of blood samples from different blood donor's individualities, thereby the leukocyte compositions for preparing many parts of dormancy (for example profound hypothermia preservation), described method is further comprising the steps of: (d) leukocyte of dormancy is preserved in the mode that can recover, in order to autotransplantation in the future.

Seventh aspect present invention provides the system's (for example, closed system) that is used for collecting from individuality isolating blood sample, and described system comprises shuttle and the leukocyte list is adopted tubing.

Therefore, the invention provides the system's (for example, closed system or functional closed system) that is used for collecting from individuality isolating blood sample, described system comprises: (a) sampling apparatus (for example, comprising syringe needle), to be used for from the described individual blood sample of collecting; (b) between the shuttle, itself and described sampling apparatus fluid communication is arranged; (c) the leukocyte list is adopted tubing, between itself and the described shuttle fluid communication is arranged; Wherein said tubing is a cecum, does not comprise the device that is used for any part of passing through the sample of fractionated is drawn back again described individuality.

Eighth aspect present invention provides and has been used for from from the isolating blood sample Selective Separation of individuality and take out leukocytic device, and described device comprises the leukocyte list device for picking that has gathering system of the present invention.

The invention still further relates to the leukocyte compositions and the leukocyte cell banks that can obtain (or having obtained) by method of the present invention.

The invention still further relates to the various therapeutic use of the method for the invention, system, device, compositions and cell bank.Correspondingly, the present invention relates to the purposes of leukocyte compositions of the present invention in treatment, for example the purposes in autotransplantation (for example restorative or therapeutic autotransplantation).

Detailed Description Of The Invention

I. Definition

When following term uses in this article, except any wideer (or narrower) implication that these terms may have in this area, unless otherwise specified, all be intended to the following implication of expression:

Term " the leukocyte list is adopted " is a term of art used herein, is used to define a kind of method, and it relates to from available from Selective Separation blood donor's the blood with take out leukocyte, give described blood donor with remaining blood recovery again.

Term " leukocyte list device for picking " is a term of art, is used to define anyly can carry out the device that the leukocyte list is adopted, no matter what mode this device adopts separate and take out leukocyte.

Term " isolating leukocyte list is adopted " is meant isolating blood sample is implemented a kind of new model that the leukocyte list is adopted with in this article.

Similarly, term " isolating blood sampling art " is meant a kind of new model of isolating blood sample being implemented the blood sampling art with in this article.

Term " isolating blood sample " is with in this article, is meant and provides the blood sample that does not have fluid communication between blood donor's the blood of this blood sample.Therefore leukocyte list device for picking and provide between the individuality of this blood sample and do not have fluid communication and/or residue blood sample not to be fed back in this individuality body, isolating blood sample is provided in the process that isolating leukocyte list adopts.

Term " autotransplantation " is meant isograft or self-transplanting with in this article, and wherein term " from body " is used for expression, and transplanting is the transplanting of carrying out to the identical biology that cellular material (for example leukocyte) is provided (being same individual)." transplanting " is meant any relating to the process in cellular material (for example leukocyte) the introducing biology, so this term comprises any type of transplanting known in the art.

Term " dormancy " is meant any dormant state or resting state with in this article, and the method that reaches this state is known in the art, and description is hereinafter arranged.Any known method comprises the profound hypothermia preservation, can use.Therefore, leukocyte can maintain quiescent condition, non-activity state or nonproliferative state.

Term " health " uses with blood donor's individuality in this article, represents that this individuality does not have leukocyte defective (according to the definition of this paper).Therefore, term " health " comprises blood donor's individuality of non-disease conditions at this paper, and this blood donor's individuality is in the state of any symptom (that is, being asymptomatic or subclinical performance) of not suffering from any disease or imbalance or not showing described disease or imbalance.Especially, term " health " comprises not suffering from blood donor's individuality of the symptom of wanting the disease for the treatment of by Method for autologous transplantation subsequently or not showing described disease in this article.

II. Blood sample

The present invention can be applied to any type of blood sample, as long as: (a) this blood sample is the isolating blood sample according to above-mentioned definition, and (b) this blood sample comprises at least a portion leukocyte of blood donor's individuality.

Can before being applied to method of the present invention, blood sample carry out various processing to it at external (ex vivo).For example, usually before use with blood sample cold preservation (chill).Other processing can comprise adds preservative agent and/or anticoagulant.

Also can before blood sampling or during the blood sampling various preparations be applied to blood donor's individuality, thereby at blood sampling pre-treatment blood sample.

Can see hereinafter the chapters and sections that are entitled as " leukocyte processing " for details at the example of processing external and/or that use in vivo.

General preferably gather 450-500ml blood at least from blood donor's individuality, this is equivalent to the blood volume of (UK blood transfusion service) unit that the blood donor provided in Britain's blood transfusion service.As possible, (for example, 2-3 is in week, in preferred 2-3 month or in 6 months or in 1 year, in 2 years or in 3 years or in the longer time) repeatedly blood sampling (for example, a plurality of 450-500ml) in a period of time.In these blood samples one or more can be divided into or be mixed into a plurality of leukocyte cell banks and preserve things.Only in Britain, annually collect the blood that surpasses 300 ten thousand units from the blood donor, it is very usual that the blood that therefrom takes out a unit is used for allograft.

The blood that takes out is very fast can be replenished (replaced), therefore can repeatedly gather the blood of a unit in 1 year from blood donor's body, in case of necessity, gathers 2-12 unit, and individual harmless to what taken a blood sample.

III. Select blood donor's individuality

General consideration

Restorative autotransplantation is that a kind of possible definitive application is in the therapy of any individuality.Therefore, the present invention can be effective to prepare the complete leukocyte cell banks that comprises Different Individual as much as possible, so that can carry out restorative autotransplantation to any specified individual whenever necessary.

For these reasons, consider the present invention is used as far as possible widely, so that can set up complete leukocyte cell banks.But depend on (being somewhat dependent upon at least) this blood donor's individuality in the health status of donating blood at that time because each part preserved the quality of thing, so the blood sample that is preferred in the inventive method is gathered from healthy is individual.

Also have other factors also to influence selection to the blood donor: for example, the blood sample that is used for the inventive method is preferably gathered more favourable when blood donor's individuality is still young, preferably at adolescence (adolescence) or grow up early stage (early adulthood).For the people, preferably in the blood sampling (preferably repeatedly blood sampling) of about 12-30 year, preferred 15-25 year.Especially preferably 16 or time blood sampling more than 17 years old, for example, 16-30 year, 17-30 year, or 18-30 year maybe can be 18-35 or 40 years old.Therefore, preferably in the host living beings maturation, or near period of maturation (maturity), but do not obtain its cell before obviously entering aging (ageing) or old and feeble (senescence) process.Especially, comparatively favourable when the immune system maturation of preferred host living beings or zoon.

But, the time that the present invention is also included within beyond the above-mentioned age bracket obtains cell, can obtain cell in any time of firm birth back (post-natal), for example the host living beings from juvenile period (juvenile) obtains cell, for example from the childhood period middle and advanced stage (mid-to late childhood) or even obtain cell from baby (infant), or obtain cell from the individuality of old (older).

From firm birth back or old host take a sample and allow to collect multiple sample, thereby increased the chance of the cell that accumulates sufficient amount.In addition, can cross moral claim (ethical requirements), inform and requirement through agreeing to take a sample as needs from juvenile host or old host sampling.

Preferably from the sampling of adolescence or adult host living beings, because the cell that is obtained, the especially cell that obtains from blood, will comprise the valuable mature T cells of larger proportion, they can discern abnormal cell colony, as cancerous cell or by the cell of viral infection.So, when using blood sample, more favourable from the blood sample that the individuality (promptly not being fetus (foetal) or neonate (neonatal)) with maturation immunity system obtains.

Therefore, the present invention relates to take place in the future or unexpectedly take place or only be purposes the occurrent incident from the individual blood sample of collecting of blood donor, described blood sample is not obtain when blood donor's individuality does not exist the direct indication that may suffer from disease, hint or suspects, and described future, incident was not the disease of having predicted or having suspected or indicated.Therefore, in some embodiments of the present invention, described blood donor's individuality is to any disease specific susceptible, or do not suffer from the danger of any disease specific, for example do not have any the indicate symptom or the performance of disease subsequently.Similarly, host living beings does not preferably suffer any damage that may cause the precognition disease.

In fact, for some application (for example, prepare leukocyte cell banks in order to carry out restorative autotransplantation subsequently), the blood sample that preferred the present invention uses obtained from this individuality before blood donor's individuality is suffered from any disease or related symptoms occurred, was more preferably at host living beings to be in good generally health level, and to obtain during preferably without any the immunocompromised host (immunocompromise) of form.In these embodiments, do not occur disease symptoms in advance or have disease or be diagnosed as suffering from disease and this disease when being the disease that to receive treatment subsequently at blood donor's individuality, that is to say at this host living beings and be in health status and be not that " after being ill recover " state (for example, be not in from the leukocyte defect part ground that will be treated or the state that fully recovers) time, the blood sample advantageous particularly obtained from this individuality.

(Predisposed) blood donor individuality of susceptible

Treatment means is constantly progressive, and also more and more deep to the understanding of lysis, this helps us to adjust orientation treatment, thereby palliates a disease and misery.Such understanding is advanced greatly owing to the technological progress of biology field.We can follow the tracks of the pathogeny of disease now on molecular level, and recognize, individual gene structure plays an important role aspect the susceptibility of some disease at them.For example, we are known, and some are individual because the relation of their genomic constitution is easy to take place some disease.

Disease majority for some individual susceptibles is the leukocyte defective, and this term is represented to use from the leukocytic situation of body in this article.Therefore this class situation comprises: individuality has been suffered from and leukocyte function imbalance diseases associated, infection or disorder, and perhaps having suffered from needs to strengthen or stimulate the active disease of endogenous leukocyte, infection or disorder." example explanation (Exemplary indications) " chapters and sections during the instantiation of leukocyte defective can vide infra.

By gene test, can identify the individuality of those susceptibles at present in leukocyte defective (for example any in the various forms such as cancer, immune dysfunction or infection).

In addition, we are for the cognition of body immune system, and especially for immune system recognition and kill and wound by the cognition of the cell of viral infection and tumor cell, in continuous progress.We are known now, immunity for the activated cell mediation, undesirable cell (offending cell) (for example by the cell of viral infection or tumor cell) must be presented the restricted tumor epi-position of HLA I class or virus epitopes and danger signal such as GM-CSF and/or TNF-α simultaneously, passs interactional with it cytotoxic T-lymphocyte (CTL) group so that immune antigen-presenting cell (APC) is costimulatory signals such as B7 and IL-12 with antigen.This presenting simultaneously causes producing activating cell clone and memory cell is cloned (summary can referring to Nature Medicine Vaccine Supplement 4 (1998) 525).Lacking under the situation of these extra signals, the HLA-I class antigen of identification undesirable cell-restricted T-cell through the processing back by broken ring or desensitization (body a kind of may be used to avoid to take place the process such as disorders such as autoimmune diseases).Inducing of this class tolerance is because nonrecognition (ignorance), anergy (anergy) or physiological disappearance (physical deletion) (Cold Spring Harbour Symp Quant Biol 2 (1989) 807; Nature 342 (1989) 564; Cell 65 (1991) 305; Nature Med 4 (1998) 525).

Clear at present, tumor cell is not automatically presented danger signal and/or costimulatory signal simultaneously.So a large amount of breedings (spawning) of tumor may cause eradicating those T cell clonies that cell-mediated antineoplastic immune power is provided.Therefore, show the patient of cancer, leukemia/lymphoma or sarcoma etc., may lose the ability of their inborn elimination tumor by this mechanism.

But, if before the diffusibility disease takes place patient, required T lymphocyte or its sample are taken out, when morbidity, relevant T cell mass can be returned to this patient so, behind collaborative the stimulation, alleviate disease through necessary T cell.Collaborative stimulation can be carried out when cell is returned to patient, also can carry out after by patient being implemented other treatment cell being returned to patient, perhaps no longer includes other stimulation except the stimulation of the natural generation of this patient.Activation/the stimulation of cell also can be external evoked before feedback is given patient.

The present invention so particularly suitable at susceptible in the case of leukocyte defective growth.Therefore it represented a kind of from the individual leukocyte that takes out of healthy, so that at this individuality afterwards whenever necessary, by transplant a kind of method (means) to this individuality from body (from gene) implantation method.Although this susceptible individual might never be accepted these cells because never the disease that need treat in this way occurs, the present invention also can be used to provide a kind of insurance forms, and the excessive risk of taking precautions against aleucemia for this individuality provides safeguard.

Similarly, do not diagnose out the individuality of susceptibility also can select, before their journey abroad, draw samples is put into leukocyte cell banks of the present invention, for future use.This class is used and can be comprised the treatment of infecting overseas.

In addition, well-known, ageing process makes more susceptible disease of individuality.As if the basic reason of this susceptibility is that the T cell in the ageing process and the number/activity of B cell significantly reduce, and causes immunologic function forfeiture (Mech Ageing ﹠amp; Dev 91 (1996) 219; Science 273 (1996) 70; Mech Ageing ﹠amp; Dev 96 (1997) 1).Disease susceptibility especially is found in elderly patients when accepting such as surgical operation in hospital, and here, they are easy to chance of occurrence and infect, and cause serious or or even fatal consequence.Blood sampling when this class individuality is younger is handled according to method of the present invention, puts into leukocyte cell banks, just can provide restorative autoplastic chance in above-mentioned situation.

This method can more be widely used in the operation back and strengthen patient's immune system, thereby reduces the probability that opportunistic infection causes post-operative complication.Therefore, the present invention can be used as prophylactic treatment, for example, be used for when elderly patients more susceptible in the disease.

IV. Leukocyte

The present invention relates to adopt art with isolating leukocyte list separates from blood sample and collects leukocyte.Be appreciated that the leukocyte list adopt do not need blood sample in the process in all leukocyte all separate and/or take out.Under many circumstances, only take out and/or isolate that the part of total white blood cells just is enough in the blood sample.Those skilled in the art are easy to the leukocytic appropriate amount of determine to need taking out, it based on factor such as isolated leukocytic purposes, sample size, blood donor's state, leukocytic feature and the concrete leukocyte list device for picking that adopted and difference is arranged.

The leukocyte of collecting in the inventive method separates from original blood sample to a certain extent and obtains.Term " isolating " is used for expression in this article, isolating leukocyte be in and be different from their a kind of physical environments the human internal environment, any concrete purity do not represented in this term.In fact, the purity absolute value is also non-key, and those skilled in the art are easy to determine suitable purity based on leukocytic purposes.

Must not represent leukocytic separation and collection in the inventive method yet, preferentially separate and collect the leukocyte of any particular type.Leukocyte of the present invention comprises any leukocyte, such as granulocyte, and lymphocyte and mononuclear cell etc.

Granulocyte comprises myelocyte (myelocyte), basophilic granulocyte, eosinophilic granulocyte and neutrophilic granulocyte.Lymphocyte comprises bone-marrow-derived lymphocyte, T lymphocyte and natural killer cell.Mononuclear cell comprises mononuclear phagocyte and other macrophage.

But the leukocyte that in some embodiments, separates and collect preferably comprises the leukocyte of one or more particular types.Preferred cell type is a lymphocyte, especially T-lymphocyte (T-cell).The special T-lymphocyte of wanting preferred maturity.

Because mature T cells adds up to 1-2.5 * 10 in every liter of human blood ⁹, so comprise 1-2.5 * 10 in the 100ml blood sample usually ⁸Mature T cells, generally this just is enough to provide can represent in the whole ripe human T-cell colony and produces the required amount of beneficial effect.But, fraction according to the total white blood cells that separates and collect with leukocyte list device for picking, and the effectiveness of any resuscitation technique that is adopted, preferably use 100ml at least, 115ml, 200ml or 300ml blood sample, more preferably surpass 400 or the blood sample of 500ml, so that when individuality generation disease, can obtain the mature T cells of sufficient amount, after returning this individuality, produce useful therapeutic effect.

Known in the art have the multiple standards technology can select concrete lymphocyte subgroup from the sample that comprises the mixed lymphocytes group.The example of this class subgroup comprises, CD3 ⁺, CD8 ⁺, CD4 ⁺And CD16/56 ⁺(NKT) T cell and CD19 ⁺The B cell.For example, any a group or any mixing or any combination may be used in method of the present invention, purposes and the compositions in this class T cell subsets, they can obtain at an easy rate by multiple known method, described method such as FACS (fluorescent activation cell sorting (Fluorescence Activated Cell Sorting)) and hemocytometer number system (haemocytometrysystems).

The present invention has also found to use widely, and except the leukocyte list is adopted art, also can adopt the separate type blood sampling art of any other form.Therefore, the present invention relates to be used to prepare the separate type blood-sampling method of stem cell compositions (and therefore producing stem cell bank).Therefore the present invention can also be applied to stem cell or progenitor cell (progenitor cell), comprises pluripotent stem cell and has entered the stem cell or the progenitor cell of concrete differentiation pathway.

V. Leukocyte is handled

Can carry out various processing to leukocyte.This class is handled and is caused, for example amplification (expansion) is a part of or representative cell subsets, improve the long-term vigor of leukocyte in the dormant stage, strengthen their treatment potentiality, remedy some or all leukocytic shortages or defective (for example, as in therapeutic autotransplantation treatment pattern) and/or make them safer when being used for autotransplantation subsequently.

Described processing can be carried out (and before or after carrying out autotransplantation) before or after the leukocyte dormancy.In addition, can after sampling, handle (being external carrying out), before making the cell dormancy or after the recovery, can handle.For example, can be, in the autotransplantation process, with a kind of other compositions and leukocyte compositions (sequentially) or use simultaneously successively to leukocytic processing.Can before being about to begin autotransplantation, carry out leukocytic processing.

Perhaps (or in addition) can be before getting blood, by using somatomedin or cytokine etc., in vivo leukocyte still handled (seeing below).

The example of transplanting pre-treatment comprises various genetic modifications, as mixes negative selection marker (for example, referring to W096/14401, it is incorporated herein by reference).This class is handled leukocyte depletion (ablation) or the titration dose-effect curve that makes after might transplanting.Other hereditary intervention can comprise, regulates or modify one or more expression of gene (for example, increasing or reduce gene expression), makes one or more gene inactivation, gene substitution and/or make one or more allogeneic gene expression).Other genetic modification comprises, the concrete T-cell of directional guide (targeting) (is seen W096/15238, it is incorporated herein by reference), and with antibody modification T-cell receptors all constituents (receptor repertoire)/expression, thereby the T-cytochimera produced.

Other processing of the present invention comprises, leukocyte is exposed to one or more stimulation molecule, for example antigen (as cancer antigen or virus antigen), antibody, T cell recognition epi-position, peptide, blood factor, hormone, somatomedin or cytokine or its combination.

For example, leukocyte can be handled with following material at external (or in getting blood before in vivo): antigen (for example, cancer antigens such as prostate specific antigen 1 or prostate specific antigen 2, her-2/new, MAGE-1, p53, Ha-ras and c-myc, or virus antigen), antibody, T cell recognition epi-position, peptide, blood factor, hormone, somatomedin or cytokine or its combination.Stimulation molecule can be synthetic molecule, recombinant molecule, perhaps can be from human body or animal body purification or isolating molecule.The useful especially stimulation molecule of this respect is selected from IFN-alpha, IFN-beta, IFN-gamma, II-1a, II-1b, II-2, II-3, II-4, II-5, II-6, II-7, II-8, II-9, II-10, II-11, II-12, II-13, II-14, II-15, GM-CSF, M-CSF, G-CSF, LT or above-mentioned two or more combination.Such processing can change leukocytic growth and/or activity and/or differentiation state, and/or can be used to distinguish or leukocyte type that Selective Separation or enrichment are required or remove undesired cell.

There are some progress to make recently and can obtain to be used for autoplastic subsequently cell.For example, the research to the hemopoietic regulatory factor causes isolating a series of factors that can influence lymphopoiesis and differentiation.These factors comprise, cytokine is (as interleukin series IL-1 to IL-18, leukotriene, tumor specific antigen such as prostate specific antigen 1 or prostate specific antigen 2, her-2/new, MAGE-1, p53, Ha-ras and c-myc), somatomedin such as TNF, TGF, FGF, EGF, GM-CSF, G-CSF, and other.This wherein has the multiple factor can buy the commodity of clinical use at present, and having some to show can increase peripheral blood lymphocyte greatly, the lymphocytic quantity of especially immature T.They are applied to blood donor's individuality before getting blood, can control of quality and/or quantity (refer to leukocyte hypotype exist quantity and characteristic), and might need not the bone marrow sampling, directly from blood donor's a large amount of required cell of peripheral body results, as immature T lymphocyte.

Other processing before transplanting comprises cultivates leukocyte (or its subgroup).For example, can cultivate leukocyte makes cell number increase.For example, can make passage with methods known in the art.This class is cultivated and can be carried out before the leukocyte dormancy and/or afterwards.

Therefore, comprise at leukocyte under the situation of T cell, can before transplanting, work in coordination with stimulation and/or before autotransplantation, make the T cellular exposure in tumor antigen (optional) with collaborative stimulating factor to the T cell.

VI. The leukocyte device

Can buy at present the leukocyte list device for picking of number of different types on the market.This class device comprises three independent assemblies usually at least: (for example comprise film or centrifugal rotor, they provide strength to make that other various compositions separate in leukocyte and the blood to (1) segregation apparatus; (2) one or more pump, be used for blood sample is transported to above-mentioned segregation apparatus, be used to take out isolating leukocyte and be used to keep and inculcate (transfusion) and reindoctrinate (retransfusion) necessary strength, (3) tubing (normally disposable), it remains on blood and various fraction thereof in the segregation apparatus with specific geometric position, it limits fixed blood circulation passage (usually be from the blood donor, get back to this blood donor's a loop by leukocyte list device for picking again) and is used to collect isolating leukocyte and/or other blood fraction or fluidic container (generally being sack).

Extensively any in the multiple commercially available leukocyte list device for picking may be used among the present invention.The concrete operations mode of leukocyte list device for picking depends on multiple factor, comprise that segregation apparatus (for example, centrifuge, filter etc.) characteristics, required leukocyte sample type, need the blood sample volume of handling, feature and state, the final use of leukocyte compositions and the characteristic that before carrying out processing of the present invention, puts on any processing of blood sample of blood donor's individuality.Therefore, those skilled in the art can determine appropriate operational parameters at an easy rate.

But preferably, selection is to the minimum leukocyte list device for picking of needs of operator's intervention and/or training.For multiple commercially available leukocyte list extraction system, the operator prepares and operates the required time and/or professional skill has nothing in common with each other.For example, reduce the complexity that the operator loads and unloads the required time of tubing and reduces these operations, can increase productivity and/or reduce the probability of operator's error.And the minimizing system is to operator's degree of dependence, then can reduce operate miss and/or reduce to the operator of these systems desired/essential qualification.

Performance-correlative factor also is related, can pass judgment on leukocyte list extraction system " collect and render a service ".A system " collect and render a service " certainly weighed with multiple mode, such as weighing with respect to the size from total cellular score in the sample with collected leukocyte part.Performance also can be estimated the influence of all kinds blood constituent based on leukocyte list mining method.For example, general hope reduces blood collection method to leukocytic at least negative effect.May also wish to weaken the platelet activation effect, to avoid the decline of sample quality in the processing procedure.

Particularly preferably be Cobe  system (Cobe BCT, Lakewood, CO, USA).

VII. The gathering system of using among the present invention

The system from individuality is collected isolating blood sample that the present invention uses can comprise shuttle (being used for collecting and holding blood sample) and the leukocyte list is adopted tubing.

Term " the leukocyte list is adopted tubing " is used for defining the tubing that chapters and sections are formerly described in this article.This tubing can comprise the blood treatment container, and in this container, leukocyte is subjected to the effect of the separating force (separation forces) in the segregation apparatus.

In the tubing of using with the leukocyte list device for picking (seeing chapters and sections formerly) that comprises the centrifugal type segregation apparatus, the blood treatment container can comprise centrifugal hoop (centrifuge loop), when in the centrifugal rotor that is equipped in segregation apparatus, this centrifugal hoop defines the vessel space that makes blood be subjected to the effect of centrifugalize power.

The system that the present invention uses can be a closed system, functional closed system, or open systems.

Term " closed system " herein, when being applied to the leukocyte list and adopting tubing, be meant a kind of aseptic and by sterile barrier and the isolated tubing of external environment, portion's all component all fully integrated (integral), (the assembled atthe manufacturing site) that interconnect (attached) and/or assemble within it in the production site.

Term " functional closed system " herein, when being applied to the leukocyte list and adopting tubing, be meant a kind of tubing that assembles in the device production site, it utilizes sterile barrier filter (as 0.22 micron filter) by the end user it to be connected with aseptic connecting device.

Term " open systems " herein when being applied to the leukocyte list and adopting tubing, only is meant that some is in the assembling of device production site, need finish the tubing of assembling by the end user according to oneself then.

Preferably, described system also comprises one or more (for example 3) leukocyte collection container.Preferred 3 or more a plurality of collection container are so that have enough samples for accepting or rejecting and also helping creating cell bank with a-type double/in triplicate sample.This allows autotransplantation scheme more flexibly.

Described system can also comprise easily can hold the container that obtains remaining blood behind the leukocyte.This remaining blood may be useful in other therapeutic scheme, and is for example useful in allos (allogenous) scheme.Described system can also comprise syringe needle or intubate (cannula), is used for blood sample is imported shuttle from individuality.

Various containers can adopt flexibility (flexible) transparent bag form easily.Partly (or all) pipelines also can adopt flexible clear materials (for example, plastics) easily.

VIII. Inducing of dormancy

Any suitable means may be used to induced dormancy.

According to a kind of preferred profound hypothermia store method, cell is preferably frozen below-160 ℃.A kind of particularly preferred dormancy means are with the boiling point of cell cryopreservation to helium (He), promptly to make an appointment with-269 ℃ or lower.

Viewpoint (Freshney ' s Tissue Culture of Animal Cells (Cultureof Animal Cells:A Manual of Basic Technique according to Freshney, Wiley Liss, 1994)), can be in proper culture medium (for example with cell suspension, comprise maximum concentration and reach 10% DMSO) in, (for example, per minute is 1 ℃ with in check speed cooling, up to-70 ℃, put into liquid/gas N then ₂).This method easily goes for the cell cooling at He/N ₂Among mixture or the He.Other realization and/or the method for keeping the cell dormancy comprise and are cooled to 4 ℃.

IX. Recovery

After the dormancy, before being used for transplanting with cell recovery.Similarly, this can be by any mode easily known in the art, and any method of cell recovery or resurrection that makes realizes.

Say that easily this can pass through, for example, cell thawing and/or dilution are realized.Resuscitation technique is known in the art.Cell thawing can followingly be operated: will hold container gentle vibration in 37 ℃ of water-baths of cell, and use then such as culture medium or serum DMSO is diluted to 1% lower concentration.

Cell can be implanted at once or implant through after the stage of recovering in culture medium.The recovery process is for rebuliding cell to be designed such as the purposes in prevention or the curative therapy.

X. Cell is built the storehouse

Leukocyte compositions of the present invention can be built the storehouse, thereby produces leukocyte cell banks.Preferably, described compositions makes leukocyte dormancy (as above-mentioned) Hou Jianku.

Any suitable cell is built the storehouse system and can be adopted, and is used for autotransplantation as long as preservation thing wherein can be recovered.This means and use some mark patterns, but this must not the form of preserving the physics additament (physical appendage) of thing with every part.

Therefore, leukocyte cell banks of the present invention can comprise many group cell storage units so that store leukocyte compositions.Usually, such cell is stored and is undertaken by the profound hypothermia preservation method, but also can adopt other preservation technology.Cell bank of the present invention can also comprise the digital information unit, be used to store identity (identity), location (location) digital information relevant with medical history with blood donor's individuality, and/or the situation relevant with the concrete preservation thing of portion (for example, from the date of the individual blood sample collection of blood donor, treatment conditions and details that contained leukocyte in this preservation thing is carried out any processing).

The digital information unit preferably comprises at least one digital computer, and it has enough digital storage capacity, can store possible and the relevant bulk information of each part preservation thing.

Leukocyte cell banks of the present invention preferably also comprises numerical data retrieval (digital data retrieval) device (arrangement), it and digital information unit join (interfaced with), can the appointed information of retrieve stored in the digital information unit.This data searcher can be incorporated in the digital computer.The digital information remote access of being undertaken by phone or the Internet can also be provided, and can visit these information in the world quickly and easily.

XI. Medical application

The present invention is applicable be hopeful to use (treated or undressed) in leukocytic treatment of body and prevention form (that is, expection has the treatment prospect).

For the purposes of the present invention, in these usable conditions, think the leukocyte defective to have occurred.

Therefore, be appreciated that the leukocyte defective (leukocyte deficiency) in the medical applications of the present invention contains multiple disease (disease), syndrome (syndrome), imbalance (disorder), disorder (condition) and infection.For example, be appreciated that, when individuality is suffered from leukocyte function imbalance diseases associated, syndrome, imbalance, disorder or is infected, and, can take place as above-mentioned generalized especially leukocyte defective when the individual endogenous leukocyte composition surface of suffering from when seeming normal but normal endogenous leukocyte activity and wishing/need to change, increases or obtains those diseases of stimulation, syndrome, imbalance, disorder or infection.Specifically, leukocyte defective described herein can be considered as the result of T cell and/or the non-specific loss of B cell, perhaps is considered as the result of concrete T cell clone group and/or B cell clone group loss or defective.

For simplicity, the leukocyte defective is referred to as in these diseases, syndrome, imbalance, disorder or infection in this article.

Be suitable for treatment of the present invention and can be divided into two big classes.One class is to utilize method of the present invention to create leukocyte compositions (promptly forming the part of leukocyte cell banks) from healthy individual blood donor's blood sample.In this application, the present invention is used to create the cell source of the healthy leukocyte tissue that is derived from individual blood donor, as long as when the leukocyte defective takes place this individuality afterwards, cell can be returned this blood donor's individuality.

In this therapy (this paper claims restorative autotransplantation), the present invention has utilized the following fact: multiple leukocyte defective be the part of a series of temporary incidents (they are with or without cause effect relation each other), so a time point before the leukocyte defective takes place is created leukocyte cell banks, has just constituted a treatment source can carrying out restorative treatment afterwards.

Second largest class is to utilize method of the present invention to create leukocyte compositions from the individual blood donor's that suffers from the leukocyte defective blood sample.External leukocyte compositions is handled then, the compositions of handling is transplanted in this individuality body again.After the processing that leukocyte compositions is carried out makes these leukocyte compositions return in blood donor's body, can alleviate or eliminate the leukocyte defective.An alternative approach of this method relates to handled individual leukocyte in vivo before the blood sampling step, these methods can relate to after the blood sampling step in external further processing.

In these treatments (claiming the therapeutic autotransplantation herein), the present invention has utilized the following fact: multiple leukocyte defective can overcome by sub-fraction in the individual leukocyte total group is handled.This class is handled and will be described in detail hereinafter, comprises the gene modification, and cell amplification (cellular expansion), selectivity are eliminated concrete cell type and stimulated with concrete molecule (as cytokine).

Above-mentioned restorative autoplastic notion can be applied to all individualities, no matter whether healthy, also is the factor of leukocyte defective index (for example age, medical history, genetic background and life style) no matter have not have to be doubted.But III joint (being entitled as " selecting blood donor's individuality ") is seen for details in the concrete colony that it allows discriminating one class to be particularly suitable for using the inventive method really.And owing to also can leukocyte defective restorative with the present invention or that the therapeutic autotransplantation is treated comprise extensively multiple known disease as above-mentioned definition, therefore, they are with the middle more detailed description of XII joint (being entitled as " example explanation ") below.

XII. Example explanation (Exemplary indications)

As described in the text, therapeutic application of the present invention and prophylactic use relate to extensive multiple disease, syndrome, imbalance, disorder and infection.

Infect

The present invention can be used for the treatment of various infection.In this case, endogenous leukocyte activity can be normal (or having normal reaction), but still wishes to change, increase or stimulate it.In other situation (infecting as HIV), endogenous leukocyte activity directly causes functional disorder because of infecting.

The infection that can treat according to the present invention or prevent comprises, bacterial infection, and fungal infection or viral infection, perhaps by any other biology, protozoacide for example, the infection that nematicide, insecticide or other parasite cause.

The preferred application is that the AIDS that causes is infected in treatment by HIV.In this case, can in individual health or uninfection, collect its CD4 ⁺Cell is also stored, when this individuality the HIV infection occurs or works as AIDS morbidity or CD4 ⁺When cell counting reduces, the cell of storing is transplanted in this individuality.This method makes it to face the individual attractive of HIV infection risk for those life style.

Cancer, leukemia and sarcoma

The present invention can also be used for the treatment of and prevent various malignant diseases (malignancies): typically refer to; any pernicious or pernicious omen (pre-malignant), the functional disorder (disturbance) in diffusion or excess diffusion (hyper-proliferative) disorder or any because body cell or the tissue or unusual or other imbalance or unusual caused disease or with they diseases associated.

Treating or preventing various forms of cancers is preferred embodiment of the invention, and it comprises any cancerous cells or the tissue of treatment or prevention body.

Therefore, the invention is not restricted to any diffusibility disease (for example, leukemia, lymphoma, cancer (carcinomas) or sarcoma), also be not limited to concrete oncogene or tumor suppressor gene epi-position such as prostate specific antigen 1 or prostate specific antigen 2, her-2/new, ras, myc, myb, fos, fas, retinoblastoma, p53 etc., thus or present other tumor cell sign epi-position that can be discerned by leukocyte with HLAI class antigen restrictive one or other similar fashion.All cancers, as leukemia, lymphoma, breast carcinoma, gastric cancer, colon cancer, rectal cancer, pulmonary carcinoma, hepatocarcinoma, uterus carcinoma, carcinoma of testis, ovarian cancer, the carcinoma of prostate and the cerebral tumor such as glioma, astrocytoma and neuroblastoma, sarcoma such as rhabdomyosarcoma and fibrosarcoma can be treated or prevent with the present invention.

Therefore, the present invention can be used for the treatment of or Breast Cancer Prevention, colon cancer, pulmonary carcinoma and carcinoma of prostate.The present invention can also be used for the treatment of or preclude blood system and lymphoid cancer (comprise Hokdkin disease (Hodgkin ' s Disease), leukemia, lymphoma, multiple myeloma, Waldenstr mShi disease), skin cancer (comprising malignant melanoma), alimentary tract cancer (comprises the incidence cancer, esophageal carcinoma, gastric cancer, cancer of pancreas, hepatocarcinoma, colorectal cancer, anus cancer), the urogenital tract cancer (comprises renal carcinoma, bladder cancer, carcinoma of testis, carcinoma of prostate), women's cancer (comprises breast carcinoma, ovarian cancer, gynecological cancer and choriocarcinoma) and brain, carcinoid in the bone, nasopharyngeal carcinoma, retroperitoneum (retroperitoneal) tumor, thyroid tumor, and soft tissue neoplasms.The present invention can also be used for the treatment of or prevent the cancer at former unknown position.

XIII. Dosiology (Posology)

Those skilled in the art are easy to determine to carry out autoplastic leukocyte compositions consumption in the medical application of the present invention.Be noted that as long as 0.01 * 10 ⁸(for example, 1-10 * 10 ⁸) mature lymphocyte (can obtain from the normal human blood sample of single a about 100ml) just is enough to strengthen (boost) experimenter's immune system, and so have a beneficial effect of Method for autologous transplantation of the present invention.Only be noted that in Britain, annually collect the blood that surpasses 300 ten thousand units from the blood donor, it is very usual that the blood that therefrom takes out a unit is used for allograft.

The leukocyte compositions of being used can be from single a blood sample, perhaps can be same blood donor's individuality is mixed group (pool) at the leukocyte compositions that many parts of different blood samples of different time points collection mix.The leukocyte compositions of being used can be all or part of of preservation thing, its part preferably, so that repeatedly (multiple dosing) uses, also can be chosen in and use (for example, the T cell number can be used the standard method amplification in vitro) again after remaining cell increased.

In the application based on the T cytoactive, the mature T cells consumption is at least 0.01 * 10 ⁸, more preferably at least 0.1 * 10 ⁸, more preferably at least 1 * 10 ⁸(as 1-10 * 10 at least ⁸).Preferred range is 0.01 * 10 ⁸-10 ¹⁰The mature T lymphocyte is as 0.1 * 10 ⁸-10 ¹⁰, 1 * 10 ⁸-10 ¹⁰Or 1 * 10 ⁹-10 ¹⁰The mature T lymphocyte.

Therefore, the required mature T cells sample of autotransplantation is at least 0.01 * 10 ⁸, usually 10 ⁸-10 ¹⁰CD3 ⁺The scope of mature T cells, preferred 2 * 10 ⁸-10 ¹⁰, more preferably 3 * 10 ⁸-10 ¹⁰CD3 ⁺, more preferably 4-5 * 10 also ⁸-10 ¹⁰CD3 ⁺Mature T cells.

Easily, every part is used for autoplastic sample and comprises 3 * 10 ⁸CD3 ⁺Mature T cells, more preferably 5 * 10 ⁸, also more preferably 1 * 10 ⁹CD3 ⁺Mature T cells.When individual physical ability provides competent blood source, also more preferably use 4-5 * 10 ⁹CD3 ⁺Mature T cells or 10 ¹⁰CD3 ⁺Mature T cells.

Preferably, the required CD3 of autotransplantation ⁺And CD8 ⁺The sample of mature T cells subgroup is at least 0.01 * 10 ⁸, usually 0.25 * 10 ⁸-0.25 * 10 ¹⁰Scope, more preferably 0.5 * 10 ⁸-0.25 * 10 ¹⁰, also more preferably 0.75 * 10 ⁸-0.25 * 10 ¹⁰, also more preferably 0.75 * 10 ⁸-0.25 * 10 ¹⁰Or 1.00-1.25 * 10 ⁸-0.25 * 10 ¹⁰Easily, be used for every duplicate samples CD3 of transplanting ⁺And CD8 ⁺Cell number be 0.2 * 10 ⁸Level, preferred 0.4 * 10 ⁸, or more preferably 1 * 10 ⁸, or also more preferably 2 * 10 ⁸, or more preferably 3 * 10 ⁸, or more preferably 5 * 10 ⁸When individual physical ability provides competent blood source, can use 1 * 10 ⁹, preferred 2 * 10 ⁹, 4 * 10 ⁹, or more preferably 1 * 10 ¹⁰CD3 ⁺And CD8 ⁺Cell.

Preferably, the required CD3 of autotransplantation ⁺And CD4 ⁺The sample of mature T cells subgroup is at least 0.01 * 10 ⁸, usually 0.1 * 10 ⁸-0.5 * 10 ¹⁰Scope, more preferably 0.65 * 10 ⁸-0.5 * 10 ¹⁰, also more preferably 0.85 * 10 ⁸-0.5 * 10 ¹⁰, also more preferably 1 * 10 ⁸-0.5 * 10 ¹⁰Or 1.8-3.6 * 10 ⁸-0.5 * 10 ¹⁰Easily, be used for every duplicate samples CD3 of transplanting ⁺And CD4 ⁺Cell number be 0.2 * 10 ¹⁰Level, preferred 0.3 * 10 ⁸, or more preferably 0.4 * 10 ⁸, 0.5 * 10 ⁸, 1 * 10 ⁸, 2 * 10 ⁸, 3 * 10 ⁸, 4 * 10 ⁸, or more preferably 5 * 10 ⁸When individual physical ability provides competent blood source, can use 1 * 10 ⁹, or more preferably 2 * 10 ⁹, or more preferably 1 * 10 ¹⁰CD3 ⁺And CD4 ⁺Cell.

Preferably, the required CD3 of autotransplantation ⁺And CD16/56 ⁺The sample of mature T cells natural killer cell subgroup is at least 0.01 * 10 ⁸, usually 0.01 * 10 ⁸-0.5 * 10 ¹⁰Scope, preferred 0.02 * 10 ⁸-0.5 * 10 ¹⁰, more preferably 0.03 * 10 ⁸-0.5 * 10 ¹⁰, also more preferably 0.5 * 10 ⁸-0.5 * 10 ¹⁰Or 0.5-2 * 10 ⁸-0.5 * 10 ¹⁰Easily, be used for every duplicate samples CD3 of transplanting ⁺And CD16/56 ⁺Cell number be 0.01 * 10 ⁸, 0.2 * 10 ⁸, 0.3 * 10 ⁸, 0.5 * 10 ⁸, 1 * 10 ⁸, 2 * 10 ⁸, 3 * 10 ⁸, 5 * 10 ⁸Level, or more preferably, when individual physical ability provides competent blood source, can use 1 * 10 ⁹, or more preferably 2 * 10 ⁹, or more preferably 1 * 10 ¹⁰CD3 ⁺And CD16/56 ⁺Cell.

In addition, the mature lymphocyte sample preferably includes the B cell, as CD19 ⁺Bone-marrow-derived lymphocyte.The mature B cell sample that is included in the T cell sample can be at least 10 ⁷, 10 ⁸Or 10 ⁹, usually 10 ⁷-10 ¹⁰The level of mature B cell, preferred 2 * 10 ⁷-10 ¹⁰Mature B cell, more preferably 3 * 10 ⁷-10 ¹⁰Mature B cell, also more preferably 4-5 * 10 ⁷-10 ¹⁰Mature B cell.

Easily, in the autograft the concrete number of B cell 3 * 10 ⁷Level, preferred 5 * 10 ⁸, more preferably 1 * 10 ⁹Mature B cell, also more preferably 4-5 * 10 ⁹Or 10 ¹⁰Mature B cell.

In addition, the lymphocyte sample can preferably include dendritic cell.The dendritic cell sample can be at least 10 ⁷, 10 ⁸Or 10 ⁹Quantity, usually 10 ⁷-10 ¹⁰The scope of dendritic cell, preferred 2 * 10 ⁷-10 ¹⁰Cell, more preferably 3 * 10 ⁷-10 ¹⁰Cell, also more preferably 4-5 * 10 ⁷-10 ¹⁰Cell.

The concrete number of dendritic cell is 3 * 10 in the autograft ⁷Level, preferred 5 * 10 ⁸, more preferably 1 * 10 ⁸, more preferably 4-5 * 10 also ⁹Or 10 ¹⁰Mature B cell.

XIV. Equivalents

Above describe the preferred embodiments of the invention in detail, therefore they be regarded as illustrating of various aspects of the present invention and be unrestricted.Those skilled in the art understand or can recognize, utilize normal experiment, can obtain the multiple equivalents (equivalent) of specific embodiments described herein, evolutionary approach (modification) or variation scheme (variation).These equivalents, evolutionary approach or variation scheme all are included in the described invention scope of claim.

Claims (20)

1. preparation is used for the method for autoplastic leukocyte compositions, may further comprise the steps:

(a) provide isolating blood sample from blood donor's individuality;

(b) with leukocyte list device for picking Selective Separation and collect leukocyte from this blood sample.

2. the process of claim 1 wherein that described leukocyte list device for picking is automatic leukocyte list device for picking.

3. claim 1 or 2 method, wherein said leukocyte list device for picking comprises closed system or functional closed system.

4. claim 2 or 3 method, wherein said leukocyte list device for picking is the centrifugal leukocyte list of a fluid device for picking successive or that be interrupted, or filtering flow formula leukocyte list device for picking successive or that be interrupted.

5. the method for arbitrary claim formerly wherein saidly comprises from cell list device for picking: (a) segregation apparatus (for example, centrifugal rotor or filter); (b) the leukocyte list is adopted plumbing installation; And (c) one or more pump, described pump is used for the sample transportation is transported to collection container by described plumbing installation with isolating leukocyte.

6. the method for arbitrary claim formerly, described method is used to prepare the used leukocyte compositions of restorative autotransplantation, and this method is further comprising the steps of:

(c) make the leukocyte dormancy (for example, making it dormancy) of collecting by the profound hypothermia preservation; With, alternatively

(d) leukocyte of dormancy is recovered (for example, making it recovery) by thawing and/or diluting.

7. the arbitrary method among the claim 1-5, described method is used to prepare the used leukocyte compositions of therapeutic autotransplantation, and this method is further comprising the steps of:

(c) handle collected leukocyte; With, alternatively

(d) make treated leukocyte dormancy (for example, making it dormancy) by the profound hypothermia preservation.

8. the method for claim 7, further comprising the steps of:

(e) leukocyte of process processing and dormancy is recovered (for example, making it recovery) by thawing and/or diluting.

9. the method for claim 6, described method is used to prepare leukocyte cell banks, wherein with described method repeated application in a series of blood samples from different blood donor's individualities, thereby the leukocyte compositions for preparing many parts of dormancy (for example profound hypothermia preservation), described method also comprises, autoplastic step is in the future preserved, prepared against to the leukocyte of dormancy in the mode that can recover.

10. system's (for example, closed system or functional closed system) that is used for collecting isolating blood sample from individuality, described system comprises: (a) sampling apparatus (for example, comprising syringe needle), to be used for from the described individual blood sample of collecting; (b) between the shuttle, itself and described sampling apparatus fluid communication is arranged; (c) the leukocyte list is adopted tubing, between itself and the described shuttle fluid communication is arranged, and wherein said tubing is a cecum, and it does not comprise and is used for and will draws back the device of described individuality through any part of the sample of fractionated.

11. the system of claim 10, wherein said tubing comprise one or more (for example 3) leukocyte collection container.

12. the system of claim 10 or 11, wherein said tubing also comprise blood treatment container (for example, centrifugal hoop).

13. also comprising, the arbitrary system among the claim 6-8, wherein said tubing be used to hold the container that obtains the later remaining blood of leukocyte.

14. the arbitrary system among the claim 10-13 also comprises and is used for importing the pin of shuttle from the blood sample of individuality.

15. be used for from isolating blood sample Selective Separation and the leukocytic device of taking-up from individuality, described device comprises the leukocyte list device for picking of the arbitrary gathering system that has among the claim 10-14.

16. the device of claim 15, wherein said leukocyte list device for picking are the arbitrary described devices among the claim 2-5.

17. leukocyte compositions, it can (or) obtain with the arbitrary method among the claim 1-9.

18. leukocyte cell banks, it can (or) use the method for claim 9 to obtain.

19. the leukocyte compositions of claim 17 is leukocyte compositions of using in treatment or prevention.

20. the leukocyte compositions of claim 17 is used for the purposes in the medicine of autotransplantation (for example, CAT therapy, perhaps restorative or therapeutic autotransplantation) in preparation.