Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
  • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
  • A61M1/02—Blood transfusion apparatus
  • A61M1/0209—Multiple bag systems for separating or storing blood components
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/14—Blood; Artificial blood
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/14—Blood; Artificial blood
  • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
  • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
  • A61M1/02—Blood transfusion apparatus
  • A61M1/0272—Apparatus for treatment of blood or blood constituents prior to or for conservation, e.g. freezing, drying or centrifuging
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
  • A61M2202/00—Special media to be introduced, removed or treated
  • A61M2202/04—Liquids
  • A61M2202/0413—Blood
  • A61M2202/0439—White blood cells; Leucocytes

Definitions

• the invention relates to a novel type of blood press pack, to processes for producing leukocyte cell banks using the pack and to various forms of therapy based thereon.
• centrifugation partitions the various components of the whole blood into three distinct layers: a blood plasma layer (of relatively low density), and red blood cell layer (of relatively high density) and a layer of intermediate density consisting predominantly of leukocytes and thrombocytes (platelets). This latter layer normally appears at the interface between an upper plasma and lower red blood cell layer after centrifugal separation, and is known as the buffy coat.
• red cells and thrombocytes can be processed separately.
• the concentrated red blood cell fraction is usually stored chilled for up to 35 days, and can be used for treating anaemic patients, victims of trauma and patients undergoing surgery.
• the plasma is generally frozen below -30°C for up to one year, and can be used to reverse anticoagulant treatment and as a component in massive transfusions.
• the thrombocytes stored at 20°C and continually agitated to prevent clotting, can be used to prevent bleeding in leukaemic patients or those undergoing chemotherapy or massive blood transfusions.
• the leukocytes present in the buffy coat fraction are usually regarded as an unwanted contaminant, since they can induce profound and possibly dangerous immunological changes if transfused inappropriately. For this reason, steps are usually taken to limit the collection of these cells during blood sampling (e.g. by the use of inline filters during blood donation).
• Such bags are known to those skilled in the art as 6/oocf (press) packs, and a wide range of different types are now commercially available (e.g. from Baxter Healthcare Ltd.).
• Such apparatus usually features one or more vertical press plates between which the bag can be introduced and which act to drive the different layers successively out of the pack when one plate is advanced towards the other. This usually results in the plasma layer being expressed first, followed by the buffy-coat. The red blood cell fraction can then be expressed last, or retained as a residual fraction in the pack.
• the bag press can be designed to express the plasma and red blood cell layers from opposite ends of the pack simultaneously, leaving the buffy coat as a residual fraction.
• bag presses are described in US 4350585, US 5874208 and US 4663032.
• a wide variety are commercially available, for example the Optipress® II from Baxter Healthcare Ltd.
• Cell banking is a service industry in which live cells are stored for later use. It has been practised for decades, and is exemplified by the storage of bovine sperm cells for the artificial insemination of cows.
• CAT Contingent autologous transplantation
• a form of therapy has recently been described (see WO 00/29551 and WO 01/88099) in which various tissues (including leukocytes) are removed from a healthy donor and stored in a tissue or cell bank for later autologous transplantation in the event that a need for such autotransplantation arises at some future date.
• This form of therapy is herein referred to as contingent autologous transplantation (CAT) therapy.
• CAT contingent autologous transplantation
• CAT therapy For any given tissue or cell type, the need for CAT therapy is likely to arise in only a fraction of the healthy population. As a result, the effectiveness of CAT therapy depends crucially on the generation of comprehensive cell and tissue banks in which deposits from a large percentage of the population are included.
• CAT therapy be facilitated by the construction of comprehensive tissue banks.
• tissue banks the nature of CAT therapy places unique and stringent demands on any such tissue bank.
• CAT therapy implies a large number of participating donors (and consequently a large number of deposits), relatively long-term storage, good retention of tissue function over time and great flexibility in ultimate therapeutic use.
• the present inventors have now recognized that contingent autologous transplantation of leukocytes requires specific combinations of blood processing techniques and cell bank construction in order to meet fulfil the unique demands imposed on a leukocyte cell bank by CAT therapy, which include inter alia the need for reliable matching of autologous material, exceptionally robust long-term storage, retention of leukocyte functionality and flexibility in ultimate therapeutic potential.
• the present invention is based, at least in part, on the discovery that blood samples collected and batch processed in the usual way from a donor can provide a convenient source of leukocytes for banking if a particular, novel type of blood press pack is used.
• a closed (or functionally closed) blood press pack comprising:
• a compressible bag for containing a blood sample during centrifugation;
• two or more leukocyte holding vessels in fluid communication with the compressible bag and disposed such that on compression of a filled and centrifuged bag in a bag press two or more discrete aliquots of a buffy coat layer are expressed into each of the two or more holding vessels.
• the term closed in the context of the packs of the invention, is used to define blood packs consisting of elements which are sterile and isolated from the outside environment by aseptic barrier(s) and in which all components are fully integral, being attached and/or assembled at the manufacturing site.
• the term functionally closed in the context of the packs of the invention, is used to define blood packs consisting of elements (e.g. tubing sets) which are assembled at the device manufacturing site and which use sterile barrier filters (e.g. 0.22 micron filters) or so-called "docking systems" for the sterile interconnection by the end user to generate a wide variety of arrays of tubing, channels, filters, satellite bags and other vessels.
• elements e.g. tubing sets
• sterile barrier filters e.g. 0.22 micron filters
• docking systems so-called "docking systems” for the sterile interconnection by the end user to generate a wide variety of arrays of tubing, channels, filters, satellite bags and other vessels.
• each of the two or more leukocyte holding vessels is in fluid communication with a leukocyte storage vessel, which storage vessel contains, or is in fluid communication with, a cryopreservation medium.
• the cryopreservation medium is simply present in liquid form within the storage vessel.
• the cryopreservation medium may be isolated from the holding vessel(s) by any convenient means, for example by: (a) a valve; (b) a breakable seal, (c) gravity in conjunction with the spatial arrangement of the holding and storage vessels; and/or (d) surface tension within a tube connecting the holding and storage vessels.
• the cryopreservation medium is in fluid communication with the storage vessel but not contained therein, for example being held in a satellite cryogenic medium vessel.
• the satellite cryogenic medium vessel may be reversibly isolated from the storage vessel by any convenient means, for example by: (a) a valve; (b) a breakable seal, (c) gravity in conjunction with the spatial arrangement of the satellite and storage vessels; and/or (d) surface tension within a tube connecting the satellite and storage vessels.
• the leukocyte storage vessels are double containment storage vessels.
• double containment as used herein in relation to the storage vessels of the invention, is a term of art defining a container having at least two barriers: a proximal barrier, in contact with the separated leukocytes, which contains the leukocyte sample and isolates it from the outside environment, and a secondary barrier which acts as a fail-safe to isolate the leukocyte sample from the outside environment in circumstances where the proximal barrier fails (for example after accidental physical or chemical trauma).
• the holding vessel serves to hold the leukocytes during the isolation and collection steps (which may take several minutes or even hours to complete) and permits stringent control over the mixing and preserving steps.
• the use of a separate holding vessel permits control over the incubation time and concentration of the leukocytes in the cryogenic preservation medium prior to freezing.
• the compressible bag of the blood pack of the invention may further comprise a serum outlet disposed such that on compression of a filled and centrifuged bag in a bag press the serum layer is expressed from the bag via the serum outlet.
• the serum outlet may be connected to a serum collection satellite vessel.
• the bag may also further comprise a red blood cell outlet disposed such that on compression of a filled and centrifuged bag in a bag press the red blood cell layer is expressed from the bag via the red blood cell outlet.
• the red blood cell outlet may be connected to a red blood cell collection satellite vessel.
• the invention provides a process for producing a leukocyte cell bank comprising the steps of: (a) providing an isolated blood sample from a healthy donor individual, wherein the sample is contained in the compressible bag of the blood press pack of the invention; (b) centrifuging the pack to produce a fractionated blood sample in which a plasma layer is separated from a red blood cell layer by a buffy coat layer; (c) compressing the centrifuged pack in a bag press thereby to express two or more discrete aliquots of the buffy coat layer containing leukocytes from the bag and into each of the two or more holding vessels; (d) cryogenically preserving each of the two or more aliquots containing leukocytes; (e) retrievably depositing each of the two or more preserved aliquots to produce a leukocyte bank.
• each of the two or more leukocyte holding vessels is in fluid communication with a leukocyte storage vessel, which storage vessel contains, or is in fluid communication with, a cryopreservation medium
• the process further comprises the step of mixing each aliquot with the cryopreservation medium within each storage vessel prior to the cryogenic preservation step.
• Particularly preferred is a process in which the aliquots containing leukocytes are transferred from the holding vessels to each of the double containment leukocyte storage vessels, when the storage vessels may conveniently be detached from the blood press pack prior to the depositing step or to the cryogenic preservation step.
• the process of the invention may be applied iteratively to a series of blood samples from different healthy donor individuals, and/or to a series of blood ' samples from the same healthy donor individual (for example to 2-12 samples taken from the same healthy donor individual over the course of one year).
• aliquots are deposited into two or more independent storage systems to produce a leukocyte bank which exhibits deposit redundancy.
• three, four, five or greater than five separate aliquots are used according to the invention.
• the independent storage systems into which the aliquots may be retrievably deposited are independent in the sense that they do not share a determinant of viability selected from: (a) power supply; and/or (b) site or location.
• a determinant of viability selected from: (a) power supply; and/or (b) site or location.
• the electricity supplies must not originate from a single generator or supplier.
• each independent storage system is sited to be geographically remote from its counterpart(s), so lessening the chances of coincidental destruction or damage by natural or man-made disasters (such as fire, flood or contamination).
• the process may further comprise the step of digitally storing information obtained from each donor individual in a digital information unit so as to permit matching of leukocyte deposit and donor for later autologous transplantation.
• the cryogenic preservation step conveniently comprises freezing to a temperature at or below about -160°C, which can be achieved using liquid nitrogen. If longer periods of storage and/or enhanced retention of functionality are required then freezing to a temperature at or below about -269°C may be effected using liquid helium.
• cryopreservation media Any of a wide range of suitable cryopreservation media may be used according to the invention, but preferred are media comprising a suitable penetrating cryoprotectant. Particularly suitable for use as a penetrating cryoprotectant is DMSO, which may be used for example at a concentration of up to 10%.
• the cryopreservation medium may further comprise an anticoagulant (such as acid citrate dextrose, EDTA, heparin or mixtures thereof), a nuclease (for example a Dnase and/or Rnase as well as a physiologically acceptable medium (for example, phosphate buffered saline).
• an anticoagulant such as acid citrate dextrose, EDTA, heparin or mixtures thereof
• a nuclease for example a Dnase and/or Rnase as well as a physiologically acceptable medium (for example, phosphate buffered saline).
• the cryopreservation medium may also further comprise a proteinaceous composition, such as blood serum or a blood serum component and/or a sugar and/or a polysaccharide (which may be particularly preferred in embodiments where plunge freezing is employed).
• compositions for use in the cryogenic preservation media of the invention comprise blood albumin (e.g. bovine serum albumin or human serum albumin).
• blood albumin e.g. bovine serum albumin or human serum albumin.
• human blood serum isolated from the blood sample of the donor individual. This can be isolated as a co-product together with the leukocytes.
• any donor may be used as a source of blood sample in the processes of the invention, provided that the donor is healthy, as herein defined.
• the invention finds particular application in relation to donor individuals which are predisposed to a leukocyte deficiency, are not in remission from a leukocyte deficiency, are juvenile, adolescent or adult, are at risk of developing a leukocyte deficiency, are human individuals between the ages of about 12 to 30 (e.g. 15 to 25) and/or have a fully-developed immune system.
• the process may further comprise the step of digitally storing information obtained from each donor individual in a digital information unit so as to permit matching of leukocyte deposit and donor for later autologous transplantation.
• the information may comprise at least that necessary to permit matching of deposit with donor, in order that later autologous transplantation can be carried out.
• the information comprises genetic information, the date at which the blood sample was collected from the donor individual, the age and sex of the donor individual, the clinical status of the donor individual, the medical history of the donor individual, biographical data identifying the donor individual, details of the processing and storage conditions used to prepare the deposit as well as data identifying the person(s) responsible for processing the sample(s).
• genetic information is stored, then this preferably comprises sequence information relating to one or more gene(s), single nucleotide polymorphism (SNP) data and/or one or more genetic fingerprint(s).
• Any suitable digital information unit may be used to store the information.
• this takes the form of at least one digital computer comprising a database.
• the database may carry data on a carrier of any convenient form.
• the information is stored independently on two or more carriers so that the database exhibits redundancy. This protects against data loss in the event of failure, corruption or loss of one of the computers or data carriers.
• the process further comprises the step of labelling the storage vessels with information sufficient to permit matching of the leukocyte deposit and donor.
• the storage vessels may be labelled with information: (a) describing the contents of the vessel (for example, sample size, number and/or volume); and/or (b) identifying the leukocyte bank; and/or (c) recording the date at which the blood sample was collected from the donor individual; and/or (d) comprising a statement that each package is for single patient use only; and/or (e) comprising instructions for opening, aseptic presentation and further storage.
• the labelling may comprise the physical attachment of an information carrier (e.g. a bar code) to the storage vessels themselves.
• the labelling may be effected by the non-physical association of the vessels with the information carrier (for example, via the correlation between the physical geometry or organization of the deposits in the bank and the entries in the database).
• the invention also contemplates treatment of the leukocytes, for example including any or all of the following: in vivo prior to provision of the blood sample, in vitro prior to separation step (b), in vitro after separation step (b) but prior to preservation step (f) and/or in vitro after preservation step (f).
• the invention contemplates a process for producing a leukocyte composition for autotransplantation into a donor individual comprising the steps of: (a) producing a leukocyte cell bank by the process of the invention; (b) matching the donor individual with a leukocyte deposit to identify an autologous leukocyte deposit; (c) retrieving a storage vessel containing an aliquot of preserved autologous leukocytes; and (d) revitalizing the preserved autologous leukocytes to produce a leukocyte composition for autotransplantation into the donor individual.
• the invention also contemplates a leukocyte composition and a leukocyte cell bank obtainable (or obtained) by the process of the invention.
• the invention contemplates the leukocyte composition of the invention for use in therapy, for example in autotransplantation (e.g. in restorative or remedial autotransplantation).
• isolated blood sample is used herein to define a blood sample which is not in fluid communication with the blood of the donor from which it originated.
• autotransplantation is used herein to define autologous transplantation (autogeneic or self-to-self transplantation), wherein the term autologous is used to indicate that the transplantation is to the same organism (i.e. the same individual) from which the cellular material (e.g. leukocytes) was removed.
• transplantation defines any procedure involving the introduction of cellular material (e.g. leukocytes) into an organism, and so any form of transplantation or grafting known in the art is encompassed.
• dormancy is used herein to define any state of suspended animation or stasis, and procedures for achieving this are well known in the art, as described below. Any of the known procedures may be used, including cryopreservation. Thus, the leukocytes may be held or maintained in a quiescent, inactive or non- proliferating state.
• healthy is used herein in relation to an individual donor to indicate that the individual is not suffering from a leukocytic deficiency (as herein defined).
• the term healthy as used herein encompasses non- diseased individual donors in a state in which the individual donor is not suffering from any disease or disorder, or is not manifesting any symptoms of said disease or disorder (i.e. is asymptomatic or is in a pre-clinical condition).
• term healthy as used herein encompasses individual donors not suffering from, or demonstrating symptoms of, the disease or disorder which it is subsequently intended to treat by the autotransplantation procedure. ll. Blood samples
• the invention may be applied to any form of blood sample, provided that: (a) the sample is isolated in the sense defined above and (b) the sample comprises at least some leukocytes from the individual donor.
• the blood sample may be subjected to various treatments ex vivo prior to use in the process of the invention. Typically, for example, the blood sample is chilled prior to use. Other treatments may include the addition of preservatives and/or anticoagulants.
• the blood sample may also be treated in vivo prior to collection by administering various agents to the donor individual before or during sample collection. Examples of treatments (which may be applied ex vivo and/or in vivo) are discussed in more detail in the section entitled "Leukocyte treatments", below. It is generally preferable to sample at least 450-500 ml of blood from the individual donor, which is the equivalent of a unit of blood as provided by a blood donor for the UK blood transfusion service. If possible a number of samples (e.g. several 450-500 ml samples) are taken over a period of time (e.g. over 2-3 weeks, preferably 2-3 months or over 6 months or a year, 2 or 3 years or more). One or more of these can then be divided or combined into a number of leukocyte cell bank deposits. The removal of a unit of blood is commonplace with over three million units of blood being taken, for allografting, from individuals annually in the UK alone.
• Restorative autotransplantation is a form of therapy that might ultimately be indicated for any individual. Consequently, the invention may be usefully applied to the generation of comprehensive leukocyte cell banks covering as large a number of different individuals as possible in order that restorative autotransplantation can be carried out in any of the represented individuals should the need arise.
• the invention be applied as broadly as possible so that a comprehensive leukocyte cell bank can be assembled.
• the quality of the individual deposits will depend (at least to some extent) on the health status of the individual donor at the time of blood sample donation, it is preferred that the blood sample for use in the processes of the invention be taken from healthy individual donors.
• the blood sample for use in the processes of the invention may advantageously be obtained from individual donors when they are young, preferably in adolescence or early adulthood.
• blood sampling preferably multiple sampling
• sampling is from the age of 16 or 17 upwards, for example in the age range 16 to 30, 17 to 30, or 18 to 30, or perhaps 18 to 35 or 40. It is thus preferred that the cells be obtained when the host organism is mature, or reaching maturity, but before the processes of ageing or senescence have significantly set in.
• the immune system of the host organism is mature or fully developed.
• cells may be obtained at any postnatal life stage e.g. from juvenile host organisms e.g. in mid-to late childhood, or even infants, or from older individuals.
• Sampling from post-natal or older hosts allows multiple samples to be collected, thereby increasing the opportunity of storing sufficient number of cells.
• sampling from juvenile or older hosts overcomes the ethical requirements such as providing informed consent.
• Sampling from adolescent or adult host organisms is preferred since the sampled cells, from blood in particular, will contain a greater proportion of valuable mature T-cells capable of recognising aberrant cell populations, such as cancer cells or virally infected cells.
• a mature immune system i.e. not foetal or neonatal.
• the invention contemplates the use of blood samples collected from donor individuals at a stage when there is no direct prediction, suggestion, or suspicion that a particular disorder or disease may develop, for use against a future possible or unpredicted event, or an event which may occur simply by chance, rather than an anticipated or suspected or predicted illness or condition.
• the donor individual is not predisposed to, or at risk from, any particular disease or disorder e.g. not exhibiting any symptoms or manifestations predictive of a subsequent disease or disorder.
• the host organism is preferably not suffering from any injuries or damage which may give rise to an anticipated or expected condition.
• the blood sample for use in the invention be obtained from the donor individual before any disease or disorder develops or manifests itself, and more preferably when the host organism is in general good health, and preferably not immunocompromised in any way.
• leukocyte deficiencies which term is used herein to indicate a condition in which the administration of autologous leukocytes is indicated. Such conditions therefore include those in which an individual has acquired a disease, infection or condition involving leukocyte dysfunction or a disease, infection or condition in which the augmentation or stimulation of endogenous leukocyte activity is indicated.
• leukocyte deficiencies are set out in the section entitled "Exemplary indications", below.
• leukocyte deficiency e.g. any of various forms of cancer, immune disorder or infection.
• a leukocyte deficiency e.g. any of various forms of cancer, immune disorder or infection.
• our knowledge of the body's immune system, and in particular the way in which it recognises and kills virally infected and tumour cells continues to advance.
• an offending cell e. g.
• a virally infected or tumour cell must co-present an HLA class I restricted tumour or viral epitope with danger signals such as GWI-CSF and/or TNF-alpha, so that the antigen presenting cells (APC) of the immune system will express co-stimulatory signals such as B7 and IL-12 in conjunction with antigen to the interacting cytotoxic T-lymphocyte (CTL) population.
• APC antigen presenting cells
• CTL cytotoxic T-lymphocyte
• HLA-I antigen-restricted T-cells which recognise offending cells are processed for destruction or desensitization (a bodily process presumably put into place to avoid the development of e.g. autoimmune disease).
• the induction of such tolerance is because of either ignorance, anergy or physical deletion (Cold Spring Harbour Symp Quant Biol 2 (1989) 807; Nature 342 (1989) 564; Cell 65 (1991) 305; Nature Med 4 (1998) 525).
• tumour cells do not automatically co-present danger and/or co-stimulatory signals. Hence, the spawning of a tumour may lead to eradication of the very T cell clones that provide cell-mediated immunity against the tumour.
• the relevant T-cell population could now be returned to the patient, after any necessary co-stimulation of the T-cells, so as to alleviate disease.
• Co-stimulation may be provided at the same time as the cells are returned to the patient, or after they are returned through further treatment (s) of the patient, or without stimulation other than that naturally produced by the patient.
• Activation/stimulation of the cells may also initially be induced in vitro prior to reinfusion.
• the present invention therefore finds particular application in the case of individuals predisposed to the development of a leukocyte deficiency. It therefore represents a means for removing leukocytes from a healthy donor individual for subsequent transplantation to that same individual in a subsequent autologous (autogeneic) transplantation procedure, when the need or desire to do so arises.
• the predisposed individual may never receive the cells because no disease to be treated by this method ever occurs, the invention nevertheless may be used to provide some form of insurance against the heightened risk of a leukocyte deficiency arising in the individual.
• individuals with no diagnosed predisposition may choose to provide samples for incorporation into the leukocyte cell bank of the invention for prospective use by themselves prior to travelling abroad. Such use might include for the treatment of infections contracted whilst abroad.
• Such an approach could be used more broadly to provide for a method of augmenting the patient's immune system after surgery in order to lessen the likelihood of post-operative complications caused by opportunistic infections.
• the invention therefore, could be used as a prophylactic therapy, e.g. for elderly patients when they are more susceptible to disease.
• the invention contemplates the use of batch centrifugation and bag pressing techniques to separate and collect leukocytes from a blood sample. It will be appreciated that the separation and/or removal of leukocytes from the blood sample during such processing need not be absolute. Rather, the removal and/or separation of a fraction of the total leukocytes present in the sample is sufficient in most circumstances. Those skilled in the art will readily be able to determine the appropriate size of the fraction to be removed, which will vary inter alia according to the use to which the isolated leukocytes are to be put, the size of the sample, the status of the donor and the nature of the leukocytes.
• the leukocytes collected in the processes of the invention are to some degree isolated from the original blood sample.
• isolated is used here to indicate that the isolated leukocytes exist in a physical milieu distinct from that in which they occur in vivo and does not imply any particular degree of purity. Indeed, the absolute level of purity is not critical, and those skilled in the art can readily determine appropriate levels of purity according to the use to which the leukocytes are to be put.
• the leukocytes which are separated and collected preferably comprise one or more specific leukocyte cell types.
• a preferred cell type is the lymphocyte, especially a T-lymphocyte (T-cell). Mature T-lymphocytes are particularly preferred.
• a 100 ml sample of blood typically contains 1-2.5 x 10 8 mature T-cells and this is generally sufficient to provide an adequate representation of the entire mature human T-cell population for the beneficial effect.
• at least 100 ml, 115 ml, 200 ml or 300 ml and even more preferably in excess of 400 or 500 ml of blood sample is used in order to obtain the appropriate number of mature T-cells to support a beneficial therapeutic effect for return to the individual if and when they become ill.
• Standard techniques are known in the art which permit selection of particular subpopulations of lymphocytes from a sample comprising a mixed population of lymphocytes.
• subpopulations are CD3 + , CD8 + , CD4 + and CD16/56 + (natural killer) T cells and CD19 + B cells.
• any one or any mixture or combination of such subpopulations of T cells can be used in the methods, uses and compositions of the invention, and they are readily obtained by means of well known methods such as FACS (Fluorescence Activated Cell Sorting) and haemocytometry systems.
• the leukocytes may be subjected to various treatments. Such treatments may, for example, result in expansion of some or all of the representative cell subsets, improve the long-term viability of the leukocytes during the dormancy period, improve their therapeutic potency, remedy a deficiency or defect exhibited by some or all of the leukocytes (as is the case, for example, in remedial autotransplantation therapeutic modalities) and/or render their subsequent use in autotransplantation safer.
• the treatments can be carried out before or after the leukocytes are rendered dormant (and before or after autotransplantation is carried out). Moreover, the treatments may be applied after the blood sample is taken (i.e. be carried out ex vivo) either prior to rendering the cells dormant or after revitalization.
• treatment of the leukocytes may be effected by co administration of a separate composition, sequentially or simultaneously with the leukocyte composition, during autotransplantation. Treatment of the leukocytes can be effected immediately prior to autotransplantation. Alternatively (or in addition) the treatments may be applied to the leukocytes while still in vivo prior to blood sampling by the administration of e.g. growth factors or cytokines (see below).
• Exemplary pre-transplantation treatments may include various genetic modifications, such as the incorporation of a negative selection marker (as described, for example, in W096/14401 , the content of which is incorporated herein by reference). Such treatment permits ablation of the leukocytes after transplantation or titration of dose versus response.
• Other genetic interventions may include regulating or modifying the expression of one or more genes (e. g. increasing or decreasing gene expression), inactivating one or more genes, gene replacement and/or the expression of one or more heterologous genes).
• Other genetic modifications include the targeting of particular T-cells (as described in W096/15238, the content of which is incorporated herein by reference), and the modification of the T-cell receptor repertoire/expression with antibodies to make T-cell chimaeras.
• Treatments contemplated by the invention include the exposure of the leukocytes with one or more stimulatory molecules, for example antigens (e.g. cancer or viral antigens), antibodies, T cell recognition epitopes, peptides, blood factors, hormones, growth factors or cytokines or combinations thereof.
• antigens e.g. cancer or viral antigens
• antibodies e.g. antibodies, T cell recognition epitopes, peptides, blood factors, hormones, growth factors or cytokines or combinations thereof.
• the leukocytes may be treated in vitro (or in vivo prior to blood sampling) with antigens (for example cancer (e.g. prostate-specific antigen 1 or prostate-specific antigen 2, her-2/new, MAGE-1 , p53, Haras and c-myc) or viral antigens), antibodies, T cell recognition epitopes, peptides, blood factors, hormones, growth factors or cytokines or combinations thereof.
• antigens for example cancer (e.g. prostate-specific antigen 1 or prostate-specific antigen 2, her-2/new, MAGE-1 , p53, Haras and c-myc) or viral antigens), antibodies, T cell recognition epitopes, peptides, blood factors, hormones, growth factors or cytokines or combinations thereof.
• the stimulatory molecules may be synthetic, recombinant or may be purified or isolated from the human or animal body.
• stimulatory molecules selected from IFN-alpha, IFN-beta, IFN-gamma, 11-1 a, ll-lb, II-2, II-3, II-4, II-5, II-6, II-7, II-8, II-9, 11-10, 11-11 , 11-12, 11-13, 11-14, 11-15, GM-CSF, M-CSF, G-CSF, LT and combinations of two or more of the foregoing.
• Such treatments may modify the growth and/or activity and/or state of differentiation of the leukocytes, and/or may serve to separate or selectively isolate or enrich desired leukocyte cell types or to purge unwanted cells.
• the leukocytes may be cultured to increase cell numbers.
• the cells may be passaged, according to methods well known in the art. Such culturing may be carried out before or after the leukocytes are rendered dormant, or both before and after dormancy is induced.
• the leukocytes include T-cells
• the T-cells may be co-stimulated prior to transplantation and/or exposed to tumour antigens (optionally together with co-stimulatory factors) prior to autotransplantation.
• the cells are frozen preferably to a temperature below -160°C.
• a particularly preferred means of achieving dormancy is to freeze the cells to the boiling point of helium (He), i.e. to about -269°C or below.
• the cells may be suspended in a suitable medium (e. g. containing up to 10% DMSO) and cooled at a controlled rate (e. g. 1°C per minute to -70°C, then into liquid/gas N 2 ).
• a suitable medium e. g. containing up to 10% DMSO
• a controlled rate e. g. 1°C per minute to -70°C, then into liquid/gas N 2 .
• a suitable medium e. g. containing up to 10% DMSO
• a controlled rate e. g. 1°C per minute to -70°C, then into liquid/gas N 2
• Such conventional procedures may be adapted to cool the cells into He/N 2 mixtures or He.
• Alternative methods of achieving and/or maintaining cell dormancy include cooling to 4°C.
• the cells are revitalised prior to use in transplantation. Again, this may be achieved in any convenient manner known in the art, and any method of revitalising or reviving the cells may be used. Conveniently, this may, for example, be achieved by thawing and/or diluting the cells. Techniques for revitalisation are well known in the art. Cells may be thawed by gentle agitation of the container holding the cells in water at 37°C, followed by dilution of DMSO to 1 % or below, e. g. with medium or serum. Cells may be implanted immediately or after recovery in culture. Revitalisation is designed to re-establish the usefulness of the cells e.g. in prophylaxis or curative therapy. VIII. Cell banking
• the leukocyte compositions of the invention may be banked, thereby creating a leukocyte cell bank.
• the compositions are banked after the leukocytes have been rendered dormant (as described above).
• Any suitable cell banking system may be employed, provided that the deposits are retrievable for autotransplantation. This implies the use of some form of labelling, but this need not be in the form of a physical appendage to the individual deposits.
• the leukocyte cell bank of the invention may comprise a plurality of cell storage units for storage of leukocyte compositions.
• cell storage is effected by cryopreservation, but other storage techniques can also be employed.
• the cell banks of the invention may further include a digital information unit for digitally storing information relating to the identity, location and medical history of the donor individual and/or the conditions associated with the particular deposit (for example relating to the date at which the blood sample was collected from the donor individual, the processing conditions and details of any treatments applied to the leucocytes contained in the deposit).
• the digital information unit preferably comprises at least one digital computer having sufficient digital storage capacity for storage of the potentially large amounts of information relating to each deposit.
• the leukocyte cell bank of the invention preferably further comprises an arrangement for digital data retrieval interfaced with the digital information unit for retrieving selected information stored in the digital information unit.
• the data retrieval arrangement may be integrated with the digital computer. Remote access of the digital information via the telephone or the internet may also be provided and may permit rapid and convenient access of the information on a global basis.
• leukocyte deficiencies in which the invention finds medical application encompass a very broad spectrum of diseases, syndromes, disorders, conditions and infections.
• a leukocyte deficiency in the special, broad sense defined above, can arise in circumstances where an individual has acquired a disease, syndrome, disorder, condition or infection involving leukocyte dysfunction as well as in circumstances where an individual has acquired a disease, syndrome, disorder, condition or infection in which the endogenous leukocyte component is seemingly normal but in which alteration, augmentation or stimulation of the normal endogenous leukocyte activity is nevertheless indicated/required.
• a leukocyte deficiency as herein defined may be deemed to have arisen either as a result of a non-specific loss of T- and or B-cells, or as a result of a loss or deficiency of a particular T- and/or B-cell clonal population.
• diseases, syndromes, disorders, conditions or infections are collectively defined herein as leukocytic deficiencies.
• the processes of the invention are employed to create a leukocyte composition (e.g. forming part of a leukocyte cell bank) from a blood sample from a healthy individual donor.
• a leukocyte composition e.g. forming part of a leukocyte cell bank
• the invention is used to create a cellular resource of healthy leukocytic tissue from an individual donor that can be restored to that donor individual should the individual acquire a leukocytic deficiency at a later date.
• the invention exploits the fact that many leukocytic deficiencies occur as part of a temporal sequence of events (which may or may not be causally interrelated), so that the creation of a leukocyte cell bank at a point in time predating onset of the leukocytic deficiency constitutes a therapeutic resource which can later be used restoratively.
• the therapeutic and prophylactic uses of the invention encompass a very broad spectrum of diseases, syndromes, disorders, conditions and infections.
• the invention may find application in the treatment of various infections.
• the endogenous , leukocyte activity may be normal (or responding normally) but its alteration, augmentation or stimulation is nevertheless desirable.
• the endogenous leukocyte activity is dysfunctional as a direct consequence of infection.
• Infections which may be treated or prevented according to the invention include bacterial, fungal or viral infections, or infections by any other organism e.g. a protozoan, nematode, insect or other parasite.
• a preferred application is the treatment of AIDS as a result of HIV infection.
• CD4 + cells can be collected from an individual when healthy or non-infected, and stored for subsequent transplantation into said individual when HIV infection manifests itself or when AIDS develops, or CD4 + cell count falls etc. Such a procedure may be attractive to an individual with a life-style likely to place them at risk from contracting HIV infection. Cancers, leukaemias and sarcomas
• the invention may find application in the treatment and prophylaxis of various malignancies: in general, any malignant or pre-malignant condition, proliferative or hyper-proliferative condition or any disease arising or deriving from or associated with a functional or other disturbance or abnormality in the cells or tissues of the body.
• Therapy or prophylaxis of various forms of cancer represents a preferred embodiment of the invention, and the treatment or prophylaxis of any cancerous cells or tissues of the body is contemplated.
• the invention is not limited to any one type of proliferative disease (e. g. leukaemias, lymphomas, carcinomas or sarcomas), nor is it restricted to specific oncogenes or tumour-suppressor gene epitopes such as ras, prostate-specific antigen 1 or prostate-specific antigen 2, her-2/new, myc, myb, fos, fas, retinoblastoma, p53 etc. or other tumour cell marker epitopes that are presented in an HLA class I antigen restricted fashion or other such way so as to be identifiable by a leukocyte.
• proliferative disease e. g. leukaemias, lymphomas, carcinomas or sarcomas
• tumour-suppressor gene epitopes such as ras, prostate-specific antigen 1 or prostate-specific antigen 2, her-2/new, myc, myb, fos, fas, retinoblastoma, p53 etc. or
• All cancers such as leukaemia, lymphoma, breast, stomach, colon, rectal, lung, liver, uterine, testicular, ovarian, prostate and brain tumours such as gliomas, astrocytomas and neuroblastomas, sarcomas such as rhabdomyosarcomas and fibrosarcomas are included for the therapy or prophylaxis by the present invention.
• the present invention finds application in the treatment or prophylaxis of breast cancer, colon cancer, lung cancer and prostate cancer. It also finds application in the treatment or prophylaxis of cancers of the blood and lymphatic systems (including Hodgkin's Disease, leukemias, lymphomas, multiple myeloma ⁇ and
• Waldenstr ⁇ m's disease skin cancers (including malignant melanoma), cancers of the digestive tract (including head and neck cancers, oesophageal cancer, stomach cancer, cancer of the pancreas, liver cancer, colon and rectal cancer, anal cancer), cancers of the genital and urinary systems (including kidney cancer, bladder cancer, testis cancer, prostate cancer), cancers in women (including breast cancer, ovarian cancer, gynecological cancers and choriocarcinoma) as well as in brain, bone carcinoid, nasopharyngeal, retroperitoneal, thyroid and soft tissue tumours. It also finds application in the treatment or prophylaxis of cancers of unknown primary site.
• leukocyte composition to be autotransplanted in the medical applications according to the invention. It should be noted that as few as 0.01 x 10 8 (e.g. 1-10 x 10 8 ) mature lymphocytes (which can be derived from a single sample of approximately 100 ml of normal human blood) are sufficient to boost the immune system of a subject and hence may have a beneficial effect according to the autologous transplantation method of the invention. It should be noted that the removal of a unit of blood is commonplace with over three million units of blood being taken, for allografting, from individuals annually in the UK alone.
• 0.01 x 10 8 e.g. 1-10 x 10 8
• mature lymphocytes which can be derived from a single sample of approximately 100 ml of normal human blood
• the removal of a unit of blood is commonplace with over three million units of blood being taken, for allografting, from individuals annually in the UK alone.
• the leukocyte composition administered may be derived from a single blood sample, or may constitute a pool of leukocyte compositions derived from a plurality of different blood samples taken from a donor individual at different times.
• the leukocyte composition administered may constitute all or a fraction of the deposited material, but preferably constitutes only a fraction thereof in order that multiple dosing can be achieved, optionally following cellular expansion of the residue (for example, T cell numbers may be increased by in vitro expansion using standard methods).
• the number of mature T-cells administered is at least 0.01 x 10 a , more preferably at least 0.1 x 10 8 , more preferably at least 1 x 10 8 (e.g. at least 1-10 x 10 8 ).
• the preferred ranges are 0.01 x 10 8 to 10 10 mature T lymphocytes, such as 0.1 x 10 8 to 10 10 , 1 x 10 8 to 10 10 or 1 x 10 9 to 10 1Q mature T lymphocytes.
• the mature T-cell sample acquired for autotransplantation is at least 0.01 x 10 8 , generally in the range of 10 8 - 10 10 CD3 + mature T-cells, preferably 2 x 10 8 - 10 10 , more preferably 3 x 10 8 - 10 10 CD3 + and even more preferably 4-5 x 10 8 - 10 10 CD3 + mature T-cells.
• each sample prepared for autotransplantation contains 3 x 10 8 CD3 + mature T-cells, more preferably 5 x 10 8 and even more preferably 1x10 9 CD3 + mature T-cells. If sufficient resources of blood are available from an individual, even more preferably still 4-5 x 10 9 CD3 + mature T-cells or 10 10 CD3 + mature T- cells may be used.
• the mature T-cell subpopulation sample acquired for autotransplantation which is CD3 + and CD8 + is at least 0.01 x 10 8 , generally in the range of 0.25 x 10 8 - 0.25 x 10 10 , and more preferably 0.5 x 10 8 - 0.25 x 10 10 , and even more preferably 0.75 x 10 8 - 0.25 x 10 10 , and even more preferably still 0.75 x 10 8 - 0.25 x 10 10 or 1.00 - 1.25 x 10 8 - 0.25 x 10 10 .
• Specific CD3 + and CD8 + cell numbers in each sample prepared for grafting is conveniently of the order of 0.2 x 10 8 , preferably 0.
• 1 x 10 9 preferably 2 x 10 9 , 4 x 10 9 , or more preferably 1 x 10 10 CD3 + and CD8 + cells may be used.
• the mature T-cell subpopulation sample acquired for autologous transplantation which is CD3 + and CD4 + is at least O.01 x 10 8 , generally in the range of 0. 1 x 10 8 - 0.5 x 10 10 , and more preferably 0.65 x 10 8 - 0. 5 x 10 10 , and even more preferably 0.85 x 10 8 - 0.5 x 10 10 , and even more preferably still 1 x 10 8 - 0.5 x 10 10 or 1.8 - 3.6 x 10 8 - 0.5 x 10 10 .
• CD3 + and CD4 + cell numbers in each sample prepared for grafting is conveniently of the order of 0.2 x 10 10 , preferably 0.3 x 10 8 , or more preferably 0.4 x 10 8 , 0.5x10 8 , 1 x 10 8 , 2 x 10 8 , 3 x 10 8 , 4 x 10 8 , or more preferably 5 x 10 8 . If sufficient resources from an individual are available, I x 10 9 , or more preferably 2 x 10 9 , or more preferably 1 x 10 10 CD3 + and CD4 + cells may be used.
• the mature T-cell natural killer subpopulation sample acquired for autotransplantation which is CD3 + and CD16/56 + is at least 0.01 x 10 8 , generally in the range of 0.01 x 10 8 - 0.5 x 10 1 °, preferably 0.02 x 10 8 - 0.5 x 10 10 , more preferably 0.03 x 10 8 - 0.5 x 10 10 , and even more preferably still 0.5 x 10 8 -0.5 x 10 10 or 0.5-2 x 10 8 to 0.5 x 10 10 .
• Specific CD3 + and CD16/56 + cell numbers in each sample prepared for grafting is conveniently of the order of 0.01 x 10 8 , 0.2 x 10 8 , 0.
• I x 10 9 or more preferably 2 x 10 9 , or more preferably 1 x 10 10 CD3 + and CD16/56 + cells may be used.
• the mature lymphocyte cell sample may preferably include B cells, such as CD19 + B lymphocytes.
• the mature B-cell sample included in the T-cell sample may be at least 10 7 , 10 8 or 10 9 , generally in the range of 10 7 -10 10 mature B-cells and preferably 2 x 10 7 - 10 10 mature B-cells, more preferably 3 x 10 7 - 10 10 mature B-cells, and even more preferably 4-5 x 10 7 - 10 i0 mature B-cells.
• B-cells in autograft is conveniently of the order of 3 x 10 7 , preferably 5 x 10 8 , more preferably 1 x 10 8 mature B-cells, and even more preferably still 4-5 x 10 9 or 10 10 mature B-cells.
• the lymphocyte cell sample may preferably include dendritic cells.
• the dendritic cell sample may be at least 10 7 , 10 8 or 10 9 in number, and generally in the range of 10 7 - 10 10 dendritic cells and preferably 2 x 10 7 - 10 10 cells, more preferably 3 x 10 7 - 10 10 cells, and even more preferably 4-5 x 10 7 - 10 10 cells.
• Specific numbers of dendritic cells in an autograft is conveniently of the order of 3 x 10 7 , preferably 5 x 10 8 , more preferably 1 x 10 8 , and even more preferably still 4-5 x 10 9 or 10 10 mature B-cells.

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