CN110241086A - Improved cell culture processes for adoptive cellular therapy - Google Patents

Abstract

The present invention relates to the improved cell culture processes for adoptive cellular therapy.New treatment cell (referred to as carrier T) is made and used in allogeneic adoptive cellular treatment background with extensive treatment benefit and the risk of GVHD is extremely low or does not have.By being changed to that there are the donor T-cells of following treatment characteristic to prepare carrier T: not including its native antigen receptor, and the treatment benefit unrelated with its native antigen specificity can be delivered.The native antigen specificity of carrier T can be height-limited, prevents it from identifying in antigen present on normal cell, can not cause GVHD, so that carrier T becomes the ideal carrier for delivering a variety for the treatment of characteristics in vivo.Carrier T makes and uses this Paradigm Change in itself not conceived previously to match the door that unrelated mode opens the treatment use of T cell with the presence or absence of HLA between donor and receptor.

Description

Improved cell culture processes for adoptive cellular therapy

The application is the divisional application of the Chinese patent application application No. is 201380030610.X, and original application is 2013 PCT International Application Serial No. PCT/the US2013/045209 submitted June 11 enters the Shen of National Phase in China on December 10th, 2014 Please.

Related application

The application is entitled " the IMPROVED METHODS OF CELL CULTURE submitted on December 8th, 2010 The part of the United States Patent (USP) No.12/963,597 (hereinafter referred to as " female case ") of FOR ADOPTIVE CELL THERAPY " after Continuous application, it is required that entitled " the IMPROVED METHODS OF CELL CULTURE FOR submitted on December 8th, 2009 The U.S. Provisional Application No.61/267 of ADOPTIVE CELL THERAPY ", 761 priority, by reference with its entirety It is incorporated herein.

Technical field

Present invention relates in general to the methods of culture cell, relate more specifically to the cell that culture is used for cell therapy.This Invention further relates to the T cell for the treatment characteristic that preparation has for adoptive cellular therapy (Adoptive Cell Therapy).

Background technique

Cell culture is the cost of cell therapy and the principal element of complexity.For current method, cell is cultivated Technique not only time-consuming cost but also high.In general, using the in vitro culture technique carried out stage by stage to prepare a large amount of cell. In the initial stage, target cell is positioned at the relatively small group in the cell composition in cell culture apparatus.? This stage, cell composition generally include: the life of source (such as peripheral blood mononuclear cells), stimulation target cell of target cell Feeder cells that are long and/or presenting antigen.Culture medium (wherein placing cell) is allowed to be generally in the culture of interference-free state Device and method is advantageous, because these cells remain relatively undisturbed state.Such device includes: standard group Knit culture plate, culture bottle and culture bag.The culture carried out in each stage is usually to comprise the steps of: allowing groups of cells The growth substrate (such as glucose) in object consumption culture medium is closed, used culture medium is removed, is used with fresh culture replacement The target cell of culture medium and the repetition process until obtaining desired amt.Often, it is needed when cell population of interest increases When additional growing surface, cell composition is transferred to the new stage for starting preparation in other devices.However, with regard to routine side For method, when the cell mass on growing surface increases, the growth rate of cell population of interest slows down.Final result is to prepare quite The cell population of interest of quantity was not only very time-consuming but also complicated.

For generating showing for T lymphocyte (EBV-CTL) with the antigentic specificity for Epstein Barr virus There is preparation method to provide an example of preparation complexity.Realizing EBV-CTL, the conventional method most preferably expanded uses mark Quasi- 24 hole tissue culturing plates, each hole have 2cm²Placement cell surface area, and due to gas transport demand and will culture Matrix product is limited in 1ml/cm².By (can be lymph matricyte system (LCL), in irradiated antigen presenting cell system with about 40 : 1 superficial density (i.e. cell/cm²Growing surface) ratio about 1 × 10⁶A PBMC/cm²With 2.5 × 10⁴A irradiated antigen is in Delivery cell/cm²) in the presence of be put into the cell composition containing PBMC (peripheral blood mononuclear cells) to start incubation.This promotes EBV-CTL group in cell composition quantitatively expands.After 9 days, in the presence of irradiated antigen presentation LCL, with 4: 1 it is new Superficial density ratio, about 2.5 × 10⁵A EBV-CTL/cm²Minimal surface density under, EBV-CTL selectively expands again Increase.It is 1ml/cm by the high specific that culture volume is limited in growth table area²So that oxygen can reach cell, this is to growth Solute (such as glucose) produces limitation.As a result, the maximum superficial density that can achieve is about 2 × 10⁶A EBV-CTL/cm²。 Therefore, maximum about 8 times (i.e. 2 × 10 of every pericyte amplification⁶A EBV-CTL/cm²Divided by 2.5 × 10³A EBV-CTL/cm²) or Below.The amplification that continues of EBV-CTL requires weekly to be transferred to EBV-CTL in 24 other orifice plates and carry out antigen to stimulate again, And culture medium and growth factor in 24 orifice plates twice in each hole are replaced weekly.Because with EBV-CTL in conventional method Superficial density reaches the possibility maximum in each hole, and EBV-CTL groups of rate of amplification is caused to slow down, so must be in longer preparation These operations are repeated in phase (being often up to 4-8 weeks), to obtain sufficient amount of EBV-CTL to survey for cell infusion and Quality Control Amount (such as sterile, identification and efficacy determinations).

The culture of EBV-CTL is only the example of the intrinsic complex cell preparation process of cell therapy.Need one kind Preparation time can be shortened while reducing the more practical cell culture processes for cell therapy of preparation cost and complexity.

We have created the new method for improving the crop growth rate in entire preparation, and thus reduction prepares answering for cell Polygamy and required time.

In adoptive cellular therapy, with native antigen specificity T cell (that is, for particular peptide T cell, it is described Particular peptide is originated from the specific target antigen presented under specific human leucocyte antigen (HLA) (HLA) allele environment) it has been applied to certainly The case where body and part HLA are matched is to treat virus infection and target tumour.In all of these situations, treatment benefit is derived from as follows It is true: (i) nave T cell Receptor recognition purpose antigen；(ii) T cell is applied to HLA equipotential needed for expression presents target peptide The receptor of gene.

In the case where allogeneic hematopoietic stem cell transplantation (HSCT), the first adoptive T cell transfer scheme is based in this way Premise: T cell contained by donor peripheral blood can in HSCT receptor mediating antitumor and/or antiviral activity.Therefore, it supplies Body lymphocyte infusion (donor lymphocyte infusions, DLI) have been widely used for provide anti-tumor immunity and Antiviral immunity power in lower degree.DLI should be comprising having the memory T of specificity thin tumour and broad range of virus Born of the same parents, although its effect is by many however, the therapy can be used successfully to treat a certain proportion of adenovirus and EBV infection Common acute virus (for example, rotavirus (RSV) and parainfluenza virus) has the low occurrence rate of T cell of specificity and of the same race The limitation of the higher occurrence rate of simplified reaction T cell.The high ratio of alloreactivity T cell and virus specific t cell Rate especially will cause problem in haploidentity (haploidentical) transplant recipient, the haploidentity transplanting by The higher incidence of graft versus host disease (GVHD) limits the tolerable dose of DLI in body, to seriously limit The dosage of the virus specific t cell received.

In order to retain the benefit of DLI and improve its safety, selectivity is inactivated or removal receptor's specificity is of the same race different The strategy of precursor reactant T cell has made assessment, including induces anergia, selective allogeneic exclusion (allodepletion) to minimize the number for the alloreactivity T cell for being applied to receptor and utilize suicide gene Internal destruction come the alloreactivity T cell missed the target.

The alternative strategy for being used to prevent and treat specific virus infection after HSCT is the in vitro expansion with antiviral activity Increase the adoptive transfer of T cell.The specific amplification of viral reaction-ive T cell has the advantages that the virus that increase can be transfused The number of specific T-cells is without increasing alloreactivity T cell.There is the enrichment antigen of reactivity for specific antigen The infusion of specific T-cells potentially improves treatment effect while reducing undesirable undershooting-effect (for example, GVHD), and It confirms, the treatment means are for treatment hematologic malignancies and solid tumor (for example, melanoma) and EBV associated malignancies (example Such as, Hodgkin lymphoma and nasopharyngeal carcinoma) it is safe and effective.

It should be noted that all therapies are required using nave T cell receptor in major histocompatibility complex (MHC) specificity of antigen is identified in the environment of molecule by nave T cell receptor (TCR).Therefore, described treatment benefit itself Dependent on use/application HLA is matched or the matched T cell in part.For example, can expand and to target melanoma cells From the expression of donor for GP100 (tumor associated antigen expressed on cancer cell) HLA haplotype (a) be directed to melanoma T cells with antigenic specificity.In this case, treatment benefit passes through natural (native or natural) T cell receptor and target Specificity interaction between antigen is to mediate.However, the interaction be only capable of there are compatibility HLA (that is, In self situation or in the case where another individual of same expression HLA) occur.This method is only capable of being extended for passing through production Life contains the cell bank of the cell line with different HLA haplotypes and controls under the case where patient matches most suitable T cell system Treat multidigit patient.

In short, in current applied all adoptive cellular therapies, treatment characteristic of the T cell for its therapeutic purposes is The native antigen specificity of donor T-cells.At least partly HLA is matched between the characteristic requirements donor and receptor, and of the same race Potential off-target effects, such as GVHD are generated in allogeneic situation.Other people propose by carrying out complicated heredity to T cell Transformation and reconstruction make it carry Chimeric antigen receptor to eliminate donor T-cells antigen receptor together, to eliminate the whole of T cell Congenital recognition capability.However, this complicates the method for preparing T cell further, this has been one of adoptive cellular therapy Main problem.

It is required to overcome the completely new approach for adoptive cellular therapy of existing complexity, to allow broadly to use In mainstream society.We disclose a new examples, and the specificity of the antigen-specific of donor T-cells is allowed to keep complete (intact), but by donor T-cells be changed to following treatment characteristic: though the native antigen specificity of donor T-cells with Its therapeutical uses is unrelated.Essentially, example transformation whether there is between donor and receptor with what is do not conceived before HLA matches the door that unrelated mode opens the treatment use of T cell.

Summary of the invention

It has been found that regularly being resettled in entire preparation process compared with current possible method by using permission The preparation process stage by stage of unconventional condition can carry out being used for cell therapy within the shorter period in a more economical way Cell preparation.The unconventional condition includes: the superficial density of the target cell of reduction (that is, number of cells/cm²), target The new ratio of cell and antigen presenting cell and/or feeder cells, and/or with increased culture volume and surface area ratio rate Use the growing surface being made of poromeric material.

Embodiment of the present invention is related to the improvement cell culture processes applied for cell therapy.It includes such as lower section Method: by using enabling cell population of interest to maintain each of more Seedling height rate in entire preparation process relative to conventional method New method is planted, time, cost and complexity needed for the target cell to reduce generation desired amt.

One aspect of the present invention depends on: carrying out culture process stage by stage and when one or more stages start Establish the condition for enabling the growth rate of cell population of interest more than current possible growth rate.In at least one rank of culture Section establishes primary condition preferably in the almost all of stage, the primary condition include: with unconventional low superficial density and With unconventional antigen presenting cell (and/or feeder cells)/target cell ratio by target cell be placed in non-breathable or On gas permeability growing surface.By the new embodiment of the application present invention in this respect, cell population of interest can be than conventional method Experience doubles more times in permitted shorter time section, so as to shorten the preparation phase.

Another aspect of the present invention depends on: carrying out culture process stage by stage and when one or more stages start Establish the condition for enabling the growth rate of cell population of interest more than current possible growth rate.In at least one rank of culture Section, preferably in the almost all of stage, set up the condition, the condition includes: with unconventional high culture volume and growth table Target cell is placed on the growing surface of poromeric material composition by area ratio rate.Pass through the application present invention in this respect new Embodiment, cell population of interest can be undergone in shorter time section more permitted than conventional method to double more times, to contract The short preparation phase.

Another aspect of the present invention depends on: it carries out culture process stage by stage and establishes the condition in each stage, so that The growth rate of cell population of interest is more than current possible growth rate.In at least one stage of culture, preferably in almost institute There is the stage, establish primary condition, the primary condition includes: with unconventional low superficial density (that is, number of cells/cm²), with The ratio of unconventional antigen presenting cell (and/or feeder cells)/target cell and unconventional high culture volume with In the presence of growth table area ratio rate, target cell is placed on the growing surface of poromeric material composition.Originally by application The new embodiment of invention in this respect, cell population of interest can be undergone more in shorter time section more permitted than conventional method Secondary multiplication, so as to shorten the preparation phase.

In some embodiments of the present invention, the Allogeneic T carrier (T- with treatment characteristic is produced Vehicle), having benefits receptor but not causes receptor by the therapeutic purposes of graft versus host disease (GVHD).

In one embodiment of the invention, therapeutic treatment is carried out by obtaining carrier T, the carrier T passes through Method comprising the following steps generate: with antigenic stimulus donor PBMC or donor Cord blood with activate to the antigen have it is natural The growth of the T cell of antigentic specificity.Thus T cells with antigenic specificity group is prepared, by (it is thin to be used to stimulation T to antigen Born of the same parents all living creatures is long) the native antigen receptor composition with antigentic specificity.T cells with antigenic specificity group, which is changed to, to be had at least It is a kind of to treat characteristic, do not include native antigen receptor and there is the treatment unrelated with the antigentic specificity of native antigen receptor Purpose, to produce carrier T group.Then, by the carrier T be delivered to can from the carrier T benefit receptor, no matter by Whether the cell of person presents the antigen that the native antigen receptor of carrier T is identified, and/or wherein the cell of receptor does not present T load The antigen that the native antigen receptor of body is identified.

In another embodiment of the present invention, therapeutic treatment is carried out by obtaining carrier T, the carrier T is logical Method comprising the following steps are crossed to generate: being had naturally to activate to antigen with antigenic stimulus donor PBMC or donor Cord blood The growth of the T cell of antigentic specificity.Thus T cells with antigenic specificity group is prepared, by (it is thin to be used to stimulation T to antigen Born of the same parents all living creatures is long) the native antigen receptor composition with antigentic specificity.T cells with antigenic specificity group, which is changed to, to be had at least It is a kind of to treat characteristic, do not include native antigen receptor and there is the treatment unrelated with the antigentic specificity of native antigen receptor Purpose, to generate carrier T group.Then, carrier T being delivered to can benefit from the carrier T and not have with carrier T The matched receptor of HLA.

In multiple embodiments of the invention, carrier T is changed to be loaded with adjuvant application recombinant protein be used to exempt from Epidemic disease therapy is changed to the treatment characteristic with chemotherapeutant for targeted therapy of cancer, is changed to antimicrobial Treat characteristic, be changed to expression assign cell tumour specificity transgenic molecules treatment characteristic, be changed to have add It is loaded with or transform as and be used to treat the treatment characteristic of autoimmune disease with recombinant protein, be changed to expression suicide gene, and/ Or be changed to be loaded with and/transform the treatment characteristic of in-vivo imaging as.

In another embodiment of the present invention, realize that preparation has desired antigen recognizing anti-by following steps The method of former specific T-cells: PBMC or Cord blood are placed in cell culture apparatus, are added and are more than into cell culture apparatus A kind of antigen, to activate the growth of more than one T cells with antigenic specificity group, each group can identify one kind of antigen, Allow a period of time that T cells with antigenic specificity is made to carry out starter population to expand, assesses culture to determine that there are at least one antigens Specific T-cells group and/or its amount determine which T cell group is suitable for continuing to be proliferated, and are only identified with suitable T cell group Antigen stimulate culture again.

In another embodiment of the present invention, realize that preparation has desired antigen recognizing anti-by following steps The method of former specific T-cells: PBMC or Cord blood are placed in cell culture apparatus, when starting into cell culture apparatus More than one antigen is added, to activate the growth of more than one T cells with antigenic specificity group, each group energy enough identifies a kind of anti- Original allows a period of time to expand T cells with antigenic specificity starter population, culture is assigned in more than one device, to every A kind of initiating antigen is only added in a device, determines which device contains the T cells with antigenic specificity group for being suitable for continuing proliferation, and Terminate the culture in the device without the T cells with antigenic specificity group for being suitable for continuing proliferation.

In multiple embodiments of the invention, donor T-cells, which are prepared into, to have only allows its identification not in normal person Exist on cell also not in the native antigen specificity of the single epitope of antigen present on normal mammalian cell.

Detailed description of the invention

Consider the following detailed description to the multiple embodiments of the present invention and the present invention can be more fully understood in conjunction with attached drawing, In the accompanying drawings:

The A of Fig. 1 show the T cells with antigenic specificity group in embodiment 1 gone through after the initial impulse in initial 7 days to Few 7 cells multiplication.

The B of Fig. 1 is shown by proving that the T in cell composition is thin determined by the tetramer analysis for embodiment 1 Born of the same parents group expands the data of amplitude at any time.

The C of Fig. 1 shows the T cells with antigenic specificity group growth rate reduction during 23 days in embodiment 1.

Fig. 2 shows a tables, and which illustrate potential (potential) of the T cells with antigenic specificity in embodiment 1 amplifications With the difference between the amplification times observed.

The A of Fig. 3 shows the presence of T cells with antigenic specificity after stimulation in embodiment 2.

The B of Fig. 3 is shown in embodiment 2 when superficial density is from 1 × 10⁶/cm²It is reduced to 3.1 × 10⁴/cm²4 are maintained simultaneously : the amplification of T cells with antigenic specificity group when the ratio of 1 T cells with antigenic specificity and antigen presenting cell.

The C of Fig. 3 is shown in embodiment 2 when superficial density is from 1 × 10⁶/cm²It is reduced to 3.1 × 10⁴/cm²Simultaneously solid The amplification of T cells with antigenic specificity group in the presence of fixed number amount antigen presenting cell.

Fig. 4 shows an example of the obtained result when continuing work described in Fig. 3, which further demonstrate,proves When the bright support for needing other cells when target cell, as long as being in the sufficient feeder cells of target cell offer and/or antigen Delivery cell, then unconventional low target cell-surface density can also originate group's amplification.

Fig. 5 is shown by with three kinds of different cell-surface density (CTL/cm²) phase starting culture come prove repeat target Cell mass expands the histogram of the ability of amplitude.

Fig. 6 shows the sectional view of the ventilation property test device for generating data.

The A of Fig. 7 shows the antigen prepared in accordance with the present invention compared with the conventional method conducted in embodiment 5 The growth curve of specific T-cells.

The B of Fig. 7 is shown for embodiment 5, and the cell survival rate of T cells with antigenic specificity prepared in accordance with the present invention is aobvious It writes and is higher than conventional method, as the forward scattering and side scatter analysis using flow cytometry are measured.

The C of Fig. 7 is shown for embodiment 5, according to the present invention the cell survival rate of prepared antigentic specificity t cell It is significantly higher than conventional method, as measured using annexin-PI (Annexin-PI) 7AAD.

The D of Fig. 7 is shown for embodiment 5, in new method of the invention the superior growth of prepared cell show with Using the identical cell specific growth rate of the cell of conventional method culture, such as by the daily fluidic cell of CFSE label cell Determined by art analysis, so that it is determined that cell rate of amplification increase is caused by being reduced by cell death.

The A of Fig. 8 shows how EVB-CTL in the case where no replacement is required culture medium reach more than can in conventional method Attainable amplification.

The B of Fig. 8 shows the condition of culture of embodiment 6 as why not changed final cell product, as using EBER is directed to What Q-PCR was assessed.

The C of Fig. 8 shows the condition of culture of embodiment 6 as why not changed final cell product, such as using for B cell mark What the Q-PCR of will object CD20 was assessed.

Fig. 9 shows an illustrative example, and wherein we have been experimentally confirmed target cell and antigen presenting cell Low-down cumulative surface density (in the case, AL-CTL merge with LCL cell generation have 30,000 cell/cm² The cell composition of superficial density) the AL-CTL groups of growths cannot be originated.

The A of Figure 10 presents the data of embodiment 8, these data cultivate two kinds of new sides of cell during being shown in 23 days How method than conventional method prepares more cells.

The B of Figure 10 shows the cell photo cultivated in test device in embodiment 8.

The C of Figure 10 is shown in embodiment 8, and two kinds of new cultural methods and conventional method are all prepared with identical performance The cell of type.

The D of Figure 10 is shown for embodiment 8, wherein with the EBV of LMP1, LMP2, BZLF1 and EBNA1 from EBV Peptide epitopes stimulation T cell simultaneously shows similar frequency with the representative culture that HLA-A2-LMP2 peptide pentamer decoration method is dyed The peptide-specific T-cell of rate.

The E of Figure 10 shows the new method and conventional method for embodiment 8, and cell maintains its dissolved cell activity and special Property and kill self EBV-LCL to mismatch the low killing rate of EBV-LCL to HLA, such as utilize⁵¹Cr release measurement is assessed.

Figure 11 shows the group's amplification and target in normal conditions using the target cell type of one aspect of the invention Cell mass expands the diagram to compare at the growth surface.

Figure 12 is shown can be by using the growing surface being made of poromeric material and more than 1 or 2ml/cm²'s One example of the obtained advantage of ratio of unconventional high culture volume and growth table area.

Figure 13 show under regular situation the amplification of cell population of interest at the growth surface in an embodiment party of the invention The new method diagram that group's amplification of target cell type compares under case, cell surface at the end of embodiment of the present invention are close Degree is much larger than conventional surface density.

Figure 14 shows another new method for preparing cell, which additionally provides further better than conventional method The advantages of.

Figure 15 shows the comparison to each preparation method described in Figure 14, the effect of to confirm new method and why It can be used for the reason of obtaining abundant efficiency in adjustment of various stages preparation method.

Figure 16 is shown as preparation carries out, and how people can adjust the preparation method in new method and obtain the one of efficiency A example.

Figure 17 shows test results, it was demonstrated that carrier T cannot be identified from the cell for mismatching allogeneic donor.

Figure 18 shows test result, show donor T-cells can be altered to create with CD34 Δ-IL7 cell because The carrier T for the treatment characteristic that sublist reaches, as determined by using flow cytometer showed.

Figure 19 A shows test result, shows that the systemic delivery of IL7 cell factor causes to detect in the kidney of mouse Cell factors more more than its tumor locus.

Figure 19 B shows test result, shows that the carrier T delivering of IL7 cell factor causes at mouse tumor position than it The higher cytokine concentrations of his organ, and show after applying carrier T, how the generation of cell factor holds at tumour Continue at least two weeks.

Figure 20 shows test result, and it is special to show that donor T-cells can be altered to create the treatment with CAR-PSCA Property carrier T, as using determined by flow cytometer showed.

Figure 21 shows test result, shows that the carrier T of the treatment characteristic with CAR-PSCA being capable of tumor eradication cell.

Figure 22 A shows the tumour cell of the neighbouring expression IL4 cell factor of carrier T with the receptor that can combine IL4.

Figure 22 B shows how carrier T can combine IL4 cell factor, and protect the IL4 cell factor of tumour cell How amount can be greatly reduced.

Figure 23 shows test result, it was demonstrated that has the treatment characteristic for expressing extracellular recombinant cytokine receptor IL4R/7 Carrier T can consume IL4 cell factor.

Figure 24 A show be loaded with chemotherapeutant carrier T will how to inflammation part migrate.

Figure 24 B shows how receptor's immune system will target carrier T positioned at tumour cell position.

Figure 24 C is shown when by receptor's immune system attack, and how carrier T at tumour cell position will discharge its load Lotus, the load are chemotherapeutant in the case.

Specific embodiment

Definition

Antigen presenting cell (APC): serve triggering target cell to specific antigen generate response cell.

CTL: cytotoxic T cell

Target cell: certain types of cell, the purpose of preparation process are to expand it quantitatively.In general, target Cell is non-adherent (non-adherent) cell, the example include: regulatory T cells (Treg), natural killer cells (NK), Tumor infiltrating lymphocyte (TIL), primary T lymphocyte and broad category of antigen-specific cellular etc. (all cells Genetic modification can also be carried out, to improve its function, internal persistence or safety).It can use feeder cells and/or antigen Cell needed for clinical application is expanded in delivery cell, the feeder cells and/or antigen presenting cell may include: PBMC, PHA mother cell, OKT3T, B mother cell, LCL and K562, (it is natural or by genetic modification come express and antigen And/or epitope and costimulatory molecules such as 41BBL, OX40, CD80, CD86, HLA and other many kinds), can use or It can not have to peptide or other related antigens and pulse is carried out to these cells.

EBV:Epstein Barr virus

EBV-CTL: a kind of T cell is identified by T cell surface receptor specificity by EBV infection cell or expression Or present the cell of the peptide from EBV.

EBV-LCL: the B lymph matricyte system through Epstein Barr virus Transformation.

Feeder cells: cell of the effect to cause target cell numbers to expand.In some cases, antigen presenting cell It may be used as feeder cells.

Growing surface: the region that culture apparatus inner cell is placed.

PBMC (peripheral blood mononuclear cells): the peripheral blood mononuclear cells obtained from peripheral blood, the peripheral blood are some mesh It marks the source of cell and feeder cells can be served as.

Responsive cell (R): the cell of response will be generated to stimulation cell.

Stand cell culture: a method of cultivating cell in the medium；That is, in addition to culture apparatus is being carried out position It is mobile to carry out conventional treatment and/or periodically to cell add fresh culture situations such as other than, without stirring or mixing.One As for, the culture medium in stationary culture is generally in stationary state.The present invention relates to stand cell culture processes.

Stimulated: the effect of antigen presenting cell and/or feeder cells to target cell.

Stimulation cell (Stimulator (S)): it will affect the cell of responsive cell.

Superficial density: the cell quantity of the per unit area on device inner cell surface placed on it.

To attempt to find the new method for the preparation for simplifying the cell population of interest for adoptive T cell therapy, a system has been carried out Column experiment, to open the door of the more efficient culture of the cell for cell therapy application.To of the invention many illustrative Example and various aspects are described, to indicate how to obtain relative to conventional method shortening preparation time and reduce complexity The ability of property.

Embodiment 1: to the proof of the limitation of conventional method.

The data of this embodiment demonstrate use every hole 2ml culture volume (that is, culture medium height is 1.0cm, culture The long-pending ratio with surface area of matrix is 1ml/cm²) 24 hole tissue culturing plate of standard (surface area in i.e. every hole be 2cm²) in preparation The limitation of the conventional culture methods of EBV-CTL.

1st stage of culture, the 0th day: by with the self EBV-LCL of antigen presentation through gamma-ray irradiation (40Gy), With 40: 1 (PBMC: LCL) ratio and 1ml/cm²Culture volume and growing surface ratio, to come from Normal donor The cell composition (about 1 × 10 of PBMC⁶A cell/ml) cultivated and originated the EBV-CTL groups of amplifications, as a result, with 45%Click culture medium (Irvine Scientific, Santa Ana, CA), 2mM GlutaMAX-I and 10%FBS supplement RPMI 1640 in establish about 1 × 10⁶A cell/cm²Cell composition superficial density.

2nd stage of culture, the 9-16 days: in the 9th day, harvesting EBV- from the cell composition that the first stage generates CTL, by them with 0.5 × 10⁶A EBV-CTL/cm²Superficial density be resuspended in fresh culture, and be irradiated it is self EBV-LCL is with 4: 1 CTL: LCL ratio (superficial density 0.S × 10⁶A CTL/cm²∶1.25×10⁵A LCL/cm²) carry out again Stimulation.In the 13rd day, the 1ml in the 2ml culture volume in each hole of 24 orifice plates is removed, with 1ml (50U/ containing recombinant human il-2 mL)(Proleukin；Chiron, Emeryville, CA) fresh culture replaced.

In 3rd stage of culture, the 17-23 days: the condition for repeating for the 2nd stage wherein added weekly IL-2 twice, in the 23rd It terminates culture.Although terminating culture, other culture ranks in similar (mimick) the 2nd stage and the 3rd stage can also be used Section continues to cultivate.

Be used as the cell line and tumour cell of target cell in cytotoxic assay: BJAB (a kind of B cell lymphoma) and K562 (a kind of chronic red system's leukaemia (chronic erythroid leukemia)) is from American Type Tissue Culture What the heart (ATCC, Rockville, MD, the U.S.) obtained.By all cell culture in containing 10% heat-inactivated fetal bovine serum (FCS), 2mM L-Glutamine, 25IU/mL penicillin and 25mg/mL streptomysin (all obtained from BioWhittaker, Walkersville, MD) 1640 culture medium of RPMI (GIBCO-BRL, Gaithersburg, MD) culture in.Containing 5%CO₂Humidifying air in 37 DEG C of culture cells.

Immunological classification:

Cell surface: green with phycoerythrin (PE), fluorescein isothiocynate (FITC), perdinin (periodin) leaf Cellulose protein (PerCP) and for CD3, CD4, CD8, CD56, CD16, CD62L, CD45RO, CD45RA, CD27, CD28, The monoclonal antibody (MAb) of allophycocyanin (APC) conjugation of CD25, CD44 (is obtained from Becton-Dickinson (Mountain View, CA, the U.S.)) cell is dyed.Using PE conjugation the tetramer (Baylor College of Medicine) and The pentamer (Proimmune Ltd, England Oxford) of APC conjugation quantifies EBV-CTL Precursor frequency.? 10,000 and 100,000 realities dyed for cell surface and pentamer are obtained on FACSCalibur flow cytometer respectively When event (live event) and utilize Cell Quest software (Becton Dickinson) carry out data analysis.

For measuring fissional CFSE label: in order to assess multiplication factor, by 2 × 10⁷A PBMC or EBV specificity CTL (EBV-CTL) is cleaned twice, and is resuspended in the 1 of 850 μ l (FBS) containing 0.1% fetal calf serum (Sigma-Aldrich) In × phosphate buffered saline (PBS) (PBS).Before dyeing, the Carboxyfluorescein diaccete succinimidyl ester of aliquot is taken (CFSE) (10mM is in dimethyl sulfoxide) (Celltrace^tmCFSE cell proliferation reagent box (C34554), Invitrogen) solution Freeze, is added in cell suspending liquid (label concentration is 1 μM) with 1 × PBS dilution 1: 1000 and by 150 μ l dilutions.In room temperature It is lower with CFSE by cell incubation 10 minutes.Then, 1mlFBS is added in cell suspending liquid, is then incubated for 10 points in 37 DEG C Clock.Then, cell is cleaned twice with 1 × PBS, counts, is stimulated with antigen as described above.

Annexin V -7-AAD dyeing: the percentage of apoptotic cell and non-viable non-apoptotic cell in the culture in order to determine us, We according to manufacturer specification (BD Pharmingen^tm, #559763, San Diego, CA) and carry out annexin -7- AAD dyeing.In short, the EBV-CTL obtained from 24 orifice plates or G-Rex is cleaned with cold PBS, by these cells with 1 × 10⁶ The concentration of a cell/ml is resuspended in 1 × combination buffer, uses annexin V-PE and 7- in the dark in room temperature (RT, 25 DEG C) AAD dye within 15 minutes.After incubation, cell is analyzed with flow cytometry immediately.

Chromium release assay: as previously mentioned, our 4 hours in standard⁵¹The cell of EBV-CTL is had evaluated in Cr release measurement Cytotoxic activity.We are made using the unmatched lymph matricyte system (EBV-LCL) through EBV conversion of self and I class and II class HLA The restricted and non-limiting killing of MHC is measured for target cell, and measures Nk Cell Activity using K562 cell line. The chromium for being utilized respectively in individual culture medium or being incubated in 1%Triton X-100 marks target cell to determine Spontaneous is (spontaneous) and maximum⁵¹Cr release.The mean percent of the Specific lytic in triplicate hole is calculated as follows Rate: [(test counting-spontaneous counting)/(the spontaneous counting of maximum count -)] × 100.

Elisa (ELIspot) measurement: IFN γ is secreted in response to antigenic stimulus using ELIspot analysis T cell frequency and function quantified.It is mixed with irradiated LCL (40Gy) or LMP1, LMP2, BZLF1 and EBNA1 peptide Close object (being diluted to 1 μ g/ml) (JPT Technologies GmbH, Berlin, Germany) or EBV peptide HLA-A2GLCTLVAML= GLC, HLA-A2CLGGLLTMV=CLG, HLA-A2-FLYALALLL=FLY and HLA-A29ILLARLFLY=ILL (Genemed Synthesis Co., Ltd, San Antonio, Texas) is to the CTL cell expanded in 24 orifice plates or G-Rex System is stimulated, and 2 μM of ultimate density is diluted to, and individual CTL is used as negative control.With 1 × 10⁶CTL is resuspended in by/ml In ELIspot culture medium [with 5% human serum (Valley Biomedical Co., Ltd, Winchester, Virginia) and 2mM L-Glutamine (GlutaMAX-I, Invitrogen, Carlsbad, CA) supplement RPMI1640 (Hycloue, Logan, UT)].In 4 DEG C with the anti-IFN-γ antibody of 10 μ g/mL (Catcher-mAB91-DIK, Mabtech, Cincinnati, OH) it is coated with 96 hole filter plates (filtration plate, MultiScreen, #MAHAS4510, Millipore, Bedford, MA) Overnight, it then cleans, is closed 1 hour in 37 DEG C with ELIspot culture medium.Responsive cell and stimulation cell are incubated over the plates It educates 20 hours, then plate is cleaned to and used the anti-IFN-γ monoclonal secondary antibody (Detector-mAB (7-B6-1- of biotin-conjugated Biotin), Mabtech) it is incubated for, then use Avidin: biotinylated horseradish peroxidase complex (Vectastain Elite ABC kit (standard), #PK6100, Vector Laboratories, Burlingame, CA) It is incubated for, is then developed (Sigma, St.Louis, MO) with AEC substrate.For each condition of culture, in triplicate into Row.The Zellnet Consulting company that each plate is sent to New York, NY is assessed.To point formed unit (SFC) and Input cell quantity is drawn.

Statistical analysis: vitro data is indicated with average value ± 1SD.It is examined using student t to determine difference between each sample Significance,statistical receives to indicate significant difference with P < 0.05.

Under these culture conditions, in originally post-stimulatory first 7 days, T cells with antigenic specificity group undergoes at least 7 times thin Born of the same parents' multiplication, as shown in Figure 1A.Therefore, indeed it is contemplated that T cell expands 128 times (such as by that will resist in cell composition weekly What the frequency of former specific T-cells was measured multiplied by total number of cells).By first, second, and third post-stimulatory tetramer sun The frequency of property cell is shown in Figure 1B.In the 0th day, there is the frequency of reactive T cell to two EBV tetramers (RAK and QAK) Respectively 0.02% and 0.01%.After single stimulation in the 0th day, to the tetramerpositive T cell in the 9th day cell composition Frequency increase to 2.7% and 1.25% from 0.02% and 0.01% respectively.Accordingly, there exist the antigen in cell composition is special The percentage of anisotropic tetramerpositive T cell reaches the increase of 135 times and 125 times, as measured by RAK and QAK.This Outside, after the single stimulation in the 0th day the 1st stage of culture, observed that the superficial density of cell in cell composition increases by the 9th day It is added to 1.1 times (data are not shown) and (has about 1.1 × 10⁶A cell/cm²).Because of the most cells in PBMC composition It is nonspecific to stimulator antigen, so observing that the whole of total cell number increases seldom, but in phase first stage of culture Between in composition the amplification times of antigen-specific cellular group be about 280 times, as is shown in fig. 1C.Regrettably, although cell The number of multiplication is identical (as measured by CSFE) during second and the phase III of culture, but culture second or The rate of amplification of this T cells with antigenic specificity does not continue during phase III, second stage be only 5.7 and in the phase III Only 4.3.Difference (the n that the table of Fig. 2 illustrates the potential amplification of T cells with antigenic specificity between the amplification times observed =3).

Embodiment 1 proves that time quantum needed for substantially preparing target cell after a week the of preparation is usually extended, because To be reduced in the rate of amplification of the follow-up phase cell population of interest of culture.

Embodiment 2: at the beginning of any Given Order section or culture each stage, can pass through reduces the thin of cell population of interest Cellular surface density increases the shortening of cell population of interest the amount of time required to realize.

We assume that compared with first time stimulates, the rate of amplification decline of cell population of interest after second of T cell stimulates The reason of be to cause activation to cause the restrictive cell condition of culture of cell death (AICD).For example, referring to Fig. 3 A, first When secondary stimulation, the 23Kda VCA specific T-cells ingredient of PBMC at most accounts for the 2% of group, and therefore antigentic specificity responsiveness The inoculum density of T cell is less than 2 × 10⁴/cm², the feeder cells that remaining PBMC serves as non-proliferative (are shown as in Fig. 3 A CFSE positive cell), these feeder cells maintain the optimal cell-cell contact for enabling Peptide-specific CTL to be proliferated.Phase Instead, in second of stimulation in the 9th day, most of T cell is antigentic specificity, although the total cell density of composition is big Cause identical, but proliferative cell density is then higher by 50 to 100 times.Therefore, when stimulating again, most cells are proliferated, thus can be fast Speed consumes and exhausts their nutrition and O₂Supply.

In order to determine whether restricted condition of culture causes suboptimum T cell growth rate, we are measured with lower thin The amplification of the activating T cell of born of the same parents' density bed board.Method is described in the embodiment 1 of front.

The EBV specific T-cells of activation are inoculated in the hole of 24 orifice plate of standard by we, and each hole has 2cm²Growth table Area causes reduced superficial density (from 1 × 10 when doubling dilution⁶/cm²It is reduced to 3.1 × 10⁴/cm²) 4: 1 are maintained simultaneously Responsive cell and stimulation the ratio between cell (R: S), as shown in Figure 3B.With 1.25 × 10⁵/cm²Starting CTL superficial density obtain Maximum CTL amplification (4.7 ± 1.1 times) is obtained, but further dilution then reduces rate of amplification, as shown in Figure 3B.We push away Surveying this restricted dilution effect may be that we, which use, within 7 days periods fixes due to caused by lacking cell-cell contact The feeder cells of quantity are (with 1.25 × 10⁵/cm²Superficial density bed board EBV-LCL) with from 1 × 10⁶To 3.1 × 10⁴Table The multiplication dilution of surface density culture EBV-CTL and assess cell amplification.It is observed that CTL amplification dramatically increases, from 1 ×10⁶/cm²Superficial density under EBV-CTL only 2.9 ± 0.8 times of amplifications until 3.1 × 10⁴/cm²Superficial density under 34.7 ± 11 times of amplifications of EBV-CTL, as shown in FIG. 3 C.Importantly, the change of this condition of culture and having not been changed cell Function or antigentic specificity (data are not shown).Therefore, the T cells with antigenic specificity group of activation, which can be realized, compares routine culture Method allows bigger amplification.It is worth noting that, the maximum surface obtained after stimulation is close regardless of initial sheet density Degree (1.7 to 2.5 × 10⁶/cm²) it is identical.

Therefore, conventional condition of culture is restrictive, this shows the ratio needs of culture volume and growth table area Increase to above conventional 1ml/cm²To allow cell population of interest to increase to above the superficial density limitation of conventional method.This Outside, by any cultivation stage start the superficial density of cell population of interest is decreased below conventional method, can obtain Reach about 34 times of better Peptide-specific CTL amplification.This has significant as a result, because preparing in cell therapy Cell quantity is often fairly limited when beginning.For example, by with reduced superficial density by the target cell of limited quantity It is distributed on increased surface area, bigger cell population of interest can be obtained within the shorter period, because relative to conventional table Group's growth rate dramatically increases for surface density.

Embodiment 3: the minimal surface density of the cell mass comprising target cell and/or antigen presenting cell can permit with The cell population of interest growth of low-down superficial density inoculation.

Fig. 4 shows an example of our the obtained results when continuing work described in Fig. 3, and the example is further It demonstrates when target cell needs the support of other cells, as long as providing sufficient feeder cells to target cell and/or resisting Original is in delivery cell, and low targeted cell surface density can originate group's amplification unconventionally.In these experiments, we continue Prove that there are about 1.0 × 10 under 8 to 1 R: S ratio⁶A target cell/cm²It is only under 1 to 32 R: S ratio About 3900 target cell/cm²Between superficial density and R: S ratio under total cell composition how to make target cell significantly It expands to being more than 50 times of initial sheet density, when amplification is to 50 times, we stop testing.

Embodiment 4: be proved by with one stage of starting of unconventionally low targeted cell surface density, allow group Body amplification terminates the stage and repeat condition and makes preparation process duplicate ability can deliver repeatable result in each stage.

We are with three targeted cell surface density (CTL/cm²) continue assessment described in embodiment 3, such as institute in Fig. 5 Show.Each specific inoculum density can realize identical amplification times always.The influence will be described in more detail below, Because these influence related with the significant shortening ability of cell population of interest preparation time.

Embodiment 5: by the training objective cell on the growing surface being made of poromeric material while increasing culture medium The ratio of volume and growth table area increases what cell population of interest can double relative to conventional method in the given stage of culture Number and increase accessible superficial density.

Cell line and tumour cell, immunological classification, CFSE label, annexin V -7-AAD dyeing, chromium release are surveyed Fixed, elisa (ELIspot) measurement, retrovirus preparation and the transduction of T lymphocyte and statistical analysis description In embodiment 1.

The construction of test device (being hereinafter generally known as " G-Rex ") is as shown in Figure 6.The bottom of each G-Rex 10 Portion 20 is made of gas permeability silica gel (silicone) film, and the thickness of the film is about 0.005 to 0.007 inch.The beauty of co-pending State patent disclosure US2005/0106717 A1 (hereinafter referred to as Wilson ' 717) is to use phase with the poromeric material of alternative Close many other information sources in one, and using the patent disclosure by the shape of gas permeability culture apparatus, feature with And those skilled in the art are informed to many advantageous other useful features of embodiment of the present invention.In this embodiment 3, G- Rex (referred to as " G-Rex40 ") has 10cm²Growing surface region, cell composition (being shown as item 30) is in the growing surface The feature of upper placement, cell composition changes in entire experiment, as described herein.Unless otherwise stated, training The volume for supporting base (being shown as item 40) is 30ml, to form 3ml/cm²Culture volume and growth table area ratio.

With 4: 1 conventional CTL: LCL ratio by the EBV specific CTL of activation and irradiated self EBV-LCL in G- It is cultivated in Rex40 device.With 5 × 10⁵A cell/cm²Superficial density EBV-CTL is inoculated in G-Rex40, will The rate of amplification of EBV-CTL group with culture volume is inoculated in than growth table area as 1ml/cm with similar face density²'s The rate of amplification of EBV-CTL in 24 orifice plate of standard is compared.It is as shown in Figure 7A (p=0.005) after 3 days, without In the case where the replacement of any culture medium, EBV-CTL in G-Rex40 is from 5 × 10⁵/cm²Increasing to intermediate value is 7.9 × 10⁶/cm² (5.7 to 8.1 × 10⁶/cm²In the range of).On the contrary, is carried out in conventional 24 orifice plates EBV-CTL that 3 days cultivate only from 5 × 10⁵/cm²Superficial density increase to the 3rd day intermediate value be 1.8 × 10⁶/cm²(1.7 to 2.5 × 10⁶/cm²In the range of). In G-Rex40, superficial density can be further increased by supplementing culture medium, and cannot pass through the training in 24 orifice plates of supplement Base or IL-2 are supported to increase cell-surface density.For example, after the 7th day supplementing culture medium and IL-2, EBV-CTL in G-Rex40 Superficial density be further increased to 9.5 × 10⁶A cell/cm²(8.5 × 10⁶To 11.0 × 10⁶/cm²In the range of) (number According to being not shown).

In order to understand the mechanism of superior cell amplification behind in G-Rex device, in culture the 5th day, we were thin using streaming The survival rate for the periphery blood T cell that born of the same parents' forward scattering analysis and side scatter analysis assessment are stimulated through OKT3.Due in culture Middle presence can interfere the remaining irradiated EBV-LCL of analysis, thus EBV-CTL cannot be assessed in this measurement.In Fig. 7 B Shown, significantly higher (survival rate in G-Rex40 is 89.2% to cell survival rate, relative to 24 orifice plates in G-Rex40 culture In survival rate be 49.9%).Then, we daily analyze culture with annexin-PI 7AAD, continue 7 days, To distinguish living cells and apoptosis/non-viable non-apoptotic cell, observe that the T cell survival rate expanded in 24 orifice plates is consistently lower than the T in G-Rex Cell survival rate, as shown in fig. 7c.These statistics indicate that, the accumulation of proliferative cell improves survival rate, this is because with 24 holes Plate is compared, and the cell number in G-Rex device increases.

In order to determine relative to 24 orifice plates in G-Rex whether there are also as caused by frequency dividing cell increase as a result, in T cell is marked with CFSE within 0th day and by T cell point in the G-Rex40 device that culture volume is 40ml and respectively Hole culture volume is in 24 orifice plates of 2ml.Daily flow cytometry proves day cell division time from the 1st day to the 3rd Number indifference.However, the cell population of interest cultivated in G-Rex40 was with the rate of the reduction rate more than the hole 2ml since the 3rd day It continues growing, shows that condition of culture has become restrictive, as shown in Figure 7 D.Therefore, big in G-Rex40 test device Caused by cell population of interest is the synergy for being reduced and being continued to multiply due to the cell mortality relative to conventional method.

Embodiment 6: by using the ratio of unconventionally high culture volume and growth table area and use is by saturating The growing surface that gas material is constituted can reduce the demand during preparing to raising culture, while it is high to obtain unconventional ground Targeted cell surface density.

This is confirmed by using the G-Rex test device of starting and amplification for EBV: LCL.For this implementation The purpose of example, G-Rex2000 refer to device as shown in Figure 8, and exception is that bottom is by 100cm²Growth table area is constituted simultaneously And provide 2000ml culture medium capacity.In the case where not changing cell phenotype, EBV-LCL is trained in G-Rex2000 It supports and expands.With 1 × 10⁵A cell/cm²Superficial density by EBV-LCL together with the complete RPMI culture medium bed board of 1000ml in G- In Rex2000, to form 10ml/cm²Culture volume and surface area ratio.In order to compare, with 5 × 10⁵A cell/cm² Superficial density in T175 bottles and the complete RPMI culture medium of 30ml is added in EBV-LCL bed board, be about 0.18ml/ to be formed cm²Culture volume and surface area ratio.As shown in Figure 8 A, the case where being replaced without any operation or culture medium Under, the amplification of the EBV-LCL cultivated in G-Rex2000 is greater than the amplification in T175 bottles.This condition of culture does not change final thin Born of the same parents' product, as assessed using the Q-PCR for EBER and B cell marker CD20, as shown in Fig. 8 B and Fig. 8 C.

Embodiment 7: when, there is no when sufficient feeder cells and/or cell antigen, target cell is then when cultivating beginning It may not expand.However, it is possible to change cell composition and it is made to contain its for serving as feeder cells and/or antigen presenting cell Its cell type, so that amplification be made to be able to carry out.

Fig. 9 shows an illustrative example, and wherein we have been experimentally confirmed target cell and antigen presenting cell Low-down cumulative surface density (it is 30,000 thin that in the case, AL-CTL and LCL cell, which are jointly formed superficial density, Born of the same parents/cm²Cell composition) the AL-CTL groups of growths cannot be caused.However, containing it by change composition serves as feeding Another cell type for supporting cell can be such that the same cell composition grows.In the case, we have rated about 0.5×10⁶A cell/cm²Lower three kinds of superficial density various forms of irradiated K562 cells feeder layer, in all situations Lower AL-CTL groups be all the initial cell composition shown in first column of histogram expand, from only 15 in 14 days, 000 cell/cm²Superficial density change to 4.0 × 10⁶A cell/cm²Superficial density.We also demonstrate, with addition Third cell type is different, and the group for increasing LCL obtains similar favourable outcome.Arbitrarily selection is for LCL's or K562 High superficial density can be with to prove when cell composition contains sufficient amount of feeder cells and/or antigen-specific cellular Using low-down cell population of interest come initial growth.When feeder cells short supply, valuableness or preparation trouble, it is proposed that The superficial density of feeder cells is decreased below 0.5 × 10⁶A cell/cm².It is proved generally speaking and as us, when thin When having antigen presenting cell and/or feeder cells in born of the same parents' composition, antigen presenting cell and/or feeder cells and target cell Increased superficial density should preferably be at least about 0.125 × 10⁶A cell/cm², to form foot in cell composition Enough superficial densities are with the amplification of initial target cell mass.In addition, continuing to expand to achieve over standard surface density limit Increase, uses the growing surface and 4ml/cm being made of poromeric material in this embodiment²Culture volume and surface area Than.

Embodiment 8: compared with other methods, the superficial density of reduced target cell, the responsive cell of change and stimulation The ratio of cell, the ratio of increased culture medium and growth table area and periodically cell distribution is existed with low superficial density culture On the growing surface being made of poromeric material, these make that more target cells can be prepared simultaneously within the shorter period Simplify preparation process.

In order to further evaluate our simplification and shorten the ability of target cell preparation, we use G-Rex test dress Set the starting and amplification for EBV-CTL.For the purpose of this embodiment, G-Rex500 refers to device as shown in Figure 6, example It is bottom is outside by 100cm²Growth table area constitutes and provides 500ml culture medium capacity.

For the initial phase of EBV-CTL preparation, we are by PBMC with 1 × 10⁶/em²Superficial density (10 will be added up to⁷ A PBMC is distributed in 10cm²On the growing surface of G-Rex40) it is inoculated in G-Rex40, and with EBV-LCL with 40: 1 PBMC: EBV-LCL ratio stimulates these cells.It prepared by CTL, in the first time stimulation for the antigentic specificity for maintaining responsiveness T cell In this 40: 1 ratio be preferred.After the initial phase of culture, in the 9th day beginning second stage, wherein by 1 × 10⁷It is a to answer The property answered T cell is transferred in G-Rex500 test device from G-Rex40.In order to originate the second stage of culture, by 200ml's CTL culture medium is placed in G-Rex500, and 2ml/cm is formed when second stage starts²Culture volume and surface area ratio The culture medium height for being higher by growing surface region of rate and 2.0cm.When second stage starts, the surface of target cell is close Degree is 1 × 10⁵A CTL/cm²And the superficial density of antigen presenting cell is 5 × 10⁵A LCL/cm², it is consequently formed unconventional 1 : 5 target cell and the ratio of antigen presenting cell.In this stage, two kinds of cell-surface densities and R: S ratio are in all screenings Donor in cause consistent EBV-CTL to expand.After four days (the 13rd day), by IL-2 (ultimate density 50U/ml) and 200ml fresh culture is directly appended in the culture, and the ratio of culture volume and surface area is adjusted to 4ml/cm².In It 16th day, collects cell and counts.CTL mean face density obtained is 6.5 × 10⁶/cm²(2.4 × 10⁶To 3.5 × 10⁷In the range of).

Compared with conventional scheme, make increased culture volume and surface using the growing surface being made of poromeric material Long-pending ratio (is greater than 1ml/cm²), reduce cell-surface density (i.e. less than 0.5 × 10⁶/cm²) and change responsive cell It is possibly realized with the ratio (less than 4: 1) of stimulation cell, so as to cause the shortening of preparation time.Figure 10 A shows embodiment 8 Compared with this G-Rex method G-Rex method described in the conventional method and embodiment 5 that embodiment 1 uses.As schemed Show, conventional method needs the target as much for delivering over 23 days with being delivered in about 10 days in any one G-Rex method Cell.After 23 days, the target that the G-Rex method of embodiment 8 can prepare 23.7 times more than the G-Rex method of embodiment 5 is thin Born of the same parents and prepare 68.4 times more than the conventional method than embodiment 1 of target cell.In addition, thin without additional antigen presentation In the case that born of the same parents stimulate, target cell continues division until the 27-30 days, as long as when cell-surface density is greater than 7 × 10⁶/cm² When just culture is separated.

Although CTL cannot be clearly seen using optical microscopy in G-Rex, CTL cell cluster can with eyes or Person is seen with inverted microscope, is shown in Figure 10 B in the appearance of the 9th, 16 and 23 day cell of culture.As shown in figure 10 c, Culture in G-Rex does not change the phenotype of amplifying cells, is wherein CD3+ cell (G-Rex more than 90% in cell composition In be 96.7 ± 1.7, relative to being 92.8 ± 5.6 in 24 holes), wherein mainly CD8+ (62.2% ± 38.3, relative to 75% ±21.7).Activation marker CD25 and CD27 and the evaluation for remembering marker CD45RO, CD45RA and CD62L are demonstrated Without substantial differences between the EBV-CTL expanded under each condition of culture.Antigentic specificity is not also influenced by condition of culture, just As measured by being analyzed using ELIspot and pentamer.Figure 10 D shows representative culture, wherein with from LMP1, LMP2, The EBV peptide epitopes of BZLF1 and EBNA1 carry out stimulation and are shown with the T cell that HLA-A2-LMP2 peptide pentamer decoration method is dyed The peptide-specific T-cell of similar frequencies is shown.In addition, amplification cell maintain they dissolved cell activity and specificity and kill Hurting self EBV-LCL, (under 20: 1 E: T ratio, 62% ± 12 relative to 57% ± 8；G-Rex is relative to 24 orifice plates), it is right HLA mismatch EBV-LCL killing rate it is low (15% ± 5 relative to 12% ± 7, E: T ratio be 20: 1), such as utilize⁵¹Cr release is surveyed It is evaluated calmly, as illustrated in fig. 10e.

Discussion to the various new methods of the improved cell preparation for cell therapy: embodiment 1-8 use has been given It (include: the reduced table of cell population of interest when manufacturing cycle starts to disclose the use of various conditions to those skilled in the art Superficial density ratio, the growing surface that is made of poromeric material between surface density, the responsive cell of reduction and stimulation cell And/or the ratio of the culture volume and growth table area increased) how can be used to accelerate and simplify grinding for cell therapy Study carefully and is prepared with the cell of clinical application.Although embodiment 1-8 is related with preparing for T cells with antigenic specificity, these new cultures Condition also can be applied to many important (or needed for Preclinical evidences of concept mouse model) with clinical meaning Suspension cell type, these cell types include: regulatory T cells (Treg), natural killer cells (NK), tumor-infiltrated lymph (all these cells can also all be lost for cell (TIL), primary T lymphocyte, the extensive antigen-specific cellular of type etc. Modification is passed, to improve its function, internal persistence or safety).Feeder cells and/or antigen presenting cell be can use to expand Increase cell, the feeder cells and/or antigen presenting cell may include: PBMC, PHA mother cell, OKT3T, B mother cell, LCL And K562, (natural or being expressed by genetic modification and antigen and/or epitope and costimulatory molecules such as 41BBL, OX40L, CD80, CD86, HLA etc.), can with or peptide and/or related antigen can not had to pulse is carried out to these cells.

Low initial sheet density unconventionally: one aspect of the present invention be have found it is thin by using lower target Cellular surface density can shorten preparation time relative to conventional method.In this way, target cell can have than conventional side Difference between method permitted bigger the smallest cell superficial density and maximum cell superficial density.Preferably, when target is thin It is close with low initial sheet again when born of the same parents' group's growth rate has started to reduce but the quantity of target cell still is not enough to terminate preparation Degree redistributes target cell on the additional growing surface being made of poromeric material.

In order to explain that the method depends on any given culture rank how using our this new cell preparation methods Lower superficial density when section starts, is described one embodiment now.Figure 11 shows and utilizes a side of the invention The group of the target cell type in face expands figure compare, that cell population of interest expands at the growth surface in normal conditions Show.In this new method, when preparatory phase starts, the superficial density of target cell is less than conventional surface density.In order to protrude this The advantages of new method, the explanation of this part do not describe the technique for initially obtaining cell population of interest." date " of culture starts from " 0 " So that those skilled in the art can more easily determine the relative time advantage of this new method.In this embodiment, routine side Each manufacturing cycle of method starts from 0.5 × 10⁶A target cell/cm²Conventional surface density, and each preparation of this embodiment is all Phase starts from much lower and unconventional superficial density 0.125 × 10⁶A target cell/cm².Therefore, it needs in this embodiment It is cultivated for 4 times of surface area needed for conventional method of surface areas (i.e. 500,000/125,000) to originate.In this embodiment, in The target cell of 14th day conventional method reaches 2 × 10⁶A cell/cm²Maximum superficial density.Therefore, 1cm²Growth area There is provided 2 × 10⁶A cell/cm², then these cells are redistributed in 4cm²Growth area on, therefore can use 0.5 × 10⁶A cell/cm²Conventional initial density (i.e. 4cm²Multiplied by 0.5 × 10⁶A cell=2 × 10⁶A cell) continue to make It is standby.So that the period is repeated another 14 days, reach maximum cell superficial density again at this time point, wherein every 4cm²Growth Surface area delivering 2.0 × 10⁶A cell, in total 8.0 × 10⁶A cell, then by these cell distributions in 16cm²Aufwuchsplate In product and manufacturing cycle is repeated, to deliver in total 32 × 10 in 42 days⁶A cell.

In the new method shown in Figure 11,500,000 target cells are not deposited on using when preparing beginning 1cm²On conventional method, but 500,000 cells are equally distributed in 4cm in the 0th day²Growth area on, with formed 125,000 low target cell/cm unconventionally²Initial sheet density.In this example, such as conventional method, new side The growth rate of method will start to reduce in the 7th day.Cell-surface density in new method is 1 × 10⁶A cell/cm².Therefore, 4 × 10 have been prepared in the culture at the time point that growth rate will reduce, this stage⁶A cell, then again by these cells It is distributed in 32cm²Growth area on, therefore can use 0.125 × 10⁶A cell/cm²Initial sheet density (i.e. 32cm² Multiplied by 0.125 × 10⁶A cell=4 × 10⁶A cell) continue second stage preparation.The period or stage for making the preparation repeat Reach maximum cell superficial density again until the 14th day, at this time point within another 7 days, wherein every 32cm²Growth table area Contain 1.0 × 10⁶A target cell, only to prepare total 32 × 10 in 14 days⁶A cell.Pay attention in each manufacturing cycle knot Shu Shi, such as conventional method, the final superficial density that new method provides is the several times of initial sheet density.However, passing through reduction Initiator cell superficial density and each stage for terminating preparation before cell has entered the growth preparation phase, shorten system with can dramatically The standby time.This embodiment describes the superficial densities by reducing target cell relative to regular growth superficial density (in this feelings 0.125 × 10 is reduced in condition⁶A cell/cm²), how within the only time of conventional method 33% (14 days to 42 days) deliver The target cell of identical quantity.

Although we use 0.125 × 10⁶A cell/cm²Initial sheet density quantitatively illustrate advantage, but ability Field technique personnel are understood that of the invention this example demonstrates any reductions lower than regular growth superficial density all will Shorten the preparation phase.In addition, those skilled in the art will appreciate that in the method given in this article and other new methods, cell The time point that the rate and cell decreased growth of growth are occurred is solely for the purpose of illustration, and the practical speed in respectively applying Rate will be changed based on various conditions (such as culture medium composition, cell type etc.).In addition, for given application, this field Technical staff will be appreciated that it is of the invention in this respect the advantages of be in any specific application since cell-surface density is reduced to Shorten lower than preparation time caused by regular growth superficial density, wherein specific routine employed in this illustrative example Superficial density can change because application is different.

Therefore, the one aspect of the method for the present invention, which now describes, is expected that by using reduced cell-surface density and makes to make The preparation phase of the standby given predicted quantitative objectives cell being present in cell composition minimizes.It should be with unconventionally low cell table Surface density deposits target cell at the growth surface, so that:

A. target cell coexists with antigen presenting cell and/or feeder cells, and if growing surface is not by gas permeability It is up to 1ml/cm that material, which constitutes the then ratio of culture volume and surface area,²If growing surface is by poromeric material structure It is up to 2ml/cm at the ratio of then culture volume and surface area², and

B. when manufacturing cycle starts, preferred superficial density condition is so that targeted cell surface density is preferably less than 0.5×10⁶A cell/cm², more preferably reduce as is also shown in fig. 4, and

C. the superficial density of target cell is preferably at least plus the superficial density of antigen presenting cell and/or feeder cells About 1.25 × 10⁵A cell/cm²。

Based on the above embodiment, if preferably proving to attempt that the surface of antigen presenting cell and/or feeder cells is close Degree is further decreased below 1.25 × 10⁵A cell/cm²It is not intended to limit the amplification of cell population of interest.It is mentioned based on proving to work as to pass through Cell population of interest can realize growth under unconventionally low density when for sufficient antigen presenting cell and/or feeder cells Target, we have selected 1.25 × 10⁵A cell/cm²。

Using the ratio of the growing surface and higher culture volume and growth table area that are made of poromeric material, It can simplify and shorten preparation.Another aspect of the present invention is discovery: using the growing surface being made of poromeric material and being surpassed Cross the culture volume/growth table area ratio rate and the amount of used growth table area increase with time of conventional ratio Duplicate manufacturing cycle, these factors will shorten the preparation phase.

An illustrative example is provided now to show how these conditions can shorten the preparation phase.Figure 12 is shown can be with By using the growing surface being made of poromeric material and more than 1 or 2ml/cm²Unconventionally high culture volume/ The discussion of the example of the obtained advantage of growth table area ratio rate.Being intended that those skilled in the art for next discussion is taken off Several selections can be provided by using this method by showing, comprising: shorten growth table area used by preparation time, reduction Amount, and/or reduce manpower and pollution danger.Those skilled in the art will recognize that Figure 12 and related discuss are only examples, And it is not intended to limit the scope of the invention.

Assume that the cell composition containing cell population of interest consumes about 1ml per " X " period in this illustrative example. Figure 12 shows two kinds of preparation processes labeled as " conventional method " and " new method ".When growing beginning, each technique starts from 0.5×10⁶/cm²Target cell superficial density.However, in new method growing surface be made of poromeric material, and And the ratio of culture volume and surface area is 2ml/cm², and the ratio of the culture volume of conventional method and surface area is 1ml/cm²。 In period " X ", the cell population of interest of conventional method has reached 2 × 10⁶/cm²Superficial density plateau and exhaust nutrition, And the additional medium volume of new method can continue to growth and desired cell-surface density 3 × 10⁶/cm².If should New method continues, then target cell reaches 4 × 10⁶/cm²Superficial density.Therefore, many advantageous selections are generated.The new side Method can terminate before the time " X " and prepare the cell more than conventional method, and it is normal for can terminating and prepare at time " X " As many as about 1.5 times of rule method of cell, or can be continued until the nutrition for exhausting culture medium and be prepared within 2 times of time Out it is as many as 2 times of conventional method of target cell, but is not necessarily to handle the device of feed supplement.In order to acquire as much in conventional method Cell, it is necessary to collect these cells and restart the technique, thus increase labour and possible risk of pollution.Because cell is treated With the cell that usually can only start from fixed quantity on the way, do not allow in conventional manner simply increases method when preparing and starting Add such option of surface area.

Figure 13 continues the embodiment of Figure 12, to show how advantageously more than one manufacturing cycle is.Figure 13 figure Show that cell population of interest in conventional method expands at the growth surface to expand with the group of target cell type in new method of the invention Increasing compares, and wherein the superficial density of the new method is more than the superficial density of conventional method.In order to protrude the present embodiment, this part Explanation in do not describe obtain cell population of interest technique." date " of culture starts from " 0 " so that those skilled in the art's energy It is enough more easily to determine the relative time advantage of the present invention in this respect.In this embodiment, two kinds of cultures are adopted in " the 0th day " With 0.5 × 10⁵A cell/cm²Conventional targeted cell surface density start.In this illustrative example, the growth table of conventional method Face is also to be made of poromeric material.However, the ratio of culture volume and growing surface is 1ml/cm in conventional method², and it is new The ratio of culture volume and growing surface is 4ml/cm in method².As shown in Figure 13, in conventional method when at about 4 days table Surface density is about 1.5 × 10⁶A cell/cm²When, the growth rate of cell population of interest start to reduce and reached 2 at the 14th day × 10⁶A cell/cm²Maximum superficial density.At this point, with 0.5 × 10⁶/cm²Superficial density by cell population of interest with 1.0ml/ cm²It is distributed in the 4cm of fresh culture²On growing surface and manufacturing cycle starts again at, reach 2 in another 14 days × 10⁶A cell/cm²Superficial density and the 28th day deliver 8 × 10⁶A target cell.In contrast, in new method, Yu great Yue 10th day to the 11st day is about 3 × 10 when superficial density⁶A cell/cm²When cell population of interest growth rate begin to decline, can To reach 4 × 10 at the 28th day⁶A cell/cm²Maximum superficial density.However, in order to accelerate to prepare, when cell population of interest still Manufacturing cycle is terminated when being so in Seedling height rate.Therefore, the 10th day to the 11st day Yu great Yue, by 3 × 10⁶A cell is with 0.5 ×10⁶/cm²Superficial density redistribute in 6cm²Fresh culture growth table area on (4.0ml/cm²) and prepare week Phase starts again at, and cell population of interest reaches 3 × 10 in about another 10 to 11 days⁶A cell/cm²Superficial density and 18 × 10 are delivered when about 21 days⁶A target cell.Therefore, compared with conventional method, the new method about 75% when It is interior to prepare the target cell more than 2 times of quantity.

We can obtain on the growing surface being made of poromeric material more than 10 × 10⁶A cell/cm²It is thin Cellular surface density, thus prove that the present invention is not limited to this density in embodiment using high superficial density.

Therefore, another embodiment of the method for the present invention is described now, wherein by using reduced cell table Surface density and minimize the preparation phase for the target cell being present in cell composition for preparing given quantity:

A. target cell is inoculated in the presence of antigen presenting cell and/or feeder cells and is made of poromeric material On growing surface region, culture volume/surface area ratio is at least 2ml/cm²,

B. preferred superficial density condition is established when manufacturing cycle starts, so that targeted cell surface density is about 0.5×10⁶A cell/cm²Conventional density in,

C. so that cell population of interest is expanded is more than about 2 × 10⁶A cell/cm²Conventional surface density, and

D. if necessary to more target cells, then target cell is redistributed additional in what is be made of poromeric material Growing surface on and repeat step a-d until obtaining enough target cells.

When using these new methods, by the way that unconventionally low surface area will be used, using target cell and/or feeding Support cell new superficial density than, using the growing surface being made of poromeric material, using unconventional high culture medium Volume/growth table area ratio carrys out Primary culture and executes multicycle preparation, these features are combined, can obtain more More benefits.The condition can be changed in any manufacturing cycle, thus to obtain it is desired as a result, for example in the preparation between contract Reach balance between short, surface area utilization, charging frequency etc..

Figure 14 shows another new method, wherein obtaining further advantage relative to conventional method.As herein Described in other illustrated embodiments, those skilled in the art will appreciate that description herein not limit the present invention Range, but be used to illustrate how realize preparation efficiency improve the advantages of.

In this example, target cell is made to double weekly under normal conditions." date " of culture starts from " 0 " so that originally Field technical staff can be easier to determine the relative time advantage of this embodiment.In addition, above-mentioned and raising is not repeated Cell and/or the related problem of Antigen Presenting Cell surface density ratio, to simplify this example.For illustrative purposes, it is preparing " the 0th day " uses starting to have 500,000 target cells and normal condition lower doubling time for 7 days cell masses.Routine side Method starts from 0.5 × 10⁶A cell/cm²Superficial density and 1ml/cm²Culture volume and surface area ratio.Such as figure It is shown, when cell population of interest reaches 2 × 10⁶A cell/cm²Superficial density when, by cell with 0.5 × 10⁶A cell/cm²'s Superficial density is distributed on additional surface area and manufacturing cycle starts again at.Start from 0.06 in the new method of this embodiment ×10⁶A cell/cm²Superficial density, growing surface region is made of poromeric material, the ratio of culture volume and surface area 6ml/cm².As shown in the figure, when cell mass will initially enter the growth platform phase, by the growth of cell redistribution Yu Geng great On surface.In the case, pay attention in conventional method cell-surface density close to the 1.5 of culture volume and the ratio of surface area Again (that is, about 1.5 × 10⁶A cell/ml) when start plateau, thus judge that cell mass initially enters plateau.Therefore, in About the 9th day, about 4.5 × 10⁶A cell/cm²Superficial density under by cell distribution in 36cm²Growth table area on And manufacturing cycle restarts.

Comparison of Figure 15 list display to each preparation method shown in Figure 14, and amplify to each stage to prove new side The efficiency of method and for where adjustment preparation method can advantageously obtain abundant efficiency in the various stages.It is noted that new method exists It is only completed just better than conventional method after the second stage of manufacturing cycle, with only 61% surface area within the only approximately half of time Demand delivers the cell close to 1.37 times.It is noted, however, that the phase III of manufacturing cycle be how to produce it is celliferous significant Increase and surface area is increase accordingly.Therefore, manufacturing cycle should be modeled, with it is expected how this method whole each week Interim adjusting initial cell superficial density and/or final cell superficial density and so that any given method is obtained optimal efficiency water It is flat.

As one embodiment, how Figure 16 can be obtained when preparing progress by changing the variable in new method if being shown Obtain an example of efficiency.For example, can make the initial sheet density in the 3rd period increases to 0.70 cell/cm from 0.06²And Final superficial density is set to change to 7.5 cells/cm from 4.5².Increase final superficial density to depend on increasing culture volume and table The ratio of area makes it be more than the 6ml/cm of starting²To bigger numerical value.Culture volume specific surface area is bigger, is maintained at quick Period (group's amplification i.e. before plateau) in growth period is longer.In the case, it is next to provide the 5 day additional time for we It completes fast growing period and the ratio of culture volume and surface area is increased to about 8ml/cm².In this way, in this embodiment In, the cell more than 3,000,000,000,000 can be prepared with reasonable surface area in 34 days.For example, we, which have produced, has about 625cm²The device for the growing surface being made of poromeric material is simultaneously tested the device.This is clearly to compare conventional method Superior cell preparation method.

Therefore, another preferred embodiment of the method for the present invention is described now, wherein desirably by using Reduced cell-surface density and the preparation phase for making to be present in the given predicted quantitative objectives cell in cell composition minimizes:

A. target cell is inoculated in the presence of antigen presenting cell and/or feeder cells and is made of poromeric material On growing surface, and the ratio of culture volume and surface area is to be at least 2ml/cm², what

B. preferred superficial density condition is established at the beginning of manufacturing cycle, so that targeted cell surface density is less than often Rule density, preferably from about 0.5 × 10⁶A target cell/cm²To about 3900 target cell/cm², and target cell and antigen It is at least about 1.25 × 10 in the total quantity of delivery cell and/or feeder cells⁵A cell/cm², and

C. make cell population of interest amplification more than about 2 × 10⁶A cell/cm²Conventional surface density, and

D. if necessary to more target cells, then target cell is redistributed additional in what is be made of poromeric material On growing surface and step a-d is repeated until obtaining enough target cells.

The disclosure makes adoptive cellular treatment neck by generating a new class for the treatment of cell for being known as carrier T It is improved in domain.Carrier T includes not carry the T cell group of intrinsic GVHD risk, is further changed into comprising one or more It can be used in the treatment characteristic of therapeutic purposes to provide treatment benefit for receptor.Because carrier T does not have starting GVHD disease Native abilities, so it becomes a kind of ideal delivery vehicles, equipment is any amount of can to fight extensive plurality of medical illness With the weapon of disease.The invention discloses make and use with treatment characteristic and without intrinsic present in art methods The method of the carrier T of GVHD risk, the treatment characteristic are used to provide the health benefits of adoptive cellular therapy for receptor.Important It is that carrier T is with the art methods mode of action for adoptive cellular therapy on the contrary, because the therapeutic purposes of carrier T are complete It is unrelated with the antigentic specificity of nave T cell receptor.By given disclosure and shown embodiment, make ability Field technique personnel recognize that the treatment characteristic of carrier T does not include the native antigen receptor of carrier T.

Carrier T is prepared by following steps: using antigenic stimulus donor PBMC or donor Cord blood, has with activation to antigen There is the growth of the donor T-cells of native antigen specificity, to generate the antigen receptor comprising having antigentic specificity to antigen T cells with antigenic specificity group.By the antigen for selecting to be not present on normal cell, can prepare to have cannot be identified normally The T cell group of the antigen receptor of cell.By ignoring the treatment benefit that can be obtained from the antigentic specificity of nave T cell, and Nave T cell is changed to following treatment characteristic: i.e. it does not include native antigen Receptor recognition ability, can produce a kind of T Carrier group, the carrier T group, which has, to be identified unrelated purposes with antigentic specificity and is not inherently prone to or even cannot Enough start GVHD.

Although carrier T may include more than one native antigen specific T-cells group, since carrier T is independent of it For therapeutic purposes native antigen specificity, it is possible to carrier T is infused into receptor, and with the serotype of receptor whether Any native antigen receptor positive matching shown with carrier T is unrelated.In addition, the key characteristic of carrier T includes that it can be used In the unmatched situation of HLA, or the nave T cell group due to obtaining carrier T does not have intrinsic GVHD risk.This makes it possible to The allogeneic library for enough establishing carrier T can be served public extensively and be not only restricted to HLA required in art methods Matching.When carrier T have HLA and receptor's mismatch native antigen it is special when, the nave T cell receptor of carrier T cannot be Cell is identified in receptor and causes GVHD.Nevertheless, carrier T can start its treatment in the completely unmatched situation of HLA Activity, because it has been changed to the treatment characteristic independent of native antigen receptor, to realize its therapeutic purposes.So And carrier T is not limited to for the unmatched situation of HLA.It is made of by generating the T cell with following native antigen receptor Carrier T still can avoid initiation GVHD disease although between the native antigen specificity of receptor and carrier T there is part HLA match Disease, the native antigen receptor have the height-limited antigentic specificity for the antigen of expression not on normal cell.For Carrier T is used under HLA matching or the unmatched situation of HLA, the native antigen specificity of preferably carrier T only allows its identification Not in antigen present on normal cell, more preferable normal cell even more preferably can only be identified not in normal lactation The single epitope of antigen present on zooblast.

Under carrier T and the unmatched situation of receptor HLA, it is contemplated that receptor can make strong immune response, will finally disappear Except carrier T.Therefore, the therapeutic purposes of carrier T can be continued by delivering the carrier T of one or more extra doses.The process can Continue as needed to obtain desired therapeutic purposes.In the preferred method, the HLA of the carrier T of each dosage is different, makes Patient immune system itself required in each carrier T for preparing attack new dosage again stress (re-prime), from And keep the interval between the carrier T of each dosage roughly the same.

Prepare the method with the T cell of native antigen receptor of height-limited antigentic specificity: in history, with wide It is general applied to the preparation T cell group of scale needed for adoptive cellular therapy.In the prior art for expanding T cell Unrealistic and uncontrollable at the preparation method of the therapeutic dose of suitable size, cell therapy is limited to only a few group by this, he Must obtain medical treatment in the mechanism of a small number of highly-specialiseds.The fundamental characteristics of carrier T is that its nave T cell feature is not intrinsic Cause receptor by GVHD in ground.Because it is preferred that the native antigen specificity of carrier T only allows its identification not deposit on normal cell Antigen, more preferable normal cell even more preferably can only identify not present on normal mammalian cell The single epitope of antigen, the widely applied foundation stone for being efficiently prepared into the method for being related to carrier T of these cells.Such T Cell is in donor PBMC or Cord blood only with frequency that is very low and can't detect sometimes appearance.Therefore, when attempting to generate When being best suited for the T cell group of carrier T, the problem that the T cell preparation method of the prior art is intrinsic is resolved.

We have found method and apparatus, entitled " the IMPROVED METHODS OF such as submitted on May 18th, 2012 The U.S. Patent application No.13/475 of CELL CULTURE FOR ADOPTIVE CELL THERAPY ", 700 (hereinafter Referred to as Vera ' 700, is incorporated herein by reference) described in, it is contradicted with art methods, to realize efficiently Prepare the T cell that there is natural characteristic but receptor is not caused to inherently suffer from GVHD.As a result, in donor PBMC or Cord blood It is satisfied with the long-term needs of the visible T cell of low frequency actually prepared.Moreover, when with to possess non-T cell natural When antigentic specificity inherently treats the new concept T cell combination of characteristic, the preparation for serving as the carrier T of bio-carrier not only becomes May, and become practical.

In an illustrative methods, cultivate start when will it is more than one selected by antigen presentation to PBMC or Cord blood (that is, original library of T cells with antigenic specificity), to stimulate more than one special T cells with antigenic specificity group (each group of expression institutes Present some antigen antigen receptor) growth.The purpose for the arrangement is that then select the most proliferative being used to prepare and/ Or most desired nave T cell group, and terminate other T cells group.With the progress cultivated after beginning, response is in a variety of antigens Multiple T cell groups may show group's amplification of different level, this depends on the original size of its group.In addition, some of which Or it all may continue to can't detect.After a period of time, assessment culture in the T cell group's of any selected antigen-reactive Acceptable growth.Such assessment can be only for a group to a kind of antigen with specificity, or is directed to other antigens Other groups with specificity.If a T cells with antigenic specificity group shows acceptable amplification, pass through only into device Adding the antigen that it is identified, to stimulate specific T cells group to will lead to remaining T cell again finally dead, and specifically it is expected T Cell mass continues to be proliferated.However, there are two types of selections: 1) only using that if more than one T cell group shows acceptable amplification The antigen that a little specific T cells react stimulates culture (thus the amplification for terminating the lower T cell group of proliferative) again, or 2) culture can be divided into more than one culture apparatus, each device receives the single antigen different from every other device, So that only have a T cell collective optimization in each device, other equal quilts other than the most culture of proliferative It terminates.Preferably, all culture apparatus are all breathables, and the common U.S. Publication in a review of such as Wilson 2008/0227176 A1 of No.2005/0106717 A1 (hereinafter referred to as Wilson ' 717) and Wilson is (hereinafter Referred to as Wilson ' 176) described in type, two patents are both incorporated herein by reference, and depend on the side of Vera ' 700 Method.

Via another example, antigen A, antigen B and antigens c can be presented to the PBMC group being placed in culture apparatus.One Section the time after, it can be estimated that in culture with the presence and/or proliferation of antigen A, B or the C group reacted.If reacted with antigen A Antigentic specificity group be uniquely do not show acceptable frequency and/or group amplification group, then can by only use antigen B and Antigens c stimulates terminated again.Alternatively, if the antigentic specificity group reacted with antigen B and antigens c substantially equally increases It grows, but not can determine which will continue to be proliferated with iptimum speed, then assign to culture in two devices, it is contemplated that a device will Finally continue to prepare, and another will be terminated.First device will receive antigen B, and second device will receive antigens c.To antigen B table The T cell for revealing antigentic specificity is proliferated in first device, but the T cell for showing antigentic specificity to antigens c is finally dead It dies.Otherwise in second device.It can be in the inspection of some time points after the culture in first device and second device occurs Measured frequency and/or group's size, to terminate the culture that there is the minimum of desired T cell group effectively to expand.Those skilled in the art It will recognize, when starting, starting has the culture of multiple antigens, and the major advantage compared to only one antigen is to increase The prospect for finding the T cell group with suitable antigen specificity and growth rate is added.In addition, being used in a device multiple anti- Former rather than multiple devices are to make PBMC or Cord blood, culture medium, cell factor, lab space, labour using an antigen It is more efficiently utilized with biological poison disposition space.

The preferred native antigen specificity of selection carrier T will now be described: although it is preferred that the native antigen of carrier T is special Property only allows its identification, and not in antigen present on normal cell, more preferable normal cell even more preferably can only be known Not not in the single epitope of antigen present on normal mammalian cell, but this is non-limiting, and art technology Many suitable characteristics of native antigen receptor can be considered in personnel.It is many to select and be characterized in suitably.For example, carrier T Native antigen specificity may include the more than one T cell group with native antigen specificity.The native antigen of carrier T is special Property can be directed to entire antigen, can also be for the single epitope of autoantigen or non-self antigen；It is reptile, amphibious dynamic Object, fish or birds；Invertebrate, as spongia, coelenterate, worm, arthropod, mollusk or spine skin are dynamic Object；Bacterium, fungi, helminth and spongia；Virus includes but is not limited to adenovirus, Epstein-Barr viral (EBV), huge Cell virus (CMV), adenovirus (Adv), respiratory syncytial virus (RSV) (RSV), human herpes virus 6 (HHV6), human herpes 7 (HHV7) of virus, BK virus, JC virus, influenza virus, H1N1 virus, parainfluenza virus, herpes simplex virus (HSV), varicella Herpes zoster virus (VZV), assays for parvovirus B 19, coronavirus, Metanpneumovirus, bocavirus (Bocavirus) or KI virus/WU virus；Or survivin, gp100, tyrosinase, SSX2, SSX4, CEA, NY-ESO-1, PRAME, MAGE-A1, MAGE-A3, MAGE-A4, Claudin-6, Cyclin-B1, Her2/neu-ErbB2, histone h1 .2, histone H 4, mammary gland pearl Albumen-A, Melan-A/MART-1, Myc, p53, ras, PSA, PSMA, PSCA, Sox2, Stromelysin-3, Trp2, WT1, Protease 3, Muc1, alpha-fetoprotein, CA-125, bcr-abl, hTERT or prostatic acid phosphatase -3.

In order to promote the suitable nave T cell all living creatures of donorcells long, those skilled in the art can be refering to U.S.'s public affairs Open No.2011/0182870 A1 (hereinafter referred to as Leen ' 870, be incorporated herein by reference), it is also contemplated that use Antigen presenting cell (APC) stimulates, and the antigen presenting cell such as Dendritic Cells, monocyte, macrophage, B be thin Born of the same parents, T cell, PBMC or artificial antigen are in the k562 of delivery cell such as transformation, and wherein any one can present desired anti- Original is to generate the expectation antigentic specificity of donor nave T cell group, to generate the native antigen specificity of carrier T；By making It is anti-with the cell lysate containing desired antigen, the purifying protein containing desired antigen, the recombinant protein containing desired antigen, coding expectation Former Plasmid DNA, the plastid rna of the coding expectation antigen and/or the peptide library containing it is expected antigen, and/or containing desired antigen Single synthetic peptide induce desired immune response in donorcells using antigen.

The preparation of T cell group preferably uses Vera ' 700 and/or method given herein to carry out, and most preferably utilizes The gas permeability culture apparatus of type described in Wilson ' 717 and/or Wilson ' 176 carries out.Those skilled in the art can It recognizes, a variety of methods in the operative body may be more or less suitable, this specific mesh applied depending on every kind 's.For example, can use a variety of superficial densities, culture medium height, culture volume and growth table area etc., and with cell because Son such as IL2, IL15, IL21, IL12, IL7, IL27, IL6, IL18 and/or IL4 stimulation and multi-frequency and concentration, can Combine any method for presenting antigen with the antigen using any source to carry out stimulated in vitro repeatedly, by including but is not limited to γ capture, Magneto separate, single cell clone and/or flow cytometry method carry out cell sorting or without cell sorting.

Embodiment 9: non-autogenous cell target cannot be identified by having the carrier T of the nave T cell receptor of identification CMV epitope NLV Mark

Within 12 days, using preceding method by NLV-CMV have native antigen specificity T cells with antigenic specificity from 0.03% frequency is expanded to 87% in PBMC.Then these cells are resisted with from the unmatched presentation target CMV of three HLA The cell of the donor of the NLV peptide of original is cultivated together.

Figure 17, which shows carrier T, cannot identify no matter whether they express NLV from the cell for mismatching allogeneic donor Peptide (" allo1 " and " allo1 pep ", " allo2 " and " allo2 pep ", " allo3 " and " allo3 pep "), but they are identified The autogenous cell (" Auto4 pep ") of NLV peptide is presented with killing, and avoids killing the autogenous cell for not presenting NLV peptide The ability of (" Auto4 ") demonstrates them with complete function.

Select and generate desired treatment characteristic: there are many selections can be used for T cells with antigenic specificity group being changed to tool There is at least one to treat characteristic.Following examples are non-limiting, but are intended to those skilled in the art and are provided following understanding: being controlled How the selection for treating characteristic depends on therapeutic purposes and treats characteristic and its therapeutic purposes and carrier T native antigen receptor why Antigentic specificity it is unrelated.

Embodiment 10: it is loaded with the carrier T that immunotherapy is used for as the recombinant protein of adjuvant application

Immunotherapy is the therapy of a kind of immune response for being designed to triggering or amplifying patient.Example includes application design For the vaccine of the immune response for the tumour antigen expressed on cancer cell or the T cell expanded in vitro or NK are delivered at activation Cell.Recombinant protein such as cell factor such as IL2, IL7, GM-CSF are by systemic administration to promote these cells to give birth in vivo Long, amplification is resident and/or is functioned, but the systemic administration of some cell factors (for example, IL2) is related to toxicity in vivo, packet Include severe catarrh, nausea, diarrhea, oedema, respiratory distress, liver and renal dysfunction and wound inducement/T cell of infusion The amplification of the regulatory T cells of function.The carrier T that application is loaded with recombinant protein (including cell factor) can be by moving to Inflammation part and these recombinant proteins are delivered directly to inflammation part (inducing by immunotherapeutic agent) to overcome these toxicity.

Those skilled in the art are able to recognize that carrier T, which can be used, carrys out these cell factors of targeted delivery instead of traditional Non-specific systemic administration.For example, having carried out experiment to prepare the truncation shape that can be generated cell factor IL7 and express CD34 Δ The carrier T of formula (its percentage that can be used for detecting transducer cell when selects transgenosis group).In this case, such as Figure 18 institute Show, the NLV epitope of CMV virus is successfully changed into the donor T-cells of 98% native antigen specificity with CD34 The carrier T of the treatment characteristic of Δ-IL7 cytokine-expressing, as determined by through flow cytometer showed.Further test shows Only the carrier T of useful retroviral vector (CD34 Δ-IL7 cell factor) transformation can generate IL7, as passed through ELISA institute Determining.

For assessment retroviral vector (the CD34 Δ-in terms of the vivo effect of IL7 cell factor and internal distribution IL7 cell factor) transformation therapeutic carrier T, mouse is divided into two groups (every group of 5 animals).At the 1st group, pass through IV whole body The IL7 cell factor of 2000ng is applied to handle mice with tumor.At the 2nd group, 10E+06T carrier is injected by single IV to handle Mouse.Then the arbitrary object of each group was put to death at the 1st week and the 2nd week, the cell factor for assessing different parts by ELISA is dense Degree, including heart, liver,kidney,spleen, peritonaeum, tumour and blood.

Figure 19 A shows the IL7 cell factor accumulation at each position in group 1.IL7 cell factor elisa assay shows to examine in kidney Higher cytokine levels are measured, and being lower than in the tumor locus level can detection limit.

Figure 19 B shows the IL7 cell factor accumulation at each position in group 2.IL7 cell factor ELiSA analysis shows, with other Organ is compared, and the cytokine concentrations of tumor locus are higher, and at least two weeks after applying carrier T, the cell at tumour The factor persistently generates.Therefore, carrier T can move to tumor locus, and preferably deliver cell factor IL7, when continuing one section Between.This clearly demonstrates that carrier T has the ability of the treatment characteristic of delivering cell factor, with prior art systemic administration Cell factor delivering method is compared and provides superior treatment benefit.As expected, carrier T has limited internal presence, As indicated by being reduced from first week to second week cytokine concentrations.This can be regarded as to the additional benefit of carrier T, Because they are simultaneously not left in receptor.Preferably, the carrier T that can according to need application extra dose, until being treated Achievement, and if without extra dose, carrier T will be removed from receptor.

Embodiment 11: donor T-cells can be transformed to prepare the carrier T with following treatment characteristic: special for targeting Determine the Chimeric antigen receptor (CAR) of antigen

There are the donor T-cells of the T cell of native antigen specificity (such as to pass through the 98% epitope NLV to viral CMV Pentamer analysis assessment), transduction has the treatment for expressing the CAR that can identify prostate stem cell antigen (PSCA) to prepare The carrier T of characteristic.The therapeutic purposes of the carrier T are to destroy prostate tumor cells.As shown in figure 20, in E2 quadrant, 57.23% Donor T-cells successfully changed with prepare with CAR-PSCA treatment characteristic carrier T, as determined by flow cytometer showed 's.In order to test the killing effect of the carrier T with CAR-PSCA treatment characteristic, by donor T-cells without alteration and pass through Donor T-cells and the carrier T for preparing are modified with CAR-PSCA to be positive (GFP+) or PSCA with 1: 1 ratio with to antigen PSCA The target cell of negative (mOrange+) is cultivated together, after culture 72 hours, is analysed by flow point to remaining PSCA positive tumor cell Quantity quantified.Figure 21 shows the experimental result at 72 hours, wherein A1 quadrant shows the number of PSCA negative cells Amount, A3 quadrant show the quantity of carrier T, and A4 quadrant shows the quantity of PSCA positive tumor cell.As expected, 72 hours Afterwards, donor T-cells without alteration do not change original culture composition.However, by contrast, expressing the carrier T of CAR-PSCA Entire PSCA positive tumor cell group can be almost removed, while being preserved from and being shown to PSCA by reservation PSCA negative cells The accurate selection of antigen.This clearly illustrates that carrier T can generate and the incoherent treatment benefit of its native antigen specificity.

Embodiment 12: donor T-cells can be transform to the carrier T that preparation has following treatment characteristic as: becoming can be The receptor of unexpected cell factor is removed in receptor

Tumour cell is by generating the immuno-suppressing cytokine for the anti-tumor effect for inhibiting endogenous T cell come from immune System invasion.Donor T-cells can be changed to prepare the carrier T with following treatment characteristic: expression provides therapeutic purposes institute Any specific cells factor acceptor needed, so that there is the treatment benefit for making tumor environment more accommodate Immunotherapy Strategy, it is described Therapeutic purposes are the specific cells factor unexpected from tumor clearance.Figure 22 A and Figure 22 B show a diagram of this method. It, can be in conjunction with the tumour of the neighbouring expression IL4 cell factor of carrier T of the treatment characteristic of IL4 with receptor in the description of Figure 22 A Cell.In the description of Figure 22 B, carrier T has been incorporated with IL4 cell factor, and protects the IL4 cell factor of tumour cell Amount substantially reduce.It should be noted that treatment characteristic, therapeutic purposes and the treatment benefit of carrier T do not include carrier T native antigen by Body and unrelated with its.It is tested to evaluate the T with the treatment characteristic for expressing extracellular recombinant cytokine receptor IL4R/7 The ability of carrier elimination IL4 cell factor.Have naturally specifically by being changed to donor T-cells to the NLV epitope of CMV virus Property prepares carrier T.24 orifice plates in 2ml culture volume in the IL4 of 2000pg/ml in the presence of culture 5E+05T carry Body, and be compared with donor T-cells.At 24,48 and 72 hours, the concentration of cell factor IL4 is evaluated by ELISA.As a result It is shown in Figure 23.It is evident that carrier T can satisfy its therapeutic purposes, because immunosupress was swollen by 72 hours periods The reduction of tumor growth factor I L4 cell factor is very prominent.On the contrary, donor T-cells are (that is, labeled as " not engineered carrier T " Histogram) presence that can reduce IL4 is not shown.

Those skilled in the art it will be appreciated that carrier T can have a variety for the treatment of characteristics can satisfy it is intended that by The therapeutic purposes of person's offer treatment benefit.A possibility that disclosed will be increased by several other embodiments now.

It is changed to the carrier T of the treatment characteristic with the chemotherapeutant for targeted therapy of cancer: a variety of different changes Learn therapeutic agent or anti-tumor drug and be used to treat different types of cancer, including breast cancer, prostate cancer, cancer of pancreas, liver cancer, Lung cancer, brain cancer, leukaemia, lymthoma, melanoma and myeloma.Most of chemotherapeutants through intravenous delivery, but Some medicaments can also be then recycled through whole body with oral administration.Quickly (most of cancers are thin for division by killing for chemotherapeutant One of main feature of born of the same parents) cell play a role.This means that chemotherapeutant can also injure in normal circumstances quickly The cell (for example, cell in marrow, alimentary canal and hair follicle) of division.This causes the most common side effect of chemotherapeutant to be bone Marrow inhibit (generation of haemocyte is reduced, therefore immunosupress occurs), catarrh (inflammation of alimentary canal internal layer (lining)) and Alopecia (hair reduction).The nausea and vomiting that chemotherapy induces is also common treatment side effect.Application is loaded with these medicines The carrier T of object is possible to offset these toxicity.This can be infused into receptor by carrier T load chemotherapeutant, from And move to inflammation (cancer) position under chemotactic gradient.In this way, chemotherapeutant is made to be placed in neighbouring tumour cell, with It is opposite that receptor is applied to systemic fashion.In the unmatched situation of HLA, receptor's immune system will attack carrier T, lead to its quilt It destroys, but still discharges chemotherapeutant at tumour cell position.Therefore, load (that is, chemotherapeutic agent) can be directly in target Site deposition, rather than applied with systemic fashion, to reduce toxicity of missing the target relevant to chemotherapy.

This method is depicted in Figure 24 A, Figure 24 B and Figure 24 C.As shown in fig. 24 a, it is loaded with the carrier T of chemotherapeutant It is migrated to inflammation part (that is, tumour cell), since the HLA between carrier T and donee's cells is mismatched, the natural of carrier T resists Original receptor nonrecognition recipient cell, to reach tumour cell without causing GVHD.As shown in fig. 24b, receptor's immune system has been It is located at the carrier T at tumour cell position through targeting.As shown in Figure 24 C, when by receptor's immune system attack, carrier T exists The position of tumour cell discharges chemotherapeutant, thus avoid the chemotherapeutant delivering method of the prior art intrinsic miss the target Toxicity.

Be changed to the carrier T of the treatment characteristic with antimicrobial: antimicrobial is that kill microorganism (such as thin Bacterium, fungi or protozoan) or inhibit these microorganisms grow substance.The usual systemic administration of these reagents and if plus Inflammation part can be reached by being downloaded to can then be delivered by delivering in the carrier T of its load in a manner of more targeting.

It is changed to the carrier T with the treatment characteristic for generating the recombinant protein applied together with immunotherapy as adjuvant: In addition to loading foreign recombinant proteins, viral (for example, adenovirus, retrovirus, slow virus) or non-viral turn also can be used Carrier T is transformed with transgene expression recombinant protein, including cell factor, chemotactic factor (CF), enzyme, tumour antigen and cell in dyeing method Factor acceptor, the recombinant protein is also designed to the adjuvant of other immunization therapy interventions, and to enhance, T cell is resident, promotes to expand Increase, induction is gone back to the nest.

It is changed to the carrier T that the treatment characteristic of transgenic molecules of cell tumour specificity is assigned with expression: Ke Yili Carrier T is transformed in an identical manner with recombinant protein such as cell factor, it can also be using virus (for example, adenovirus, reverse transcription Virus, slow virus) or non-viral transfection method transformation carrier T so that it transgenically expresses Chimeric T cell receptor (CAR).

It is changed to the T for being loaded with or being transformed into Ju You for treating the treatment characteristic of the recombinant protein of autoimmune disease Carrier: autoimmune disease is originating from body to the inappropriate immune response normally present in substance and tissue in body.It changes Certain part mistake of body as pathogen and is attacked its own cell by Yan Zhi, immune system.It can be limited to certain organs.Load Have by inhibit the carrier T of the recombinant protein (such as IL10, TGFB, IL13 cell factor) of inflammation application can by by these Recombinant protein is delivered directly to inflammation part and divides indiscriminately to which load to be directly delivered to the position of this needs Bulk weight histone and overcome these autoimmunity effects.

Carrier T can be transform to expression suicide gene as:, can be to carrier T in order to quickly and completely eliminate infused cells It is incorporated to and can trigger the safety switch or suicide gene for generating toxicity.It is from herpe simplex disease by the suicide gene most preferably verified The thymidine kinase (HSV-tk) of malicious I.The enzyme makes nontoxic prodrug Ganciclovir phosphorylation, then by endogenous kinases by its phosphorylation For GCV triphosphoric acid, lead to chain termination, it is single-stranded to be broken after being incorporated to DNA, to kill dividing cell.Some I-II phases are studied Show that the application of Ganciclovir can eliminate safely the cell of transfer being transformed through HSV-tk in vivo.Recently, induction type Fas, Fas correlation death domain-containing albumen (FADD) and caspase 9 have been considered as alternative non-immunogenic suicide base Cause.These each molecules may act as suicide switch, the FKBP variant knot when with FK binding protein (FKBP) variant fusion Dimer chemically inducer (CID), AP1903 are closed, is to have proven to safe synthetic drug in healthy volunteer.Application The small molecule causes to promote the crosslinking of apoptosis target molecule and activation.Up to 90% transduction has the T cell of induction type Fas or FADD sudden and violent It is exposed to CID and undergoes apoptosis later.Although there is prospect, elimination 90% may be insufficient to assure that in vivo through transducer cell through losing Pass the safety of engineered cells.The transgene expression for speculating the CD20 molecule usually expressed in B cell is for T cell therapy Suicide gene.The strategy depends on the clinical application of Humanized anti-cd 20 antibodies (Rituximab), the Humanized anti-CD 20 Antibody (Rituximab) is widely used in both the normal B cells for eliminating expression CD20 antigen and neoplastic B cell.Therefore, transgenosis The subsequent internal application for expressing the infusion and Rituximab of the T cell of h CD20 should efficiently eliminate the T cell group of infusion, but this Also normal B cell will be eliminated.Therefore, carrier T can be transformed with express one or a combination set of these different suicide genes with The elimination and delivering of control load.

It is changed to the carrier T for being loaded with and/or transforming as the treatment characteristic of in-vivo imaging: Positron Emission Tomography (PET) it is a kind of medicine imaging technique, generates the 3-D image or function course picture of body.The system detection is by emitting The gamma-rays pair that the radionuclide (tracer introduces body by bioactive molecule) of positive electron issues indirectly.Then lead to Cross the 3-D image of tracer concentration in computer analysis building body.Since carrier T can move to tumor locus, Ke Yixiang Carrier T loads radioactive isotope so that carrying out vivo detection and determining the position of tumor locus.

Similarly, iodo- 123 (123I or I-123) are the radioactive isotopes of the iodine used in nuclear medicine, including Single photon emission computerized tomography,SPECT (SPECT).It is the isotope for being most suitable for the diagnosis research for thyroid disease.It is right In for 24 hours for the test of (hour) iodine uptake, the half-life period of about 13.3h (hour) is ideal, and 123I is in parathyroid tissue Have the advantages that other with the diagnosing image aspect of metastasis of thyroid carcinoma.Due to being generated by enzyme thyroid peroxidase (TPO) Hydrogen peroxide and the selectivity capture of the iodine that carries out in " iodine retention ", iodine can be safely used for being imaged or treating thyroid gland Tumour.In this way, carrier T can be transformed with thyroid peroxidase (TPO) can be used for imaging and/or killer T carrier to retain Iodine.

Those skilled in the art by many technologies it will be appreciated that can generate controlling for any give for carrier T The treatment characteristic of purpose is treated, the technology includes but is not limited to:

A) genetic modification, such as retrovirus, adenovirus, adeno-associated virus or slow virus are carried out with viral vectors, and/ Or

B) genetic modification is carried out by non-virus carrier, including the use of by physically and/or chemically technology (such as using turning The electroporation and/or lipofection method of stand and transposase (for example, Sleeping Beauty) and/or Piggybac technology) DNA the and/or RNA carrier being incorporated to, and/or

Genetic modification is carried out comprising into one or more transgenosis, the transgenosis is improved carrier T migration, is incorporated to certainly Gene is killed, receptor's Immune Reconfiguration (for example, cell factor generation) is improved and/or causes directly antiviral or anti-tumor effect (example Such as, Chimeric antigen receptor) or inhibit immune response to treat autoimmune disease, and/or

D) progress genetic modification is described to improve carrier T migration by expressing one or more chemokine receptors Chemokine receptors such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2、CXCR3-A、CXCR5、CXCR6、CX₃CR1 and/or XCR1 to improve the migration of carrier T, and/or

E) genetic modification is carried out to express one or more cell factors by carrier T or by expression or overexpression Costimulatory molecules CD80, CD86,41BBL, OX40L improve receptor's Immune Reconfiguration, the cell factor such as GM-CSF, TNF α, INF γ, IL2, IL8, IL15, IL7, IL12, IL21 or IL26, and/or

F) genetic modification is carried out to induce carrier T death, the suicide by expressing one or more suicide genes Gene such as thymidine kinase TK gene, CD20, CD19 or iCaspase9, and/or

G) genetic modification is carried out to cause direct antiviral or anti-tumor effect, is including but not limited to expressed one or more A transgenosis, such as Chimeric antigen receptor (CAR), the single chain variable fragment (scFv) by being isolated from specific antibody identify Tumor targets, the specific antibody be connected as follows: i) extracellular spacer, such as by using from the area IgG-FC CH2CH3 sequence；Or ii) cross-film component, the including but not limited to sequence of CD28, CD4, CD3 or CD8；Iii) CD3 ζ intracellular domain (endodomain)；Or iv) pass through expression native ligand, such as the cell factor or cell factor receptor of coding CD3 ζ intracellular domain Body, and/or

H) carry out genetic modification to inhibit immune response to treat autoimmune disease, for example, by expression generate one or The transgenosis of more immuno-suppressing cytokines, or pass through expression competition ligand, such as CTLA-4, PD1, the cell factor Such as IL4, IL6, IL10, IL13, TFG β.

Those skilled in the art are able to recognize that the therapeutic purposes of carrier T can be widely, including but not limited to following It is any:

A) DNA, RNA, recombinant protein, peptide or aptamer (aptamer) are carried as bio-carrier

B) compound is carried as bio-carrier, and/or

C) compound with therapeutic purposes, including but not limited to chemotherapeutic agent, small point are carried as bio-carrier Son, nano particle, Hormone agonists or antagonist, antivirotic, antifungal agent, antiparasitic, and/or

D) no therapeutic purposes are carried as bio-carrier but has the compound of secondary harvest, the harvest includes but unlimited In the internal identification and imaging that will make it possible to identify disease metastasis site.

Quote in this application every application, patent and article and (including every awarded in every application, patent and article Weigh the course of the review of patent；The file of reference " application "), U.S. Publication No.2005/0106717 A1 and 2008/ in a review The every file or bibliography quoted in 0227176 A1 correspond to or require any of these applications and patentss excellent Each piece of the PCT and foreign application or patent that first weigh and cited or reference the file in the cited file of every application Each piece pass through reference be clearly incorporated herein.

It is any of above will not be with content explicitly disclosed herein by the theme for being limited to be incorporated to that is incorporated to of citation It contradicts.It is any of above to be also limited to claim contained in document by being incorporated to for citation and be not incorporated by reference into this Text.It is any of above that this is not incorporated by reference into by any definition for being also limited to provide in the literature that is incorporated to of citation Text clearly states except being included herein.

In order to explain claim of the invention, explicitly points out and save the 6th section of regulation not based on 35 U.S.C.112, remove It is non-to record specific term " meaning " or " ... the step of " in the claims.

It would be recognized by those skilled in the art that can many modifications may be made without departing from hair described herein to present disclosure Bright spirit.Therefore, it is not intended that limiting the scope of the invention to the embodiment and embodiment.But it is of the invention Range is explained by appended claims and its equivalent variations.

The following contents corresponds to the original claims in parent application, is now incorporated to this as part of the specification Place:

1. a kind of method for being used to prepare the T cell group with desired native antigen specificity, which comprises

A. PBMC or Cord blood are added into cell culture apparatus,

B. culture medium and more than one antigen are added into the cell culture apparatus to activate more than one antigen-specific Property T cell group growth, every kind of T cells with antigenic specificity group to one of described antigen have native antigen specificity,

C. a period of time is allowed to make at least one T cells with antigenic specificity group response at least one antigen And starter population expands,

D. culture is assessed with determination at least one T cells with antigenic specificity group there are situation and/or amount,

E. determine which kind of T cells with antigenic specificity group is suitable for continuing to be proliferated, and

F. the culture is stimulated again with the antigen of the T cells with antigenic specificity group identification only by being considered as suitable for continuing proliferation Object.

2. method described in 1, wherein the method the result is that being prepared for single T cells with antigenic specificity group.

3. method described in 1, wherein the T cells with antigenic specificity group for being suitable for continuing proliferation, which has, is only capable of identification not On normal cell there are antigen native antigen specificity.

4. method described in 1, wherein the T cells with antigenic specificity group for being suitable for continuing proliferation, which has, is only capable of identification not On normal cell there are antigen native antigen specificity.

5. method described in 4, wherein the T cells with antigenic specificity group for being suitable for continuing proliferation, which has, is only capable of identification not On normal cell there are antigen single epitope native antigen specificity.

6. method described in 3, wherein the T cells with antigenic specificity group for being suitable for continuing proliferation, which has, is only capable of identification not On normal cell there are antigen and can identify including reptile, amphibian, fish, birds, invertebrate, Bacterium, fungi, helminth, spongia and/or virus antigen including antigen native antigen specificity.

7. method described in 1, wherein the cell culture apparatus includes poromeric material.

8. method described in 7, wherein the PBMC or Cord blood are placed on the poromeric material.

9. method described in 1, wherein the floor level face of the culture medium and the highest level face of the culture medium it Between exist be more than 2.0cm distance.

10. method described in 1, wherein the PBMC or Cord blood are less than 500,000cm²Superficial density it is resident.

11. a kind of method for being used to prepare the T cell group with desired native antigen specificity, which comprises

A. PBMC or Cord blood are added into cell culture apparatus,

B. culture medium and antigen are added into the cell culture apparatus has native antigen special the antigen to activate The growth of the T cells with antigenic specificity of the opposite sex,

C. between the floor level face of the culture medium and the highest level face of the culture medium exist be more than 2.0cm away from From,

D. the PBMC or Cord blood are placed on poromeric material,

E. the native antigen specificity is only capable of the antigen that identification does not present on normal cell.

12. method described in 11, wherein the T cells with antigenic specificity, which has, is only capable of identification not on normal cell There are antigen native antigen specificity.

13. method described in 12, wherein the T cells with antigenic specificity, which has, is only capable of identification not on normal cell There are antigen single epitope native antigen specificity.

14. method described in 12, wherein the T cells with antigenic specificity has and is only capable of identification and does not deposit on normal cell Antigen and at least can identify including reptile, amphibian, fish, birds, invertebrate, bacterium, fungi, The native antigen specificity of antigen including the antigen of helminth, spongia or virus.

15. method described in 11, wherein the PBMC or Cord blood are with less than 500,000 cell/cm²Surface it is close Degree is placed.

Claims (7)

1. a new class for the treatment of cell for being known as carrier T, wherein the carrier T includes with antigen of the identification from fungi The human T-cell of nave T cell receptor, and the human T-cell is transformed into at least one treatment characteristic.

2. carrier T described in claim 1, wherein the treatment characteristic is expression Chimeric antigen receptor.

3. carrier T described in claim 1, wherein the treatment expresses suicide gene with cell.

4. carrier T as claimed in claim 3, wherein the suicide gene is thymidine kinase or phase from herpes simplex virus I Close death domain-containing albumen or caspase 9.

5. carrier T described in claim 1, wherein the treatment has the day for an epitope for only identifying the antigen with cell Right antigen receptor.

6. carrier T as claimed in claim 2, wherein the Chimeric antigen receptor identifies tumor targets.

7. carrier T as claimed in claim 6, wherein the human T-cell has been subjected to virus transfection.