CN100379852C - Compositions and methods for restoring immune responsiveness in patients with immunological defects - Google Patents

Abstract

The present invention relates generally to methods for stimulating, activating, and maintaining or increasing the polyclonality of expressed TCRs in a population of T cells. In the various embodiments, cells are stimulated with a surface, wherein the surface has attached thereto one or more agents that ligate a cell surface moiety of at least a portion of the T cells and stimulates at least a portion of the T cells, yielding enhanced proliferation, cell signal transduction, and/or cell surface moiety aggregation. In certain aspects methods for stimulating a population of cells such as T-cells, by cell surface moiety ligation are provided by contacting the population of cells with a surface, that has attached thereto one or more agents that ligate a cell surface moiety thereby inducing cell stimulation, cell surface moiety aggregation, and/or receptor signaling enhancement. Also provided are methods for producing T-cells for the use in diagnostics and the treatment of a variety of indications, including cancer, viral infection, and immune related disorders. Compositions of cells having increased polyclonality produced by these processes are further provided.

Description

Be used in immune deficiency patient body, recovering the composition and the method for immune response

Background of invention

Invention field

Generally speaking, the present invention relates to be used to stimulate the method for T cell, in particular, relate to the method for the polyclone of the TXi Baoshouti (TCR) that provides expressed in the T cell mass, thereby recover the immunological competence of described T cell.The invention still further relates to cell composition and uses thereof, described composition comprises the irriate cell of polyclone increase.

The explanation of association area

The whole antigenic ability that the T cell recognition is relevant with multiple cancer or infectious organism is given by its T cell antigen receptor (TCR), and TCR is made up of α chain and β chain or γ chain and δ chain.In order to produce the huge diversity of TC R, the protein of forming these chains is by adopting the machine-processed dna encoding of uniqueness.TCR α and β or γ and δ chain are by disulfide linkage link to each other (Janeway, Travers, Walport, Immunobiology, the 4th edition, 148-159 page or leaf, Elsevier ScienceLtd/Garland press, 1999).α/β or gamma/delta heterodimer are compound with constant CD3 chain on cytolemma, and this mixture identification and MHC molecule bonded specific antigen peptide, or may discern the part that does not rely on the MHC restriction in the situation of gamma delta T cells.The specific huge multifarious generation of TCR is closely similar with the immunoglobulin (Ig) diversity of resetting generation by somatic cell gene.The β chain gene comprises and surpasses 50 variable (V), 2 diversity (D), surpasses 10 connection (J) sections and 2 constant (C) sections.The α chain gene comprises about 70 V sections, surpasses 60 J sections, no D section and 1 C section.In the cytocerastic process of T, the gene rearrangement of β chain D to J having taken place, follows and reset to DJ by the V constant gene segment C in thymus gland.This functional VDJ β exon through transcribe with montage after be connected C β.For the α chain, V α constant gene segment C is reset to J α constant gene segment C and has been generated functional exon, then through transcribe with montage to C α.

The ability of V, D and J constant gene segment C gang has been introduced the multifarious macroelement of combination at random in the TCR set.The accurate site that V, D are connected with the J section is variable, thereby has improved the local amino acid whose diversity in junction.The accurate nucleotide position that connects can differ nearly 10 residues, causes being changed thereby produce codon in the junction of these sections by V, D and the terminal deleted nucleotide of J constant gene segment C.Add when being not extra Nucleotide by arbitrary constant gene segment C coding when the junction between the constant gene segment C of linking to each other, in rearrangement process, further increased diversity.(mutability that produces by this method is called " N district diversity ".) (Janeway, Travers, Walport, Immunobiology, the 4th edition, the 98th and 150 page, Elsevier ScienceLtd/Garland press, 1999).

By indivedual T cells in the assessments T cell aggregation adopted which TCRV β chain and V β gene after the number of the random nucleotide of insertion, can partly measure T cell complete or collected works' diversity level.Generally speaking, when circulation T cell aggregation comprises the T cell of expressing four corner TCR V β chain, and when those indivedual V β chains be when utilize inserting Nucleotide the gene recombination incident of extensive arrangement is derived, immune T cell branch will have all maximum capacities of the potential antigen of identification.When the scope of the TCRV β chain of being expressed by circulation T cell aggregation is restricted or dwindles, and when expressed TCR utilized by the coded chain of the recombination with the insertion of finite nucleus thuja acid, the width of immunne response potential dwindled with regard to corresponding.Its consequence is to respond to extremely multiple antigenic ability to reduce, and causes infected and cancered risk to raise.

The spectral pattern analysis is the TCR V β, the V α that are used for measuring the T cell aggregation, V γ or the V δ gene rate of utilization of latest developments and the method (as U.S. Patent number 5,837, described in 447) of the cytocerastic regrouping process Nucleotide of T insertion level.The spectral pattern analysis can be used for measuring the width or the narrow degree of T cellullar immunologic response potential.

It is central event in the t cell activation that α β TCR and antigen presenting cell (APC) are gone up combining of institute's bonded antigen peptide in the MHC molecule, and it betides the immunology cynapse of point of contact between T cell and the APC.In order to keep t cell activation, the T lymphocyte needs second costimulatory signal usually.Stimulate altogether that normally t helper cell is needed in order to generate the cytokine levels that enough brings out clonal expansion.Bretscher, Immunol.Today 13:74,1992; June etc., Immunol.Today15:321,1994.Main costimulatory signal when combining with the CD28 on the T cell, B7 family part (CD80 (B7.1) or CD86 (the B7.2)) member on the antigen presenting cell (APC) after activating takes place.

Stimulate the method for some T cell subclass amplification to have the potentiality that generate the multiple T cell composition that can be used for immunotherapy.Improve polyclone, reactivity and the quantity of T cell by effective stimulus, have the immunotherapy of the success of helping.

Human T-cell's the multiple technologies of can be used for increasing mainly rely on the use of helper and/or exogenous growth factors such as interleukin-2 (IL-2).IL-2 and anti-cd 3 antibodies one are used from the CD8 that stimulates T cell proliferation, advantage pcr T cell ⁺Subgroup.Think two kinds of apc signals all be best t cell activation, amplification and again behind the infusion survival of T cell long-period needed.To having proposed major issue as the demand of helper, because the life-span of APC is shorter relatively about the long-term cultivation system with the APC of MHC coupling.Therefore, in the long-term cultivation system, constantly the origin source obtains APC and replenishes.For the treatment of the affected immune deficiency of helper, the necessity of renewable supply helper is a problem very much.In addition, when the treatment virus infection, if helper carries virus, cell may pollute whole T cell mass in long-term cultivation so.

The existing method in this area has been utilized anti-CD3 and the anti-CD28 T cell that increases.Yet these methods had not all been described polyclone and the advantageous results thereof of using these or similar approach to improve the T cell mass.In addition, the suitability through amplification T cell is only limited to the some diseases state.And, have method inclination now in clone's property of further deflection T cell mass, but not improve and/or keep the polyclone of T cell mass.In order to obtain effect in the maximum body, in theory, exsomatize or body in generate, should be in the immunne response that can maximumly coordinate (orchestrate) through the activated T cells group at cancer, communicable disease or other morbid state.The invention provides and generate more highly activating and the method for purer T cell of greater amt, the polyclone of described T cell in the TCR expression is improved.

Summary of the invention

One aspect of the present invention provides a kind of external method of the T of recovery cell mass polyclone, and it comprises:

(a) provide a group cell, wherein its part comprises the T cell at least;

(b) described cell mass is exposed to first kind of reagent and second kind of reagent, wherein said first kind of reagent comprises anti-CD 3 antibodies, or its Fab and described second kind of reagent comprises anti--CD28 antibody, or its Fab;

Wherein described cellular exposure is enough to increase polyclone in the time of described first kind of reagent and second kind of reagent; Wherein the increase of polyclone comprises the conversion of at least a V 'beta ' family gene from monoclonicity or few clone's property to polyclone, and described V 'beta ' family gene is selected from by V β 4,9,11,13,14,15 and 22 groups of forming,

Recover the polyclone of T cell mass thus.

Provide T cell mass that aforesaid method recovered polyclone to be used for recovering application in the medicament of immunne response on the other hand at the non-responsiveness individuality in preparation.

Another aspect provides a kind of composition, and it comprises T cell mass and the pharmaceutical excipient that has recovered polyclone according to aforesaid method.

One aspect of the present invention provides the TCR that is used to recover from non-responsiveness patient's T cell mass to express the method for polyclone, be used to recover patient's immune response, it comprises: a group cell is provided, wherein at least a portion comprises the T cell, cell mass is exposed to the surface of having adhered to one or more reagent, described reagent connects the cell surface part of at least a portion T cell and stimulates at least a portion T cell, with described cell cultures for some time at least a TCR V β that is enough to increase TCR expression aspect, V α, V γ, and/or the polyclone of V δ family, thereby the polyclone of recovery T cell mass.

In one embodiment of the invention, according to the measurement of V β, V α, V γ and/or the V δ spectral pattern feature of at least a V β, V α, V γ and/or V δ family gene, recover to comprise that the T cell mass is transformed into few clone's property, is transformed into polyclone or is cloned the transformation that sex reversal becomes polyclone by the widow by monoclonicity by monoclonicity.In another embodiment provided herein, change and to comprise the polyclone T cell number of expressing at least a V β, V α, V γ and/or V δ family gene brought up to and be enough to be used in treatment.

Another aspect of the present invention provides the method that is used for recovering at the non-responsiveness individuality immune response, the TCR of wherein individual T cell expresses polyclone, and individuality reduces with respect to immunne response is arranged, comprise: obtain cell mass by individuality, wherein at least a portion comprises the T cell; Cell mass is exposed to the surface of having adhered to one or more reagent, and described reagent connects the cell surface part of at least a portion T cell and stimulates at least a portion T cell; With described cell cultures for some time to the polyclone that is enough to increase at least a TCR V β, V α, V γ and/or V δ family; And the part of the stimulation of T cell beaten in the non-responsiveness individuality, recover the immune response of non-responsiveness individuality thus.In certain embodiments, the polyclone of inculcating the T cell was kept after inculcating 3 to 6 months to 1 year in vivo at least.

In one embodiment, the non-responsiveness individuality suffers from cancer.Cancer can be any of following group: melanoma, non Hodgkin lymphoma, Hodgkin, leukemia, plasmoma, sarcoma, neurospongioma, thymoma, mammary cancer, prostate cancer, colorectal cancer, kidney, renal cell carcinoma, carcinoma of the pancreas, nasopharyngeal carcinoma, esophagus cancer, the cancer of the brain, lung cancer, ovarian cancer, cervical cancer, multiple myeloma, hepatocellular carcinoma, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CML), large granular lymphocyte leukemia (LGL), and lymphocytic leukemia (CLL).In a preferred embodiment, cancer is a B cell lymphocyte leukemia.

In another embodiment, the non-responsiveness individuality has infected infectious organism.Infectious organism can comprise virus, such as single strand RNA virus or single-stranded DNA viruses, human immunodeficiency virus (HIV), first type, B-mode or hepatitis C virus, hsv (HSV), human papillomavirus (HPV), cytomegalovirus (CMV), EB (EBV), parasite, bacterium, pulmonary tuberculosis mycobacterium (M.tuberculosis), pneumocystis pneumoniae (Pneumocystis carinii), candiyeast (Candida) or aspergillus tubigensis (Aspergillus) or its combination.

In another embodiment, the non-responsiveness individuality suffers from the congenital heredity disease, such as Reconstruction in Sever Combined Immunodeciency (SCID) or common variable immune deficiency (CVID).In certain embodiments, individuality be since with the treatment of related to cancer non-responsiveness.In certain embodiments, individuality be since with hematopoietic stem cell transplantation, bone marrow transplantation, Cord blood, allogeneic, from body or heterogenous cell transplanting, chemotherapy, radiotherapy, cytotoxic reagent treatment, immunosuppressant treatment (as ciclosporin, reflunomide, or the like) relevant treatment and non-responsiveness.In also having an embodiment, the non-responsiveness individuality suffers from immune deficiency or autoimmune disease.In also having an embodiment, the non-responsiveness individuality suffers from the chronic disease that influences kidney, liver or pancreas.In a specific embodiment, individuality suffers from diabetes.In one embodiment, the non-responsiveness individuality is subjected to aged influence.In another embodiment, the non-responsiveness individuality was accepted gene therapy or was related to the other therapies of the gene transfer that causes T cell complete or collected works deflection.

In another embodiment, the non-responsiveness individuality suffers from and change of T cell collection or deflection diseases associated, includes but not limited to: rheumatoid arthritis, multiple sclerosis, insulin-dependent diabetes mellitus, bronzed disease, celiac disease, chronic fatigue syndrome, inflammatory bowel, ulcerative colitis, regional ileitis, Fibromyalgia, systemic lupus erythematous, psoriasis, the Sjogren syndrome, hyperthyroidism/Graves disease, hypothyroidism/Hashimoto thyroiditis, insulin-dependent diabetes mellitus (I type), myasthenia gravis, endometriosis, scleroderma, pernicious anemia, the thorough syndromes of Gourde(G) Paasche, wegener disease, glomerulonephritis, aplastic anemia, any of multiple cytopenia, paroxysmal nocturnal hemoglobinuria, myelodysplastic syndromes, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, Fanconi anemia, the Ai Wensi syndromes, the Factor IX inhibitor syndrome, the factors IX inhibitor syndrome, systemic vasculitis, dermatomyositis, polymyositis and rheumatic fever.Method and composition as herein described can be used for treating and is characterized as the low blood disease of blood counting.

In another embodiment, the non-responsiveness individuality suffers from and relevant neurological disorder or the cardiovascular disorder of T cell collection deflection.

In another embodiment, cell composition of the present invention is applied to the autoimmunization patient.One embodiment of the invention provide the method that is used for recovering at the non-responsiveness individuality immune response, and wherein the non-responsiveness individuality suffers from autoimmune disease.In certain embodiments, autoimmune disease includes but not limited to rheumatoid arthritis, multiple sclerosis, insulin-dependent diabetes mellitus, bronzed disease, celiac disease, chronic fatigue syndrome, inflammatory bowel, ulcerative colitis, regional ileitis, Fibromyalgia, systemic lupus erythematous, psoriasis, Sjogren syndrome, hyperthyroidism/Graves disease, hypothyroidism/Hashimoto thyroiditis, insulin-dependent diabetes mellitus (I type), myasthenia gravis.In another embodiment, the non-responsiveness individuality was accepted the treatment of chemotherapy.In also having an embodiment, the non-responsiveness individuality was accepted the treatment of cytotoxic reagent.In another embodiment, the non-responsiveness individuality was accepted the treatment of immunosuppressor.In one embodiment, the non-responsiveness individuality suffers from the blood disease relevant with cytopenia, includes but not limited to: aplastic anemia, myelodysplastic syndromes, Fanconi anemia, idiopathic thrombocytopenic purpura and autoimmune hemolytic anemia.

One aspect of the present invention provides the composition that comprises T cell mass and pharmaceutical excipient, the polyclone of T cell mass has obtained recovery according to methods described herein described in the T cell mass, described composition is used for recovering immune response at the non-responsiveness individuality, and wherein immunne response is individual to be reduced Ge Ti T cell polyclone with respect to having.

In certain embodiments, after using chemotherapy agents such as fludarabine, external source bundle radiation-therapy (XRT), endoxan or antibody such as OKT3 or CAMPATH to carry out the treatment of T cell ablation, the patient is used composition of the present invention.In another embodiment, B cell ablation treatment such as with the reagent such as Rituxan of CD20 reaction after, the patient is used composition of the present invention.The dosage that is applied to the patient in the above-mentioned treatment will be along with the recipient of the accurate essence of symptom to be treated and treatment and is changed.Can be applied to the measurement of people's dosage according to the accepted convention in this area.For example, the dosage of CAMPATH generally is for adult patients in 1 to about 100mg scope, uses the every day in period between 1-30 days usually.Although may use the heavy dose up to 40mg/ days in some situation, preferred per daily dose is 1-10mg/ days (being described in U.S. Patent number 6,120,766).

On the one hand, the invention provides the composition that comprises T cell mass and pharmaceutical excipient, the polyclone that TCR expresses in the T cell mass has obtained recovery according to methods described herein, be used for recovering immune response at the non-responsiveness individuality, wherein immunne response is individual to be reduced Ge Ti T cell polyclone with respect to having.

On the other hand, the first kind of reagent that has adhered to the first kind of cell surface part that connects the T cell described in the method provided by the invention on the surface; And the second kind of reagent that has adhered to the second section that connects described T cell on same surface or the second kind of surface, wherein first kind with induce described T cell proliferation being connected of second kind of reagent.In one embodiment, first kind of reagent comprises that anti-cd 3 antibodies and described second kind of reagent comprise the part in conjunction with accessory molecule on the described T cell surface.In another embodiment, described accessory molecule is CD28.In another embodiment, first kind of reagent comprises that anti-cd 3 antibodies and described second kind of reagent comprise anti-CD28 antibody.In also having an embodiment, described first kind and second kind of reagent are attached to described surface or described second kind of surface by covalency.In other embodiments, described first kind and second kind of reagent are by direct absorption or be attached to described surface or described second kind of surface indirectly.

On the other hand, adhered to cell surface part that connects at least a portion T cell and one or more reagent that stimulate at least a portion T cell described in the method provided by the invention on the surface, described t cell activation is induced in the connection of wherein said one or more reagent.In one embodiment, one or more surfaces have been used among the present invention.In another embodiment, three kinds or more reagent are with cis or trans being attached on the described surface.

The accompanying drawing summary

Fig. 1 is the synoptic diagram that TCRV β chain spectral pattern is analyzed, illustration the typical spectral pattern feature of polyclone, few clone and mono-clonal T cell mass.

Fig. 2 has shown un-activation T cell, usefulness OKT3 and the IL-2 activated T cells by the spectral pattern analysis to measure and has used XCELLERATE ^TMThe T cell aggregation of method activated T cells relatively.

Fig. 3 is that T cell from B-CLL patient is at XCELLERATE ^TMBefore activating and spectral pattern analysis afterwards, shown XCELLERATE ^TMMethod is corrected observed T cell shortage in the patient.

Fig. 4 is with Goroshov remoulding indes (Gorochov G., Neumann A.U., KereveurA., Parizot C., Li T.,, Katlama, C.,, Karmochkine M.,, Raguin G.,, AutranB., with Debre P., Nat.Med.4:215-221,1998) figure of T cell aggregation " disturbance " aggregate level of 8 donors of diagram.

The column diagram of Fig. 5 a and 5b has shown the CD4 that is untreated from 2 normal donors ⁺And CD8 ⁺The flow cytometry of the TCRV β cell surface expression of several V 'beta ' families on the T cell, and with from 2 B-CLL donors be untreated and through XCELLERATED ^TMThe CD4 that handles ⁺And CD8 ⁺The T cell compares.The figure illustrates the per-cent of expressing representative proteinic CD8 of TCR V 'beta ' family (5a) and CD4 (5b) cell in its surface.

The linear graph of Fig. 6 has shown XCELLERATE ^TMMethod has been improved the lymphocyte recovery in accepting the myelomatosis people who transplants.

Detailed Description Of The Invention

Before setting forth the present invention, recognize that definition illustrated in some term that hereinafter will use may be helpful.

Term " biocompatible " refers to the characteristic to the survivaling cell totally nontoxic when being used for this paper.

Term " stimulation " refers to the primary response that induces by the connection of cell surface part when being used for this paper. For example, in the content of acceptor, this kind stimulation needs the connection of acceptor and signal transduction event subsequently. As for the T cytositimulation, this kind stimulation refers in one embodiment the connection of the T cell surface part of the transduction of inducement signal subsequently event, such as in conjunction with the TCR/CD3 compound. In addition, the stimulation event may also raise or reduce cell surface molecule such as the expression of acceptor or adhesion molecule by active cell, and the secretion of perhaps raising or reducing molecule is such as downward modulation β-tumour growth factor (TGF-β). Therefore, even when lacking direct signal transduction event, the connection of cell surface part still may cause the again tissue of cytoskeletal structure, or the merging of cell surface part, and wherein every kind of part all can be used for strengthening, modify or change cell response subsequently.

Term " activation " refers to induce the cell state of measurable morphology, phenotype and/or changes of function after enough cell surfaces partly connect when being used for this paper. In the content of T cell, this kind activation can be the state that the T cell has fully stimulated to induce cell proliferation. T cell activation also may be induced generation and/or the secretion of cell factor, and raises or reduce cell surface molecule such as the expression of acceptor or adhesion molecule, the secretion of perhaps raising or reducing some molecule, and carry out and regulate or molten cytological effect thing function. In the content of other cell, this term refers to rise or the downward modulation of specific physical and chemical process.

Term " target cell " refers to be intended to when being used for this paper partly connect any cell of stimulating by cell surface.

" antibody " comprises (as humanized), mouse, mouse-people, mouse-primate and chimeric of polyclone and monoclonal antibody (mAb), primateization when being used for this paper; And can be complete molecule, its fragment (such as scFv, Fv, Fd, Fab, Fab ' and F (ab) '₂Fragment) or the polymer of complete molecule and/or fragment or aggregation; And can be naturally occurringly maybe can produce, as producing by immunity, synthetic or genetic engineering; " antibody fragment " refers to be derived or the fragment relevant with antibody by antibody when being used for this paper, and described antibody conjugated antigen and can being showed by derivation in some embodiments is convenient to the architectural feature removing and absorb, as by mixing galactose residue. This comprises such as Fab ', F (ab) '₂, scFv, variable region of light chain (V_L), weight chain variable district (V_H) and combination.

Term " protein " comprises protein, glycoprotein and other cell-derived modified protein, polypeptide and peptide when being used for this paper; And can be polymer or the aggregation of complete molecule, its fragment or complete molecule and/or fragment; And can be naturally occurringly maybe can produce, as producing by synthetic (comprising chemistry and/or enzymatic) or genetic engineering.

Term " reagent ", " part " or " in conjunction with the reagent of cell surface part " refer to the molecule in conjunction with the designated cell group when being used for this paper. Reagent can be in conjunction with any cell surface part, such as upper other binding site that exists of acceptor, antigenic determinant or target cell group. Reagent can be protein, peptide, antibody and antibody fragment thereof, fusion, synthetic molecules, organic molecule (such as little molecule), etc. In specification and in the content of T cytositimulation, antibody is used as the prototype example of this kind reagent.

Term " cell surface part " can refer to any other binding site that cell surface receptor, antigenic determinant or target cell group exist when being used for this paper.

Term " in conjunction with the reagent of cell surface part " and " cell surface part " are interpreted as when being used for this paper usually to show a cover complementation/anti-complementary molecule of specific bond than high affinity.

" costimulatory signal " refers to cause with primary signal the signal of T cell proliferation and/or activation when being used for this paper, connect such as TCR/CD3.

" separation " comprises any means (as by filtration, affinity, buoyant density or magnetic attachment) with a kind of component and the abundant purifying of another kind of component when being used for this paper.

" surface " refers to have reagent to adhere to any surface on it when being used for this paper, includes but not limited to: metal, glass, plastics, copolymer, colloid, lipid, cell surface, etc. Any surface can both keep the reagent combination or adhere on it in essence.

" monoclonicity " has monospecific T cell mass at the content middle finger of T cell mass according to the definition of spectral pattern analysis (measuring TCR V β, V α, V γ or V δ chain hypervariable region complete or collected works) when being used for this paper. When V β, V α, V γ and/or the V δ spectral pattern sign of the TCR of appointment V β, V α, V γ and/or V δ family have single dominant peak (seeing Fig. 1), think that the T cell mass is monoclonal (or single special). The spectral pattern analysis is distinguished specific size but not the rearrangement variable gene of sequence. Therefore, being appreciated that single peak may represent expresses limited number and resets in the TCR variable gene (V β, V α, V γ or V δ) any T cell mass, and described rearrangement TCR variable gene comprises the combination of 4 kinds of any or 4 kinds of nucleotides in may nucleotides (adenine (a), guanine (g), cytimidine (c) or thymidine (t)) in the bonding land. In certain embodiments of the invention, may expect that the Cloning and sequencing particular bands is to measure the sequence that represents the rearrangement variable gene that exists in the band of length-specific.

" few clone property " when being used for this paper the content middle finger of T cell mass according to spectral pattern analysis (measuring TCR β chain hypervariable region complete or collected works) but the T cell mass of definition with multiple narrow antigentic specificity. When the V β spectral pattern of the TCR of appointment V 'beta ' family characterizes the advantage peak (seeing Fig. 1) that has between about 2 and about 4, think that the T cell mass is few clone.

" polyclone " has multiple and wide antigentic specificity in the content of T cell mass according to the definition of spectral pattern analysis (measuring the super variable region complete or collected works of TCR β chain) when being used for this paper T cell mass. When the V β spectral pattern feature of the TCR of appointment V 'beta ' family has a plurality of peaks, 5 or more advantages peak and when having Gaussian distribution in most of situation when (referring to for example Fig. 1,2 and 3) think that the T cell mass is polyclonal typically.

" recover or the enhancing polyclone " when being used for this paper, to refer to analyze defined according to spectral pattern analysis or similarity analysis such as flow cytometry or sequence, in the expressed TCRV β of T cell mass, V α, V γ and/or V δ gene, characterized by monoclonal and to be transformed into that few clone characterizes or (in other words polyclone characterizes, be transformed into few clone's property or polyclone by monoclonicity), or cloned to characterize by the widow and be transformed into polyclone and characterize (in other words, clone sex reversal by the widow and become polyclone). Usually at least a TCR V β, V α, V γ and/or V δ family, observe the T cell mass and be transformed into few clone's sign or polyclone sign by monoclonal V β, V α, V γ and/or V δ expression sign. In one embodiment of the invention, 2,3,4 or 5 kind of V 'beta ' family in observe this kind transformation. In certain embodiments of the invention, 6,7,8,9 or 10 kind of V 'beta ' family in observe this kind transformation. In another embodiment of the invention, 11,12,13 or 14 kind of V 'beta ' family in observe this kind transformation. In another embodiment of the invention, in 15-20 kind V 'beta ' family, observe this kind transformation. Also have in the embodiment of the present invention, in 20-24 kind V 'beta ' family, observe this kind transformation. In another embodiment, in all V 'beta ' families, observe this kind transformation. The ability that the functional meaning that recovers or increase T cell mass polyclone is the immune potentiality of T cell mass or replys whole antigens is restored or improves. Aspect some, the TCR of some T cells may not can participate in method in this paper (expressing the T cell of downward modulation such as TCR) in the group of the present invention. Yet, approaching by the T cell of methods described herein activation and the factor of their secretions by extreme, these T cells can then raise its TCR and express, thereby cause the polyclone of T cell mass further to increase.

Term " animal " or " mammal " are contained all mammals when being used for this paper, comprise the people. Preferably, animal of the present invention is people experimenter.

Term " exposure " instigates it to be in state or the situation that extremely approaches or directly contact when being used for this paper.

Term " propagation " refers to grow or breed by generating new cell when being used for this paper.

" immune response or response " refers to the activation of immune system cell when being used for this paper, include but not limited to the T cell, thereby induce the specific effect thing function of specific cells. Effector function can include but not limited to propagation, secrete cytokines, secretory antibody, expression adjusting and/or adhere to molecule and induce cytolytic ability.

" immune stimulatory is replied " refers to realize the activation and any stimulation of inductive of immune system cell effector function when being used for this paper.

" immunne response dysfunction " refers to inappropriate activation and/or the propagation or the shortage of immune system cell when being used for this paper, and/or the inappropriate secretion or the shortage of cytokine, and/or inappropriate or insufficient the inducing of other effector function of immune system cell, express, adhere to and/or homing receptor and cytolytic inducing such as regulating.

Term " prevention " or " suppressing the development of cancer or cancer cell " refers to being prevented of cancer when being used for this paper or the outbreak of cancer obtains sluggishness.

Term " existence of treatment or minimizing cancer or cancer cell " or " existence of treatment or minimizing tumour or tumour cell " refer to that when being used for this paper cancer or growth of tumor are inhibited, and this is by reflecting as gross tumor volume or malignant cell number.Can use the many technology in this area to measure dwindling of cancer, comprise that the proteic measurement of M-, the detection based on PCR, RNA and DNA hybridization detects or original position PCR or hybridization, etc.Can measure gross tumor volume by multiple currently known methods, as obtaining two-dimensional with dial plate caliper (dial caliper).

" development of prevention or inhibition communicable disease " refers to being prevented of communicable disease when being used for this paper or the outbreak of communicable disease obtains sluggishness, or existing spreading of infecting obtains reverse or stable.

" improvement " is defined as when being used for this paper and makes its better, improvement (TheAmerican HeritageCollege Dictionary, the third edition, Houghton Mifflin company, 2000).

" particle " or " surface " when being used for this paper, can comprise colloidal particle, microsphere, nanoparticle, bead, or the like.The surface can be to make part in conjunction with on it or integration any surface wherein, comprises cell surface (for example K562 cell), and is biocompatible, promptly treats the target cell totally nontoxic of stimulation.In a plurality of embodiments, commercially available surface such as bead or other particle all be available (as the Miltenyi particle, Miltenyi Biotec, Germany; Agarose beads, PharmaciaFine Chemicals, Sweden; DYNABEADS ^TM, Dynal company, New York; PURABEADS ^TM, Prometic Biosciences, from the magnetic bead of Immunicon, Huntington Valley, PA, from the microsphere of Bangs Laboratories company, Fishers, IN).

" paramagnetic particle " refers to adaptation defined above magnetic field and localized particle when being used for this paper.

Term " communicable disease " refers to any disease of being caused by infectious organism when being used for this paper.Infectious organism can comprise that virus is (as single strand RNA virus, single-stranded DNA viruses, human immunodeficiency virus (HIV), the first type, B-mode, and hepatitis C virus, hsv (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human papillomavirus (HPV)), parasite is (as protozoon and metazoan pathogenic agent, such as plasmodium species (Plasmodia species), leishmania species (Leishmania species), Schistosoma species (Schistosoma species), trypanosoma species (Trypanosoma species)), bacterium is (as mycobacterium (Mycobacteria), mycobacterium tuberculosis (M.tuberculosis) particularly, salmonella (Salmonella), streptococcus (Streptococci), intestinal bacteria (E.coli), staphylococcus (Staphylococci)), fungi is (as the candiyeast species, the aspergillus tubigensis species), pneumocystis pneumoniae, and Protein virus (known prion-infected animal and cause itch, be the neural a kind of propagable degenerative disorders of sheep and goat, and mad cow disease (BSE) or " mad cow disease ", cat family spongiform encephalopathy with cat.Four kinds of prion diseases of known infected person are the special cortico-striatal spinal degeneration (CJD) in (1) Kuru disease, (2) Creutz Fil, (3) Ge-Shi-Sha disease (GSS) and (4) fatal familial insomnia (FFI)." Protein virus " is included in the people of used any animal-particularly and raises and train the Protein virus that livestock causes the form of ownership of all or any these or other disease when being used for this paper.

Stimulate, activate and recover the polyclone of T cell

The polyclone increase through stimulating and generating by inducing the activated cell surface partly to be connected among the present invention through activated T cells.The polyclone increase through stimulating and being by the activated T cell group and using accessory molecule to generate, as Application No. 08/253,694,08/435 in conjunction with the ligand stimulation T cell surface of accessory molecule through activated T cells, 816,08/592,711,09/183,055,09/350,202 and 09/252,150 and the patent No. 6,352,694,5,858,358 and 5,883, described in 223.

Generally speaking, can partly connect by cell surface and realize t cell activation and polyclone recovery, such as stimulating TXi Baoshouti (TCR)/CD3 mixture or CD2 surface protein.Many anti-people CD3 monoclonal antibodies have realized commercialization, for example clone BC3 (XR-CD3; FredHutchinson DKFZ, Seattle, the State of Washington), by the OKT3 and the monoclonal antibody G19-4 of the hybridoma preparation that derives from American type culture collection.Similarly, the stimulation form of anti-CD2 antibody is known and obtainable.Usually use the combination of at least two kinds of anti-CD2 antibody of difference to realize with of the stimulation of anti-CD2 antibody through CD2.The stimulation combination of the existing anti-CD2 antibody of describing comprises: antibody combined T11.1 of T11.3 or T11.2 antibody (people such as Meuer, Cell 36:897-906,1984), and 9.6 antibody (discerning identical epi-position) associating 9-1 antibody (people such as Yang with T11.1, J.Immunol.137:1097-1100,1986).Also can use other antibody with the identical epi-position of any antibodies mentioned above.Can prepare and identify the combination of other antibody or antibody by standard technique.Can also realize stimulating by contacting following reagent: superantigen (as staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB), toxic shock syndrome toxin 1 (TSST-1)), intracellular toxin or by multiple mitogen include but not limited to phytohemagglutinin (PHA), phorbol tetradecanoic acid acetic ester (PMA) and ionomycin, lipopolysaccharides (LPS), T cell mitogen and IL-2.

In order further to activate and increase the polyclone of T cell mass, use common stimulation or accessory molecule, such as CD28 in conjunction with the ligand stimulation T cell surface of accessory molecule.Therefore, one of skill in the art will recognize that and to comprise that with the molecule crosslinked any reagent of CD28 the native ligand of anti-CD28 antibody or its fragment or CD28 all can be used for stimulating the T cell.The exemplary anti-CD28 antibody or its fragment that can be used in the content of the present invention comprise monoclonal antibody 9.3 (IgG2 _a) (Bristol-MyersSquibb, Princeton, New Jersey), monoclonal antibody KOLT-2 (IgG1), 15E8 (IgG1), 248.23.2 (IgM), clone B-T3 (XR-CD28; Diaclone,

, France) and EX5.3D10 (IgG2 _a) (ATCC HB11373).Exemplary native ligand comprises the B7 protein families, such as B7-1 (CD80) and B7-2 (CD86) (people such as Freedman, J.Immunol.137:3260-3267,1987; People such as Freedman, J.Immunol.143:2714-2722,1989; People such as Freedman, J.Exp.Med.174:625-631,1991; People such as Freedman, Science262:909-911,1993; People such as Azuma, Nature 366:76-79,1993; People such as Freedman, J.Exp.Med.178:2185-2192,1993).

Can include but not limited to CD54, Ox-40, LFA-1, ICOS, 41-BB and CD40 with other example among the present invention in conjunction with the T cell surface accessory molecule of the ligand stimulation of accessory molecule.

In addition, native ligand in conjunction with homologue, no matter be natural or by chemistry or recombinant technology synthetic, all can use according to the present invention.Other reagent can comprise natural and the synthetic part.Reagent can include but not limited to other antibody or its fragment, somatomedin, cytokine, chemokine, soluble receptors, steroid, hormone, mitogen such as PHA or other superantigen.

Amplification T cell mass

In one aspect of the invention, the T cell amplification that can exsomatize by the cell mass that stimulates at least a portion wherein to comprise the T cell.In one embodiment of the invention, can stimulate the T cell with single agents.In another embodiment, can stimulate the T cell with two or more reagent, a kind of primary signal of inducing, and other reagent is induced one or more costimulatory signals.Can be with soluble form, be attached to cell surface or be fixed in the accessory molecule that surface described herein is used the part that can be used for stimulating single signal or stimulate primary signal and stimulated secondary signal.Be attached to lip-deep part or reagent as " agency " antigen presenting cell (APC).In a preferred embodiment, primary and secondary reagent is fixed on the surface jointly.In one embodiment, provide molecule such as the CD3 part of primary activation signal and costimulatory molecules such as the CD28 ligand coupling on same surface, particle for example.In addition, as indicated above, can with a kind of, two kinds or more thorniness swash molecule and be used for identical or different surface.

Before amplification, obtain T cell source by the experimenter.Term " experimenter " is intended to comprise the survival organism (as Mammals) that can cause immunne response therein.Experimenter's example comprises people, dog, cat, mouse, rat and genetically modified organism thereof.Can obtain the T cell by many sources, comprise peripheral blood lymphocytes, marrow, thymus gland, slicer, tumour, lymph node tissue, digestive tube associated lymphoid tissue, mucosa associated lymphoid tissue, spleen tissue or any other Lymphoid tissue and tumour.Can be by T clone and by obtaining the T cell from body or allogeneic source.Can also obtain T cell, for example mouse, rat, non-human primates and pig by the xenogenesis source.In certain embodiments of the invention, many technology such as the glycan body that can use those of skill in the art to know separates by the blood acquisition T of a unit cell of gathering from the experimenter.In a preferred embodiment, obtain cell by single blood sampling composition art or white corpuscle extraction method by the circulation blood of individuality.Single blood sampling composition art generally includes lymphocyte from product, and it comprises T cell, monocyte, granulocyte, B cell, other has nuclear leukocyte, red corpuscle and thrombocyte.In one embodiment, the cell of can rinsing collecting by single blood sampling composition art is to remove the blood plasma part and to place suitable damping fluid or substratum to be used for subsequently procedure of processing in cell.In one embodiment of the invention, clean cell with phosphoric acid buffer (PBS).In alternate embodiment, scavenging solution is calcic not, and may not contain magnesium, is not all also to be many divalent cations even perhaps do not contain.To recognize easily as those of ordinary skills, can realize cleaning step by the method that those skilled in the art will know that, such as use according to the specification sheets of manufacturers semi-automatic " flowing through " whizzer (Cobe2991 cellular processor for example, Baxter).After the cleaning, cell can be resuspended in multiple physiologically acceptable damping fluid, such as the PBS that does not for example have calcium, no magnesium.Perhaps, can remove in single blood sampling composition art sample undesired part and cell directly is resuspended in substratum.

In another embodiment, by cracking or eliminate red corpuscle and eliminate monocyte and separate the T cell, for example pass through PERCOLL by peripheral blood lymphocyte ^TMGradient centrifugation.Can select technology further to separate the specific T cells subgroup by plus or minus such as CD28 ⁺, CD4 ⁺, CD8 ⁺, CD45RA ⁺, and CD45RO ⁺The T cell.For example, in a preferred embodiment, by puting together bead such as DYNABEAD with the anti-CD28 of anti-CD3/ (promptly 3 * 28) M-450CD3/CD28T is incubated together and separates the T cell, and the time is enough to the key player on a team and selects desirable T cells.In one embodiment, the time is about 30 minutes.In another embodiment, time range is 30 minutes to 36 hours or reaches wherein all round valuess for more time.In another embodiment, the time is at least 1,2,3,4,5 or 6 hour.In another preferred embodiment, the time is 10 to 24 hours.In a preferred embodiment, soaking time is 24 hours.In order to separate the T cell by the leukaemic, use longer soaking time can improve cell yield such as 24 hours.In compare the less any situation of T cell with other cell type, can use longer soaking time to separate the T cell, such as separating tumor infiltrating lymphocyte (TIL) by tumor tissues or by the non-responsiveness individuality.In addition, use longer soaking time to improve and catch CD8 ⁺The efficient of T cell.For example, can use CD3/CD28 to put together the magnetic bead grain (as DYNABEADS

M-450CD3/CD28T Cell Expander) just selecting CD3 ⁺, CD28 ⁺The T cell.In one aspect of the invention, can use antibody to make up the enrichment that realizes by the negative T of selection cell mass at negative selection cell unique surface mark.A kind of preferred method is by using at the negative magnetic immunosorption of the monoclonal antibody mixture of bearing the cell surface marker that exists on the selection cell or cell sorting and/or the selection that flow cytometry carries out.For example, in order to come enrichment CD4 by negative selection ⁺Cell, the monoclonal antibody mixture typically comprises the antibody at CD14, CD20, CD11b, CD16, HLA-DR and CD8.

Another aspect of the present invention provides have been eliminated before amplification or the T cell mass or the composition of the cell mass of multiple mark are expressed in enrichment, described mark such as CD62L, CD45RA or CD45RO, cytokine (as IL-2, IFN-γ, IL-4, IL-10), cytokine receptor (as CD25), pore-forming protein, adhesion molecule (as VLA-1, VLA-2, VLA-4, LPAM-1, LFA-1) and/or the molecule of going back to the nest (selecting albumen as L-).In one embodiment, by antibody or other part/binding reagents, eliminate or just selecting to express the cell of any of these mark at mark.Those of ordinary skills will be easy to identify multiple concrete grammar that is used to eliminate or just selecting to express the cell sample of expectation mark.

Eliminate for monocyte mentioned above, can eliminate the monocyte group by Blood Preparations before the amplification of exsomatizing by several different methods (is CD14 ⁺Cell), comprises bead or pillar, perhaps utilize the activate the phagocytic capacity of these cells so that eliminate or by adhering on the plastics with anti-CD14 bag quilt.Therefore, in one embodiment, the present invention uses size to be enough to the paramagnetic particle of being engulfed by engulfing property monocyte.In certain embodiments, paramagnetic particle is commercial bead, for example the commodity Dynabeads by name that is produced by Dynal AS ^TMExemplary Dynabeads in this respect ^TMBe M-280, M-450 and M-500.On the one hand, by being eliminated other non-specific cell by paramagnetic particle with " haveing nothing to do " protein (as serum protein or antibody) bag.Irrelevant protein and antibody comprise not can selectively targeted T cell to be amplified those protein and antibody or its fragment.In certain embodiments, irrelevant bead comprises the bead that wraps quilt with sheep anti mouse antibodies, goat anti-mouse antibody and human serum albumin.

In brief, this monocyte is eliminated and can followingly be carried out: will use the glycan body can eliminate monocytic one or more irrelevant or non-antibody link coupled paramagnetic particles (bead: cells ratio is about 20: 1) in 22-37 ℃ of pre-incubation about 30 minutes-2 hours together by the PBMC of separation of whole blood or through the peripheral blood of single blood sampling composition art with any amount, magnetic elimination subsequently is attached to or engulfs the cell of paramagnetic particle.Also can carry out pre-incubation in being low to moderate under 3-4 ℃ the temperature.Can use this area standard method to carry out this separation.For example, can use any magnetism separate method, comprise that multiple commodity are (as DYNAL

Magnetic particle concentrating instrument (DYNAL MPC

)).Can monitor the essential elimination of affirmation by the several different methods that those of ordinary skills know, comprise before the described elimination and the flow cytometry of CD14 positive cell afterwards.

Can also be after cleaning step the freezing T cell that is used to stimulate, this does not need the monocyte removal process.Wish not accept opinion and limit, the step of thawing freezing and subsequently provides more consistent product by granulocyte or the monocyte of eliminating in the cell mass to a certain degree.After eliminating blood plasma and hematoblastic cleaning step, can be with cell suspension in refrigerating fulid.Although many refrigerating fulids and parameter are known in this area and are useful in this content, a kind of method comprises uses PBS or other the suitable cell medium contain 20%DMSO and 8% human serum albumin, then cell is refrigerated to-80 ℃ and be stored in the gas phase of nitrogen storage tank with 1 ℃/minute speed.

Can irritation cell group as described herein, such as the anti-cd 3 antibodies or the anti-CD2 antibody that are fixed in the surface by contact, perhaps by the contact ionophoric protein kinase C activation agent of Combined with Calcium (as liver moss statin (bryostatin)).In order to stimulate the accessory molecule of T cell surface altogether, used part in conjunction with accessory molecule.For example, can contact CD4 with anti-cd 3 antibodies with anti-CD28 antibody being suitable for stimulating under the condition of T cell proliferation ⁺Cell mass.Similarly, in order to stimulate CD8 ⁺The propagation of T cell, can use anti-cd 3 antibodies and anti-CD28 antibody B-T3, XR-CD28 (Diaclone,

, France), other method (people such as Berg, TransplantProc.30 (8): 3975-3977,1998 generally known as this area; People such as Haanen, J.Exp.Med.190 (9): 1319-1328,1999; People such as Garland, J.Immunol Meth.227 (1-2): 53-63,1999).

The primary stimulus signal and the costimulatory signal of T cell can be provided by different schemes.For example, provide the reagent of various signals to may reside in the solution or be coupled on the surface.When being coupled to the surface, reagent can be coupled to same surface (promptly in " cis " mode) or different surfaces (promptly in " trans " mode).Perhaps, a kind of reagent can be coupled on the surface, and another kind of reagent exists in the solution.In one embodiment, provide the reagent of costimulatory signal to be incorporated on the cell surface, provide the reagent of primary activation signal then to exist in the solution or be coupled on the surface.In certain embodiments, two kinds of reagent can all exist in the solution.In another embodiment, reagent can be soluble form, then with surface-crosslinked, such as the cell of expressing the FC acceptor or antibody or with other binding reagents of binding reagents.In a preferred embodiment, in sphere or semispherical surface, the prototype example is bead or cell with two kinds of immobilization of reagents, or same bead is " cis ", or different bead is " trans ".For example, the reagent that primary activation signal is provided is anti-cd 3 antibodies, is anti-CD28 antibody and the reagent of costimulatory signal is provided; Two kinds of reagent are to equate that molecular amounts is fixed on the same bead altogether.In one embodiment, for the amplification of T cell and the growth of T cell, use 1: 1 ratio with every kind of antibodies on bead.Of the present invention aspect some, viewed amplification is compared when using 1: 1 ratio, is using certain ratio will resist CD3: the CD28 antibodies is observed the T cell amplification on bead the time to be increased.In a specific embodiment, viewed amplification is compared during with 1: 1 ratio of use, observes about 0.5 to about 3 times increase.In one embodiment, be incorporated into the anti-CD3 of bead: CD28 antibody ratio ranges is 100: 1 to 1: 100, and covers wherein all round valuess.In one aspect of the invention, compare with anti-cd 3 antibodies, more anti-CD28 antibodies is on particle, i.e. CD3: the CD28 ratio is less than 1.In certain embodiments of the invention, be incorporated into anti-CD28 antibody on the bead to the ratio of anti-cd 3 antibodies greater than 2: 1.In a specific embodiment, used 1: 100 with bead bonded CD3: CD28 antibody ratio.In another embodiment, used 1: 75 with bead bonded CD3: CD28 antibody ratio.In also having an embodiment, used 1: 50 with bead bonded CD3: CD28 antibody ratio.In another embodiment, used 1: 30 with bead bonded CD3: CD28 antibody ratio.In a preferred embodiment, used 1: 10 with bead bonded CD3: CD28 antibody ratio.In another embodiment, used 1: 3 with bead bonded CD3: CD28 antibody ratio.In also having an embodiment, used 3: 1 with bead bonded CD3: CD28 antibody ratio.

Can use 1: 500 to 500: 1 and any integer-valued particle pair cell ratio to stimulate T cell or other target cell between the two.As what those of ordinary skills recognized easily, the ratio of particle pair cell may depend on the size of particle with respect to target cell.For example, the small size bead can only be in conjunction with a few cell, and can be in conjunction with many than macrobead.In certain embodiments, also can use 1: 100 to 100: 1 and any integer-valued cell that the particle ratio is stimulated the T cell between the two, and in other embodiments, described ratio comprise 1: 9 to 9: 1 and any round values between the two.Cause the anti-CD3 of T cytositimulation and anti-CD28 coupling particle that the ratio of T cell can as indicated abovely be changed, yet some preferred value comprises at least 1: 5,1: 4,1: 3,1: 2,1: 1,2: 1,3: 1,4: 1 to 6: 1, and a preferred ratio is at least 1: 1 particle/T cell.In one embodiment, used 1: 1 or lower particle pair cell ratio.In another embodiment, particle pair cell ratio can be according to stimulating fate to change.For example, in one embodiment, first day particle pair cell ratio is 1: 1 to 10: 1, and every day or add extra particulate every other day reach 10 days in cell, and whole ratio is 1: 1 to 1: 10 (according to the cell counting of adding day).In a specific embodiment, the particle pair cell ratio that stimulated first day is 1: 1, and is adjusted to 1: 5 in the 3rd day and the 5th day in stimulation.In another embodiment, every day or to add particle to the whole ratio that stimulated first day every other day be 1: 1 and to stimulate the 3rd day and the 5th day be 1: 5.In another embodiment, first day particle pair cell ratio of stimulation is 2: 1, and is adjusted to 1: 10 in the 3rd day and the 5th day in stimulation.In another embodiment, every day or to add particle to the whole ratio that stimulated first day every other day be 1: 1 and to stimulate the 3rd day and the 5th day be 1: 10.Those skilled in the art will recognize that multiple other ratio also may be applicable to the present invention.Particularly, ratio will according to granular size and cell be big or small and type changes.

It may be favourable using some method to separate the T cell for keep T cell mass long-time stimulus after initial activation and stimulation with stimulator after about 12 to about 14 days.For example, by such as using Coulter counter (coulter counter) to check T cell size or measuring the T cell volume, regular (as every day) monitoring T cell proliferation speed.In this respect, the mean diameter of dormancy T cell is about 6.8 microns, and at initial activation with after stimulating, when having the stimulation part, the mean diameter of T cell will increase at the 4th day and surpass 12 microns, and begins to descend at about the 6th day.When the T average cell diameter drops to about 8 microns, can activate once more and stimulate the T cell to induce further T cell proliferation once more.Perhaps, can monitor T cell proliferation speed and T cell stimulation time again by measuring cell surface molecule such as the existence of inductive CD154, CD54, CD25, CD137, CD134 on activated T cell.

In order to induce CD4 ⁺And/or CD8 ⁺The long-time stimulus of T cell mass, possible essential usefulness stimulant such as anti-cd 3 antibodies and anti-CD28 antibody (B-T3, XR-CD28 (Diaclone,

, France)) or monoclonal antibody ES5.2D8 activate again or stimulate again the T cell for several times with generate with the naive T cell faciation than quantity increase about 10 times to about 1000 times CD4 ⁺And/or CD8 ⁺Cell mass.For example, in one embodiment of the invention, stimulate the T cell as described in example 1 above 2-3 time.In another embodiment, stimulate T cell 4 or 5 times as described in example 1 above.Use method of the present invention, might make with stimulate before compare the polyclone increase the T cell number reach about 100 times to about 100,000 times.In addition, in culture supernatants, secrete the cytokine (as IL-2, IFN-γ, IL-4, GM-CSF and TNF-α) of a large amount of (substantial) levels by the T cell of the inventive method amplification.For example, compare, stimulate the CD4 of amplification altogether by using anti-CD3 and anti-CD28 with the IL-2 stimulation ⁺The GM-CSF of T cell secreting high levels in substratum and TNF-α.Can perhaps supernatant liquor can be used directly in and keep cell in the cultivation by these cytokines of culture supernatants purifying.Similarly, the T cell by the inventive method amplification is grown in culture supernatants and cytokine can be used for the body of sustenticular cell.

In one embodiment, carry out the T cytositimulation with the anti-CD3 and the anti-CD28 antibody that for example are fixed in jointly on the bead (3 * 28 bead), the time is enough to make cell to reply stationary state (low or do not have propagation) (after the initial impulse about 8-14 days).Remove stimulus signal by cell then, clean cell and it is inculcated back in patient's body.As what embodiment proved, the cell when by method of the present invention stimulation period being finished becomes " super derivable ", confirms as the ability by antigenic ability of these cell responses and displaying memory sample phenotype thereof.Therefore, after the stimulation again of inculcating back external source or body endoantigen, show with unique phenotypic characteristic to be replying strongly of feature through activated T cells, such as keep CD154 express, increase cytokine production, or the like.

In other embodiment of the present invention, with cell such as T cell unite through pack by or the bead puted together, bead isolation and cell, culturing cell then subsequently.In alternate embodiment, before cultivation, do not separate through pack by or the bead puted together and cell but cultivate together.In another embodiment, at first applying pressure concentrates bead and cell, causes cell surface partly to connect, thereby inducing cell stimulates and/or the polarization of activation signal.

For example,, contact T cells contacting to be prepared, can connect the cell surface part by the paramagnetic bead (3 * 28 bead) that allows to have adhered to anti-CD3 and anti-CD28 when the T cell is the target cell group time.In one embodiment, in the preferred PBS of damping fluid (no divalent cation such as calcium and magnesium), merge cell (for example 10 ⁴To 10 ⁹Individual T cell) and bead (DYNABEADS for example

M-450CD3/CD28T paramagnetic bead, ratio 1: 1).In addition, those of ordinary skills can be easy to recognize and can use any cell concn.For example, target cell is may be very in sample rare, and only accounts for 0.01% of sample, and perhaps entire sample (promptly 100%) may comprise the purpose target cell.Therefore, any cell number all belongs in the scope of the present invention.In certain embodiments, may expect significantly to reduce particle and cytomixis volume (promptly improving cell concn) together, to guarantee cell and particulate maximum contact.For example, in one embodiment, the concentration of use is about 2 * 10 ⁹Individual cell/ml.In another embodiment, the concentration of use surpasses 1 * 10 ⁸Individual cell/ml.In another embodiment, the cell concn of use is 10,15,20,25,30,35,40,45 or 50 * 10 ⁶Individual cell/ml.In also having an embodiment, the cell concn of use is 75,80,85,90,95 or 100 * 10 ⁶Individual cell/ml.In other embodiments, spendable concentration is 125 or 150 * 10 ⁶Individual cell/ml.The use of high density can cause the increase of cell yield, cell-stimulating and cell amplification.In addition, the use of high cell concentration can be more effective catch may faint expression purpose target antigen cell, such as the negative T cell of CD28.These cell masses may have therapeutic value and will expect acquisition.For example, the use of high cell concentration can more effective selection have the CD8 that more weak CD28 expresses usually ⁺The T cell.

In a related embodiment, may wish to use low cell concn.By remarkable dilution T cell and particulate mixture, the interaction between particle and the cell is minimized.This can select great expression to expect antigenic cell in conjunction with particulate.For example, with CD8 ⁺The T cell is compared, CD4 ⁺The T cell is expressed the CD28 of higher level and is obtained more effective seizure and stimulation in the concentration of dilution.In one embodiment, the cell concn of use is about 5 * 10 ⁶/ ml.In other embodiments, used concentration can be about 1 * 10 ⁵/ ml is to about 1 * 10 ⁶/ ml and any between the two round values.

The damping fluid that is used for suspension cell can be any damping fluid that is applicable to particular cell types.When using some cell type, damping fluid can be included in keeps complete necessary other component of cell in the treating processes, as 1-5% serum.In another embodiment, can in cell culture medium, merge cell and bead.For example can be by rotation, stirring or any mixing means cell mixing and bead, time range be 1 minute to a few hours.Then by pressure such as placing magnetic field to concentrate the container of bead and cell.For example remove substratum and unconjugated cell, and clean and be attached to bead or other surperficial cell, be resuspended in the substratum that is suitable for cell cultures then by peristaltic pump suction.

In one embodiment of the invention, mixture can be cultivated 30 minutes to a few hours (reaching about 3 hours) to about 14 days or any between the two round values (in hour and minute).In another embodiment, mixture was cultivated 21 days.In one embodiment of the invention, bead and T cell were cultivated about 8 days together.In another embodiment, bead and T cell were cultivated 2-3 days together.As indicated above, may need several stimulation cycle to make the incubation time of T cell can reach 60 days or more.The condition that is suitable for the T cell cultures comprises that the suitable culture medium that may comprise the propagation and the necessary factor of surviving is (as bottom line minimum medium (MinimalEssential Media) or RPMI substratum 1640 or X-vivo 15; (Bio Whittaker)), comprise serum (as tire ox or human serum) or interleukin-2 (IL-2), Regular Insulin or any other cell growth additive that those skilled in the art will know that.Substratum can comprise RPMI1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, and add amino acid and VITAMIN, perhaps do not contain serum or replenish an amount of serum (or blood plasma) or the one group of hormone limiting and/or the cytokine of growth of T cell and amplification q.s.Have only experimental substratum to comprise microbiotic such as penicillin and Streptomycin sulphate, will beat into the intravital cell culture medium of experimenter and then not contain.Supporting to keep target cell under the necessary condition of growth, for example suitable temperature (as 37 ℃) and atmosphere (add 5% CO as air ₂).

In another embodiment, can so change or adjust be exposed to stimulant such as through the anti-CD28 of anti-CD3/ (promptly 3 * 28) bag by the time of bead to obtain the desirable T cells phenotype.Perhaps, can before stimulation, use many selection technology to select the desirable T cells group.May need more helper cell group (T _H), CD4 typically ⁺But not CD8 ⁺Cytotoxicity or regulatory T cells are because T _HThe amplification of cell can improve or recover whole immune response.Although many specific immune responses are by CD8 ⁺T cells with antigenic specificity mediation, its directly cracking or kill target cell, yet most of immune response demands CD4 ⁺The help of T cell, it expresses important immune modulatory molecules, such as for example GM-CSF, CD40L and IL-2.In preferred auxiliary situation, keep or improve CD4 by the CD4 mediation: the method for CD8 ratio all as described herein may be particularly advantageous.Improve CD4 ⁺The quantity of T cell can increase the amount that imports the intravital CD40L by cell expressing of patient, and potential improves the visibility (improving the APC function) of target cell.(all are mainly by CD4 by increasing expression of GM-CSF or IL-2 ⁺The T cell expressing) the number of inculcating cell can be observed similar effect.Perhaps, not too needing the auxiliary and needs increase CD8 of CD4 ⁺In the situation of T cell number, also can use XCELLERATE as herein described ^TMMethod (seeing embodiment 1) is by for example preselected CD8 before stimulating and/or cultivating ⁺Cell.These situations may be present in the situation that preferred IFN-γ level increases or target cell lysis increases.Can also change the time and the type that are exposed to stimulant and have desirable T CR complete or collected works as expressing the T cell of the V 'beta ' family gene of expecting with amplification.

In order to finish the separation of different T cell masses, can change the particulate time that is exposed to.For example, in a preferred embodiment, separate the T cell by being incubated with 3 * 28 beads such as Dynabeads M-450, the time is enough to the key player on a team and selects desirable T cells.In one embodiment, soaking time is about 30 minutes.In another embodiment, the time is at least 1,2,3,4,5 or 6 hour.In also having an embodiment preferred, the time is 10-24 hour or more.In a preferred embodiment, soaking time is 24 hours.In order to separate the T cell by the cancer patients, use longer soaking time can improve cell yield such as 24 hours.

In certain embodiments, stimulate and/or proliferation time can be 10 all or following, 8 all or following, 4 all or following, 2 all or following, 10 days or following, 8 days or following (4 all or below comprise the scope of 4 thoughtful 1 day (24 hours) interior if having time or all values between these two numerals).In some embodiments, for example may need to use limiting dilution or cell sorting to clone the T cell, it may be essential wherein swashing the time than lunge.In some embodiments, stimulate and amplification can carry out 6 days or following, 4 days or following, 2 days or below, and be few in other embodiments, and preferred 4-6 hour or time (these scopes comprise any round values between two numerals) still less to 24 hours or time still less.When the T cytositimulation carried out the short period, the number of T cell mass may not can significantly increases, but group will provide T cell after the more powerful and healthy activation, and they can continue to breed in vivo and more closely be similar to natural effector T cell aggregation.Because the auxiliary operability of T cell usually is that selective amplification is rich in CD4 at the limiting factor in the antibody response of proteantigen ⁺The T cell mass or its selectivity is beaten into the intravital ability of experimenter is extremely beneficial.Other advantage of these enriched populations is conspicuous, and reason is that the activated helper cell of discerning bone-marrow-derived lymphocyte institute antigen-presenting discharges contact of two class stimulation-physics and cytokine generation, and this causes B cell proliferation and differentiation.

In a plurality of embodiments, those of ordinary skills can understand by eliminating stimulus signal in the cell depends on used surface type.For example, if use the paramagnetic bead, then magnetic resolution is a feasible selection.Isolation technique is described in detail in the specification sheets of paramagnetic bead manufacturers (for example DYNAL company, Olso, Norway).In addition, if the surface be even as big as with the bead of cellular segregation, then can use filtration.In addition, multiplely inculcate filter commercialization, comprise that 20 microns and 80 microns are inculcated filter (Baxter).Therefore, need only the mesh size of bead greater than filter, this filtration is exactly highly effective.In a related embodiment, bead can pass filter, but cell can keep, and so just can separate.In a specific embodiment, used biocompatible surface is in degraded (promptly being biodegradable) in culture between exposure period.

Although can be easy to obtain employed antibody in the methods described herein such as ATCC by open source, can be by the antibody of standard technique generation at T cell accessory molecule and CD3 mixture.The methodology that is used for generating the used antibody of the inventive method is well known in the art and more detailed description is arranged in this article.

Lip-deep part is fixed

As mentioned above, method of the present invention is preferably used the part of mating surface.The surface can be to make part in conjunction with on it or integration any surface wherein, and is biocompatible, promptly treats the target cell totally nontoxic of stimulation.Biocompatible surface can be biodegradable or abiotic degradable.The surface can be natural or synthetic, and synthetic surface can be a polymkeric substance.The surface can comprise the protein of collagen, purifying, peptide, polysaccharide, glycosaminoglycan, extracellular matrix composition, liposome or the cell of purifying.Polysaccharide can comprise for example Mierocrystalline cellulose, agarose, dextran, chitosan, hyaluronic acid or alginate.Other polymkeric substance can comprise polyester, polyethers, polyanhydride, polyalkyl alpha-cyanacrylate (salt), polyacrylamide, poe, polyphosphonitrile, polyvinyl acetate, segmented copolymer, polypropylene, polytetrafluoroethylene (PTFE) or polyurethane(s).Polymkeric substance can be lactic acid or multipolymer.Multipolymer can comprise lactic acid and oxyacetic acid (PLGA).Abiotic degradable surface can comprise polymkeric substance such as poly-(dimethyl siloxane) and poly-(ethane-acetic acid ethyenyl ester).Biocompatible surface comprises the protein of for example glass (as bio-vitric), collagen, chitin, metal, hydroxylapatite, aluminate, bioceramic material, hyaluronic acid polymer, alginate, acrylic ester polymer, lactic acid polymer, glycolic acid polymer, lactic acid/glycolic acid polymer, purifying, the peptide or the extracellular matrix composition of purifying.The surface that comprises other polymkeric substance can comprise glass, silica, silicon, hydroxylapatite, hydrogel, collagen, propenal, polyacrylamide, polypropylene, polystyrene, nylon, perhaps many plastics or synthetic organic polymer, or the like.The surface can comprise biological structure, such as liposome or cell surface.The surface can be lipid, plate, bag, ball, fiber, screen cloth or particulate form.Particle can comprise micelle, microsphere, nanoparticle, bead, or the like.In a plurality of embodiments, can commodity in use surface such as bead or other particle (as the Miltenyi particle, Miltenyi Biotec, Germany; Agarose beads, Pharmacia FineChemicals, Sweden; DYNABEADS ^TM, Dynal company, New York; PURABEADS ^TM, Prometic Biosciences).

When using bead, bead can be any size that can realize that target cell stimulates.In one embodiment, big or small preferably approximately 5 nanometers of bead are to about 500 microns.Therefore, the concrete purposes of bead is depended in the selection of bead size.For example, if bead is used to eliminate monocyte, then select small size so that monocyte is taken in (as diameter 2.8 μ m and 4.5 μ m or any size that can be swallowed up, such as nano-scale); Yet when the filtering separation bead was passed through in expectation, the typical case used size to be not less than the bead of 50 μ m.In addition, when using the paramagnetic bead, the bead size scope is typically about 2.8 μ m to about 500 μ m, and more preferably about 2.8 μ m are to about 50 μ m.At last, can select to use the super paramagnetic nano particulate that can be as small as about 10nm.Therefore, as conspicuous in above discussing, in fact can adopt any granular size.

Can be by known in the art and and obtainable several different methods is adhered to reagent or coupling or be incorporated in the surface.Reagent can be native ligand, protein ligands or synthetic ligands.Adhere to can be covalently or non-covalently, electrostatic or hydrophobic, and can realize by multiple attachment means, comprise chemistry for example, machinery, enzymatic, electrostatic or make the alternate manner that part can irritation cell.Adhering to of reagent can be direct or indirect (as carrying the baby).For example, at first can be with the anti-surface (directly adhering to) that is attached at part, perhaps can anti-ly be attached to the surface with avidin or Streptavidin or in conjunction with first anti-second, be used in conjunction with biotinylated part (adhering to indirectly).Can will be attached to the surface at the antibody of part by antiidiotypic antibody.Another example comprises that use is attached to surperficial albumin A or Protein G or other non-specific antibody binding molecule and comes binding antibody.Perhaps, can part be attached to the surface, such as commodity in use linking agent (Pierce, Rockford, Illinois) or other means and surface-crosslinked by chemical means.In certain embodiments, part is covalently bound on the surface.In addition, in one embodiment, according to the specification sheets of manufacturers with commercial through tosyl group activatory DYNABEADS ^TMOr contain the DYNABEADS of epoxy surface reaction group ^TMBe incubated with the desired polypeptides part.In brief, these conditions typical case relates in 4 to 37 ℃ of temperature range insulations in the phosphate buffered saline buffer of pH4 to pH9.5.

On the one hand, reagent such as some part can be single source or multiple sources and also can be antibody or its fragment, and on the other hand, when using the T cell, stimulating part altogether is B7 molecule (as B7-1, B7-2).These parts are coupled on the surface by any different attachment means discussed above.Wait to be coupled to lip-deep B7 molecule and can separate, or use the standard recombinant dna technology that can generate and separate costimulatory molecules described herein and expression system and obtain from the cell of expressing costimulatory molecules.Also can use and when being coupled to cell surface, keep B7 molecule fragment, mutant or the variant that in the T cell, triggers the ability of costimulatory signal.In addition, one of skill in the art will recognize that and useful any part in activation and inducing T cell subclass propagation can also be fixed in bead or culture vessel surface or any surface.In addition, when the covalent attachment on part and surface is a kind of preferred method, can also use absorption or seizure by the secondary monoclonal antibody.For example, then can be easy to measure the amount of the specific ligand of adhering on the surface by flow cytometry if the surface is a bead; If the surface is tissue culture ware, screen cloth, fiber, bag, then can measure by enzyme-linked immuno-sorbent assay (ELISA).

In a specific embodiment, the stimulation form of B7 molecule or anti-CD28 antibody or its fragment are attached to identical solid phase surface with reagent such as the anti-cd 3 antibodies that stimulates the TCR/CD3 mixture.Except anti-cd 3 antibodies, can also use other antibody in conjunction with the acceptor of imitation antigen signals.For example, can be with anti-CD2 antibody and B7 molecular combinations, particularly anti-cd 3 antibodies and anti-CD28 antibody combination bag by bead or other surface.

When being coupled to the surface, reagent can be coupled to same surface (promptly in " cis " mode) or different surfaces (promptly in " trans " mode).Perhaps, a kind of reagent can be coupled on the surface, and another kind of reagent exists in the solution.In one embodiment, provide the reagent of costimulatory signal to be incorporated on the cell surface, provide the reagent of primary activation signal then to exist in the solution or be coupled on the surface.In a preferred embodiment, two kinds of immobilization of reagents are on bead, or same bead is " cis ", or different bead is " trans ".For example, the reagent that primary activation signal is provided is anti-cd 3 antibodies, is anti-CD28 antibody and the reagent of costimulatory signal is provided; Two kinds of reagent are to equate that molecular amounts is fixed on the same bead altogether.In one embodiment, for CD4 ⁺The growth of the amplification of T cell and T cell, use 1: 1 ratio with every kind of antibodies on bead.Of the present invention aspect some, viewed amplification is compared when using 1: 1 ratio, is using certain ratio will resist CD3: the CD28 antibodies is observed the T cell amplification on bead the time to be increased.In a specific embodiment, viewed amplification is compared during with 1: 1 ratio of use, observes about 0.5 to about 3 times increase.In one embodiment, anti-CD3: the CD28 antibodies is 100: 1 to 1: 100 in the ratio ranges of bead, and covers wherein all round valuess.In one aspect of the invention, compare with anti-cd 3 antibodies, more anti-CD28 antibodies is on particle, i.e. CD3: the CD28 ratio is less than 1.In certain embodiments of the invention, be incorporated into anti-CD28 antibody on the bead to the ratio of anti-cd 3 antibodies greater than 2: 1.In a specific embodiment, used 1: 100 with bead bonded CD3: CD28 antibody ratio.In another embodiment, used 1: 75 with bead bonded CD3: CD28 antibody ratio.In also having an embodiment, used 1: 50 with bead bonded CD3: CD28 antibody ratio.In another embodiment, used 1: 30 with bead bonded CD3: CD28 antibody ratio.In a preferred embodiment, used 1: 10 with bead bonded CD3: CD28 antibody ratio.In another embodiment, used 1: 3 with bead bonded CD3: CD28 antibody ratio.In also having an embodiment, used 3: 1 with bead bonded CD3: CD28 antibody ratio.

Reagent

The reagent that the present invention pays close attention to comprises protein ligands, native ligand and synthetic ligands.Can and cause that connecting and assemble the reagent that produces signal then includes but not limited to lectin (phytohemagglutinin (PHA) for example in conjunction with cell surface part under certain conditions, lens culinaris agglutinin (lentil lectin), concanavalin A), antibody, antibody fragment, peptide, polypeptide, glycopeptide, acceptor, B-cell receptor and TXi Baoshouti part, extracellular matrix components, steroid, hormone (tethelin for example, reflunomide, prostaglandin(PG), tetra-iodo thyronine), bacterium part (such as lipopolysaccharides), mitogen, superantigen and derivative thereof, somatomedin, cytokine, adhesion molecule (is selected albumen such as L-, LFA-3, CD54, LFA-1), chemokine, and small molecules.Can be by natural origin such as cell, blood products and tissue or by the cellular segregation reagent of external breeding, reorganization preparation, chemosynthesis or other method by those skilled in the art will know that.

In one aspect of the invention, when hope stimulated the T cell, useful reagent comprises respectively can and start activation or the part of propagation in conjunction with CD3/TCR mixture, CD2 and/or CD28.Therefore, the term part comprises those protein as " natural " part of cell surface proteins, and such as the B7 molecule for CD28, and artificial part is such as the antibody at cell surface proteins.Can generate these antibody and fragment thereof according to routine techniques, such as hybridoma method and recombinant DNA and protein expression technology.Useful antibody and fragment can comprise the people derived from any species, perhaps can be the forms that adopts from the chimeric protein of the sequence that surpasses a kind of species.

Method well-known in the art can be used for generating to the special antibody of part, polyclonal antiserum or monoclonal antibody.Can also be as generating the antibody that design has desired characteristic through the immunoglobulin (Ig) (Ig) of genetic modification or Ig fragment.For example, and unrestricted, antibody can comprise as chimeric fusion rotein having at least one variable (V) plot structure territory and at least one reorganization IgG from the constant region structural domain of second kind of different mammalian species from first kind of mammalian species as example.Modal is that chimeric antibody has mouse variable region sequences and human constant region sequence.Can be by being transplanted in the constant region of the V district framework region of derived from human and derived from human and with this mouse/people's gomphosis immunoglobulin " humanization " derived from complementary determining region murine antibody, that give antigen-binding specificity (CDR).Can also merge the antibody that comprises different specific C DR and generate multiple specific (dual or triple specificitys etc.) antibody.Can be follow-up with gentleness reduction disulfide linkage and alkanisation or generate the fragment of these molecules by the genetic recombination engineering by proteolytic digestion or optional proteolytic digestion.

If antibody is to be greater than or equal to about 10 ⁴M ^-1, preferably be greater than or equal to about 10 ⁵M ^-1, more preferably be greater than or equal to about 10 ⁶M ^-1, and still more preferably be greater than or equal to about 10 ⁷M ^-1Avidity constant K a specific combination antigen, then they are defined as " immunologic opsonin ".Use for example people (Ann.N.Y.Acad.Sci.USA 51:660 such as Scatchard of routine techniques, 1949) described or by surperficial excimer resonance (surface plasmon resonance) (BIAcore, Biosensor, Piscataway, New Jersey) can be easy to measure the avidity of binding partners or antibody.Consult as people such as Wolff Cancer Res.53:2560-2565,1993.

Generally can prepare antibody (consulting as people such as Harlow Antibodies:A Laboratory Manual, 1988, cold spring harbor laboratory) by any multiple technologies that those of ordinary skills know.In so a kind of technology, be used as antigenic part immune animal to generate polyclonal antiserum.Suitable animal comprises rabbit, sheep, goat, pig, ox, and can comprise less mammalian species, such as mouse, rat and hamster.All right as U.S. Patent number 6,150,584,6,130,364,6,114,598,5,833,985,6,071,517,5,756,096,5,736,137 and 5,837, generate antibody of the present invention described in 243.

Immunogen can comprise cell, purifying or partially purified ligand polypeptide or its variant or the fragment or the ligand peptide of expressing part.Can generate ligand peptide by proteolysis cutting or chemosynthesis.Can select to be suitable for the peptide of immunity to determine more likely in host animal, to produce the aminoacid sequence that antigen is replied by one-level, secondary or the tertiary structure of the methods analyst part known according to those skilled in the art, thereby the peptide of selecting to be used for immunity (is consulted as Novotny, Mol.Immunol.28:201-207,1991; Berzoksky, Science 229:932-40,1985).

Immunogenic preparation can comprise with ligand polypeptide or its variant or fragment or the another kind of immunogenic protein of peptide covalent coupling, such as keyhole limpet hemocyanin or bovine serum albumin(BSA).In addition, can be with the emulsification in adjuvant of peptide, polypeptide or cell (consulting people such as Harlow, Antibodies:ALaboratory Manual, 1988, cold spring harbor laboratory).Generally speaking, after injecting for the first time, according to the preferred plan of animal species, animals received one or many booster immunization.Can by regular blood sampling animal, separation of serum and in immunoassay such as Ouchterlony measures serum analysis monitor immunne response with assessment specific antibodies titre.In case determine antibody titers, the animal of can regularly taking a blood sample is with the accumulation polyclonal antiserum.Can the albumin A of suitable solid phase upholder or the affinity chromatography of ligand polypeptide or peptide have for example been passed through to use coupling by the polyclonal antibody of these antiserum(antisera) purifying specific combination ligand polypeptides or peptide then.

For example, can use technology (Nature 256:495-497,1975 of Kohler and Milstein; Eur.J.Immunol.6:511-519,1976) and improve the monoclonal antibody for preparing specific combination ligand polypeptide or its fragment or variant.Can generate hybridoma is immortal eukaryotic cell lines, and the antibody of its generation has the expection specificity to ligand polypeptide or its variant or fragment.With the part immunogen immune animal of preparation as indicated above, for example rat, hamster or preferred mouse.Can be by merging and will be splenocyte-immortalization available from the lymphocyte of immune animal-modal through myeloma cell's fusion partner (preferably the animal with immunity is a homologous) of medicine sensitization.Can with splenocyte and myeloma cell and film merge promotor such as polyoxyethylene glycol or nonionic detergent is mixed together several minutes, be inoculated in low density then and support hybridoma but not the selective medium of myeloma cell's growth.Preferred selection substratum is HAT (xanthoglobulin, aminopterin-induced syndrome, a thymidine).After the enough time, in normally about 1 to 2 week, observe population of cells.Separate monocoenosis, and to the antibody test that generates by cell to ligand polypeptide or its variant or segmental binding affinity.Preferred the antibody that generates has high affinity and specific hybridoma to part antigen.The present invention pay close attention to the monoclonal antibody specific combination ligand polypeptide that generates or its variant or segmental hybridoma.

Can separate monoclonal antibody by the supernatant liquor of hybridoma culture.The another kind of method that is used to generate mouse monoclonal antibody is that hybridoma is expelled in the peritoneal cavity of homology mouse.Mouse generates the ascites that contains monoclonal antibody.Can eliminate impurity from antibody by routine techniques such as chromatography, gel-filtration, precipitation or extracting.

Can generate human monoclonal antibodies by many technology.Method includes but not limited to that Epstein-Barr virus (EBV) transforms the human peripheral blood cell and (consults U.S. Patent number 4,464,456), external immune human B cell (is consulted as people such as Boerner, J.Immunol.147:86-95,1991), merging the splenocyte of transgenic mice of carrier's immunoglobulin gene of the immunity of hanging oneself and the splenocyte that merges the transgenic mice that carries immunoglobulin gene (inserting by yeast artificial chromosome (YAC)) of the immunity of hanging oneself (consults as U.S. Patent number 5,877,397; People such as Bruggemann, Curr.Opin.Biotechnol.8:455-58,1997; People such as Jakobovits, Ann.N.Y.Acad.Sci.764:535-35,1995) or separate from human normal immunoglobulin V district phage library.

Can generate the chimeric antibody and the humanized antibody that use among the present invention.Chimeric antibody has at least one and (consults as people such as Morrison derived from the variable region structural domain of second kind of different mammalian species with at least one derived from the constant region structural domain of first kind of mammalian species, Proc.Natl.Acad.Sci.USA 81:6851-55,1984).More commonly, can at least one (be consulted as people such as Shin such as making up chimeric antibody in the carrier of being cloned into the sequence that comprises at least one the human constant region of encoding derived from the polynucleotide sequence of the variable region of mouse, rat or hamster monoclonal antibody derived from the variable region structural domain of non-human monoclonal antibodies by encoding, Methods Enzymol.178:459-76,1989; People such as Walls, Nucleic Acids Res.21:2921-29,1993).The human constant region of selecting may rely on the desired effector function of antibody specific.The another kind of method that is used to generate chimeric antibody known in the art is homologous recombination (U.S. Patent number 5,482,856).Preferably, the carrier transfection is used for the stably express chimeric antibody in eukaryotic cell.

Further genetic modification inhuman/people's chimeric antibody to be to generate " humanization " antibody.This antibody has the numerous CDR derived from the immunoglobulin (Ig) of non-human mammal species, the variable framework region of at least one individual and at least one human normal immunoglobulin constant region.Compare with non-human monoclonal antibodies or chimeric antibody, humanization may generate the antibody that binding affinity reduces.Therefore, those skilled in the art can use one or more strategies to design humanized antibody.

In certain embodiments, may preferably use antigen-binding fragments of antibodies.These fragments comprise Fab fragment or F (ab ') ₂Fragment can prepare by papoid or pepsic proteolytic digestion respectively.Can Fab and Fc fragment be separated by affinity chromatography, for example use immobilization albumin A or fixed ligand polypeptide or its variant or fragment.Be used to generate the segmental another kind of method of Fab and comprise gentle reduction F (ab ') ₂Fragment and alkanisation (consulting as Weir Handbook of Experimental Immunology, 1986, Blackwell Scientific, Boston) subsequently.

Inhuman, the people of any Ig molecule mentioned above or humanization heavy chain and variable region of light chain can be built into strand Fv (sFv) fragment (single-chain antibody).Consult as people such as Bird Science 242:423-426,1988; People such as Huston, Proc.Natl.Acad.Sci.USA 85:5879-5883,1988.Can generate multi-functional fusion rotein by the polynucleotide sequence of the polynucleotide sequence that in same reading frame, connects coding sFv and the multiple effect protein of encoding.These methods are known and disclosed in this area, for example EP-B1-0318554, U.S. Patent number 5,132,405, U.S. Patent number 5,091,513 and U.S. Patent number 5,476,786.

Being used to select specific combination ligand polypeptide or its variant or segmental another kind of method is that phage display (is consulted as people such as Winter Annul.Rev.Immunol.12:433-55,1994; People such as Burton, Adv.Immunol.57:191-280,1994).Can make up people or mouse immune globulin variable zone gene combinatorial libraries in the phage vector that can screen (consults as U.S. Patent number 5 to select specific combination ligand polypeptide or its variant or segmental Ig fragment (Fab, Fv, sFv or its polymer), 223,409; People such as Huse, Science 246:1275-81,1989; People such as Kang, Proc.Natl.Acad.Sci.USA 88:4363-66,1991; People such as Hoogenboom, J.Molec.Biol.227:381-388,1992; People such as Schlebusch, Hybridoma 16:47-52,1997 and the reference quoted).

Using method

Except method mentioned above, can be used for a plurality of fields by methods described herein stimulation and/or activated cell.The T cell of polyclone increase of the present invention can be beaten into and suspect or observe in any individual body of T cell aggregation deflection.The T cell of polyclone increase of the present invention can be beaten in the donor body so that extensive and effective immunoprotection to be provided.In content of the present invention, composition described herein and method can be used for treating the non-responsiveness individuality, as have the individuality of the T cell aggregation (or natural generation or by medicine or treatment artificial induction) of immunologic defect or deflection described herein.

In certain embodiments, the non-responsiveness individuality is born non-responsiveness, promptly is attributed to the reason of congenital existence, all any diseases as described herein or disorder.In other embodiments, individuality is by inducing the non-responsiveness state that reaches, for example at the result of many treatments of any disease described herein.In this background, individual possibility non-responsiveness, perhaps in other words, the T cell aggregation that may have immunologic defect or deflection is because the result of any other treatment of chemotherapy, treatment, cytotoxic reagent, the immunosuppressor usually implemented in fields of implantation or the T cell aggregation that causes change described herein or deflection.In certain embodiments, individual non-responsiveness is chemotherapy, radiation or melts the result of agent such as CAMPATH, anti-cd 3 antibodies, cytotoxin, fludaribine, ciclosporin, FK506, rapamycin, Mycophenolic Acid, steroid, FR901228 and irradiation such as treatment, antibody or other immunity of reagent such as ciclosporin, azathioprine, methotrexate, mycophenolate and FK506.These medicines or suppress to rely on the Phosphoric acid esterase calcineurin (ciclosporin and FK506) of calcium, or the p70S6 kinases (rapamycin) wanted of suppressing the signal of growth factor-induced overstated (people such as Liu, Cell 66:807-815,1991; People such as Henderson, Immun.73:316-321,1991; People such as Bierer, Curr.Opin.Immun.5:763-773,1993; Isoniemi (seeing above)).

The immunne response of natural generation is that any given antigen is selected significantly epi-position of some immunity, these epi-positions is had specific T cell obtain activation, amplification and mediate immunne response.Unfortunately, many other potential epi-positions fail to participate in the competition in the t cell activation process in vivo, but in immunne response, keep un-activation/do not participate in, thus the possibility of pathogenic agent/tumour uniform immunological surveillance improved.By activating and improve the polyclone of donor T cell, these not too significant T cells with the TCR that can reply target antigen can access and order about and reach the state that response improves, and make them become potential player in the immunne response.This has widened has not homospecific a complete set of T cell to challenge any immunology damage in the immunity system.The protection of the escape variant that this method thereby be used to takes place when helping at narrow immunne response as binding mode.

Methodology as herein described can be used for the CD28 of selective amplification polyclone increase aspect the TCR expression ⁺, CD4 ⁺, CD8 ⁺, CD45RA ⁺, and/or CD45RO ⁺The T cell mass, thereby be used for the treatment of communicable disease, autoimmune disorder, many cancers, blood disease (as cytopenia), with transplant the arbitrary of coexistence (as hematopoietic stem cell transplantation) or panimmunity defect state or situation and be used for immunotherapy.As a result, can generate the T cell mass of expressing TCR, described TCR is polyclonal aspect antigen reactivity, but at CD4 ⁺Or CD8 ⁺The aspect is homologous in essence.In addition, this method allows the T cell number is expanded to is enough to rebuild individual total CD4 ⁺Or CD8 ⁺(individual total lymphocyte count is about 5 * 10 to the T cell mass ¹¹).The T cell mass that can also genetic transduction generates thus also is used for immunotherapy, perhaps can be used for the method for analyzed in vitro infectious agents.For example, can obtain tumor-infiltrated lymphocyte populations, and stimulate the T cell amplification to enough numbers by the individuality of suffering from cancer.The T cell mass that can genetic transduction generates thus is with expressing tumor necrosin (TNF) or other protein (for example many cytokines, inhibitors of apoptosis (as Bc1-2), protection cell avoid arbitrary (as the scFv) of gene such as RevM10 that HIV infects or intrakines etc., targeted molecular, adhere to and/or go back to the nest molecule and multiple antibody or its fragment) and give individuality.

CD4 of the present invention ⁺A kind of concrete purposes of T cell mass is that the individual HIV of treatment infects.Long-term HIV infects and finally causes CD4 ⁺The T lymphocyte number significantly descends.This decline causes deep immune deficiency state then, makes the patient be easy to suffer a series of life-threatening opportunistic infections.With CD4 ⁺The T cell number is supplemented to normal level may be able to return to level of significance with immunologic function.Therefore, method as herein described provides amplification CD4 ⁺The T cell number is to the means that are enough to rebuild this colony in the HIV infected patient and increase its polyclone.The T cell of may also must avoiding infection in the long-time stimulus process perhaps may be wished to make the T cell forever to resist HIV and be infected.Exist many technology can make T cell resistance HIV infect or before giving back infected individual, can not generate virus the T cell.For example, can be with CD4 before amplification ⁺The T cell is cultivated with inhibition HIV with one or more anti-retroviral agents and is duplicated or virus generation (, consulting as people such as Chow Nature 361:650-653,1993 as the medicine of target reversed transcriptive enzyme and/or viral machine-processed other parts).

Several method can be used for genetic transduction T cell and suppresses the molecule that HIV infects or duplicates to generate.For example, in a plurality of embodiments, can genetic transduction T cell to generate transdominant inhibitor, " molecule phantom target ", antisense molecule or toxin.These methodologies are more detailed is described in Application No. 08/253,751,08/253,964 and PCT publication No. WO95/33823.

In one embodiment, can treat malignant tumour such as non Hodgkin lymphoma (NHL) and B cell lymphocytic leukemia (B-CLL).Use the preliminary study of amplification back T cell (to consult people such as Liebowitz though in NHL, tested, Curr.Opin.Onc.1O:533-541,1998), T cell mass of the present invention provides significantly enhancing immunity treatment and reactive enhancing polyclone feature of achieving.As shown in Figure 3, to several V 'beta ' families, B-CLL patient has mono-clonal or the few clonal expression of TCR in the T cell mass.At 12 days XCELLERATE ^TMAfter the method, these T cell masses have been recovered the polyclone that TCR expresses.In addition, there is particular difficulty in B-CLL patient, comprises that the low leukemia cell of reaching of relative T cell number bears height in the peripheral blood, is accompanied by common T cellular immunization and suppresses.T cell mass of the present invention can especially be united stem cell (CD34 in this disease of treatment ⁺) effect of remarkable improvement is provided during transplantation therapy.Therefore, will be favourable with the anti-CD28 co-immobilization of anti-CD3x bead increase T cell function and anti-CLL T cytoactive.

The present invention also provides and has been used in animal prevention, suppresses or reduce composition and the method that cancer or malignant cell exist, and comprises that the experimenter that animal is used effective anticancer activates back polyclone T cell.

What the present invention paid close attention to, the inductive immunne response at, perhaps wait to prevent, suppress, or the cancer that reduces existence can include but not limited to melanoma, non Hodgkin lymphoma, Hodgkin, leukemia, plasmoma, sarcoma, neurospongioma, thymoma, mammary cancer, prostate cancer, colorectal cancer, kidney, renal cell carcinoma, carcinoma of the pancreas, nasopharyngeal carcinoma, esophagus cancer, the cancer of the brain, lung cancer, ovarian cancer, cervical cancer, multiple myeloma, hepatocellular carcinoma, acute one-tenth lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CML), large granular lymphocyte leukemia (LGL), and lymphocytic leukemia (CLL).In one embodiment, cancer is a B cell lymphocytic leukemia.

The compositions and methods of the invention also are used in the individuality of having accepted chemotherapy, cytotoxic reagent or described herein and any immunosuppressant treatment that those skilled in the art will know that and recover immune response.In another embodiment, the compositions and methods of the invention can be used for the individuality that hematopoietic stem cell transplantation has been accepted in treatment (promptly recovering immune response).In certain embodiments, will accept Cord blood, allogeneic, transplant with the individuality of present composition treatment from body or heterogenous cell.

In another embodiment, method and composition of the present invention is used in and has accepted gene therapy or relate in the individuality of any therapy of the gene transfer that can cause the deflection of T cell aggregation and recover immune response.In particular, the transgenosis by retrovirus-mediated method in the former generation T lymphocyte can be induced in the cell that gene obtains revising and be activated and transduction/selection dependent T CR V β deflection.Yet, cell-stimulating behind the use methods described herein modification gene and stimulation (CD3/CD28 as described herein stimulates altogether) prevent TCR V β set in CD4 and two kinds of T cells of CD8 subclass change (deflection).

In certain embodiments, the compositions and methods of the invention are used in the individuality of suffering from numerous disease and recover or otherwise improve immune response, described disease is relevant with the immunity function imbalance, the T cell aggregation that comprises change includes but not limited to that disease is such as rheumatoid arthritis, multiple sclerosis, insulin-dependent diabetes mellitus, bronzed disease, celiac disease, chronic fatigue syndrome, inflammatory bowel, ulcerative colitis, regional ileitis, Fibromyalgia, systemic lupus erythematous, psoriasis, the Sjogren syndrome, hyperthyroidism/Graves disease, hypothyroidism/Hashimoto thyroiditis, insulin-dependent diabetes mellitus (I type), myasthenia gravis, endometriosis, scleroderma, pernicious anemia, the thorough syndromes of Gourde(G) Paasche, wegener disease, glomerulonephritis, aplastic anemia, multiple cytopenia arbitrary, paroxysmal nocturnal hemoglobinuria, myelodysplastic syndromes, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, Fanconi anemia, the Ai Wensi syndromes, the Factor IX inhibitor syndrome, the factors IX inhibitor syndrome, systemic vasculitis, dermatomyositis, polymyositis and rheumatic fever.Method and composition as herein described can be used for treating and is characterized as the low blood disease of blood counting.

In certain embodiments, the compositions and methods of the invention can be used for treating and the relevant neurological disorder of T cell aggregation deflection.In another embodiment, composition described herein is used to treat cardiovascular disorder.

Can stimulation as described herein and amplification T cell to induce or to strengthen to the response of agent of causing a disease such as virus (as the human immunodeficiency virus), bacterium, parasite and fungi.Pathogenic agent comprises any disease that is caused by infectious organism.Infectious organism can comprise that virus is (as single strand RNA virus, single-stranded DNA viruses, human immunodeficiency virus (HIV), the first type, B-mode, or hepatitis C virus, hsv (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human papillomavirus (HPV)), parasite is (as protozoon and metazoan pathogenic agent, such as the plasmodium species, the leishmania species, the Schistosoma species, the trypanosoma species), bacterium is (as mycobacterium, mycobacterium tuberculosis particularly, salmonella, streptococcus, intestinal bacteria, staphylococcus), fungi is (as the candiyeast species, the aspergillus species), pneumocystis pneumoniae, and Protein virus (known prion-infected animal and cause itch, it is the neural a kind of propagable degenerative disorders of sheep and goat, and mad cow disease (BSE), or the cat family spongiform encephalopathy of " mad cow disease " and cat.Four kinds of prion diseases of known effect people are the special cortico-striatal spinal degeneration (CJD) in Kuru disease, Creutz Fil, Ge-Shi-Sha disease (GSS) and fatal familial insomnia (FFI).When being used for this paper, " Protein virus " is included in the people of used any animal-particularly and raises and train the Protein virus of the form of ownership that causes all or any these or other disease in the livestock.

Thereby can stimulation as described herein and amplification T cell in the non-responsiveness individuality, induce or strengthen response, for example suffer from the individuality of congenital heredity disease, such as Reconstruction in Sever Combined Immunodeciency (SCID) or common variable immune deficiency (CVID).In one embodiment, thus can stimulation as described herein with amplification T cell owing to induce in the individuality of the treatment relevant and non-responsiveness or strengthen response with bone marrow transplantation, chemotherapy, radiotherapy or other cancer therapy.In one embodiment, thus can stimulation as described herein and amplification T cell in suffering from the non-responsiveness individuality of immune deficiency or autoimmune disease, induce or strengthen response.In also having an embodiment, thereby the T cell that can stimulate and increase is induced in suffering from the non-responsiveness individuality of the chronic disease that influences kidney, liver or pancreas or is strengthened response.In a special embodiment, T cell of the present invention is used to induce or strengthen response in suffering from the individuality of diabetes.In another embodiment, T cell of the present invention is used to induce and strengthen response in the individuality that influenced by old-age group.

The present invention also provides by the method for mixing the specific subgroup of T cell mass selective amplification T cell.Particularly, the invention provides CD4 behind specific enrichment ⁺And CD8 ⁺The T cell mass that two positive T cell ratios are much higher.

Another embodiment of the invention provides by CD4 ⁺T cell mass selective amplification T _H1The method of cell mass.In this method, stimulate CD4 altogether such as monoclonal antibody 9.3 with anti-CD28 antibody ⁺T cell and induce T _H1The specific cell factor comprises the secretion of IFN-γ, causes T _H1Cell is with respect to T _H2The enrichment of cell.

The present invention also provides by CD4 ⁺T cell mass selective amplification T _H2The method of cell mass.In this method, stimulate CD4 altogether such as monoclonal antibody B-T3, XR-CD28 with anti-CD28 antibody ⁺T cell and induce T _H2The secretion of the specific cell factor causes T _H2Cell is with respect to T _H1(consult for example people such as Fowler, Blood 84 (10): 3540-9, on November 15th, 1994 in the enrichment of cell; People such as Cohen, Ciba Found Symp 187:179-93,1994).

The present invention also provides and has been used for the method that selective amplification is expressed the T cell mass of specific V β, V α, V γ or V δ gene.For example, in this method, plus or minus selects to express the T cell of specific V β, V α, V γ or V δ gene, further increases/stimulates according to method of the present invention then.Perhaps, can plus or minus select to express specific purpose V β, V α, V γ or V δ gene through stimulating the T cell of amplification, and further stimulate and increase.

In another example, directly blood is extracted extremely independently disposable apparatus by the patient, two or more immobilized antibodies (as anti-CD3 and anti-CD28) or other component wherein are housed, thereby before cell is applied to the experimenter, stimulate the needed acceptor of t cell activation (as being fixed on frosting or the separable particulate).In one embodiment, disposable apparatus can comprise the container (as plastics bag or bottle) with suitable pipe connection, is applicable to syringe and aseptic anchoring device combing/ grappling.This device will have the solid surface that is used for fixing t cell activation component (as anti-CD3 and anti-CD28 antibody); Can be the surface of container self or plug-in unit, normally plane, etching plane, irregular surface, porous pad, fiber, clinical acceptable/the iron content fluid of safety, bead, etc.In addition, when using self-contained system, the experimenter can keep being connected with device, and perhaps device can be discerptible with the patient.In addition, can perhaps use removable warmer incubation under physiological temp at the room temperature using appts.

Since the apparatus and method that are used to gather with processing blood and blood products are well-known, those skilled in the art will be easy to recognize by technology provided herein, can be easy to design the multiple device or the repacking existing apparatus that satisfy above requirement.Therefore, be not subjected to the restriction of the particular that this paper proposes as these apparatus and method, but comprise and to keep aseptic and blood is remained on any device or the method for fluid form, complement activation reduces in the described blood, and is fixed by blood or blood product before being applied to the experimenter or separate the necessary component of t cell activation (as anti-CD3 and anti-CD28 antibody or its part).In addition, those of ordinary skills can be easy to recognize that multiple blood products can be used for uniting apparatus and method as herein described.For example, method and apparatus will be used for after thawing, be applied to the T cell from the T clone of the blood derived cell of the whole blood of freezing preservation, peripheral blood lymphocytes, other freezing preservation or freezing preservation of fast activating before the experimenter.In another example, apparatus and method are used in the T cellular product of the stripped amplification of raising preliminary election process that is applied to the experimenter or the activity of T clone, thereby height activated T cells product is provided.At last, as what will readily recognize that, aforesaid method and the device can be used for simultaneously experimenter and donor from body or allochthonous cell therapy.

Method of the present invention also can be used with effect in enhancement antigen reactivity and the enhancing body with vaccine.In addition, consider that the T cell by the present invention's amplification has the relatively long transformation period in vivo, these cells can be used as the perfect carrier of gene therapy, carry its intended purposes nucleotide sequence and potential and return cancer, disease or infection site.Therefore, by expanded cells of the present invention can combined vaccine, one or more cytokines, one or more therapeutic antibodies, etc. deliver to the patient.In fact, any therapy of benefiting from more strong T cell mass is all belonged in the scope of using method described herein.

The invention provides the composition and the method for the T cell of the polyclone increase that TCR expresses, be used for prevention, suppress, or reduce existence: melanoma such as, but not limited to following cancer, non Hodgkin lymphoma, Hodgkin, nasopharyngeal carcinoma, leukemia, plasmoma, sarcoma, neurospongioma, thymoma, mammary cancer, prostate cancer, colorectal cancer, kidney, renal cell carcinoma, carcinoma of the pancreas, esophagus cancer, the cancer of the brain, lung cancer, ovarian cancer, cervical cancer, multiple myeloma, hepatocellular carcinoma, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CML), lymphocytic leukemia (CLL), large granular lymphocyte leukemia (LGL), with other knurl known in the art.

Perhaps, polyclone T cell composition of the present invention can be used for inducing or strengthens response at infectious organism.Infectious organism can comprise virus, such as single strand RNA virus or single-stranded DNA viruses, human immunodeficiency virus (HIV), first type, B-mode or hepatitis C virus, hsv (HSV), human papillomavirus (HPV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), parasite, bacterium, mycobacterium tuberculosis, pneumocystis pneumoniae, candiyeast or aspergillus or its combination.

In another embodiment of the invention, polyclone T cell composition as herein described can be used for inducing or strengthens response to correct congenital heredity disease or immunodeficient disease such as Reconstruction in Sever Combined Immunodeciency (SCID) or common variable immune deficiency (CVID).In certain embodiments, polyclone T cell composition as herein described can be used for inducing or strengthens response to correct the immune deficiency that causes because of the treatment relevant with bone marrow transplantation, chemotherapy, radiotherapy or other cancer therapy.In another embodiment, polyclone T cell composition as herein described can be used for inducing or strengthens response to correct immune deficiency or autoimmune disease.In also having an embodiment, polyclone T cell composition as herein described can be used for inducing or strengthens response to correct the chronic disease that influences kidney, liver or pancreas.In also having an embodiment, polyclone T cell composition as herein described can be used for inducing or strengthens response with the treatment diabetes.In some embodiments, polyclone T cell composition as herein described can be used for inducing or strengthens response to correct and old and feeble relevant immune deficiency.

In another embodiment, the T cell composition of demonstration TCR expression polyclone of the present invention increase can be used for the other therapies that the associating tradition is used for the treatment of these communicable diseases and cancer.

Pharmaceutical composition

T cell mass of the present invention can be used separately, perhaps as the pharmaceutical composition of uniting thinner and/or other component such as IL-2 or other cytokine or cell mass.In brief, pharmaceutical composition of the present invention can comprise target cell group described herein, and one or more medicinal or physiology acceptable carrier, thinner or vehicle are closed in parallel connection.These compositions can comprise buffer reagent such as neutral buffered salt, phosphate-buffered saline or the like; Carbohydrate such as glucose, seminose, sucrose or dextran, N.F,USP MANNITOL; Protein; Polypeptide or amino acid are such as glycine; Antioxidant; Sequestrant such as EDTA or gsh; Adjuvant (as aluminium hydroxide); And sanitas.Preferably composition of the present invention is mixed with and is used for intravenously and uses.

Can be to be treated to be suitable for the mode of (or prevention) disease use pharmaceutical composition of the present invention.Quantity of using and frequency will be by such as the decisions of factors such as the type of status of patient and patient disease and severity, yet suitable dosage may be determined by clinical trial.

By use of the present invention tried composition in animal body the inductive immunne response may comprise by can kill tumor and the cellullar immunologic response and the helper cell of the cytotoxic T cell mediation of infected cell reply.Thereby also may induce mainly by activating the humoral immunoresponse(HI) that the B cell causes the helper cell mediation of antibody generation.Multiple technologies can be used for analyzing the type by present composition recovery or inductive immunne response, and this area has a detailed description to this; As people such as Coligan, Current Protocols in Immunology, John Wiley ﹠amp; Sons Inc., 1994.

When indication " immunology significant quantity ", " antitumor significant quantity ", " tumor suppression significant quantity " or " therapeutic dose ", the doctor can determine the exact quantity of the present composition to be administered after the individual difference of considering age, body weight, tumour size, infection or metastasis degree and status of patient.Typically, in adoptive immunotherapy research, use about 2 * 10 to the patient ⁷To 2 * 10 ¹¹Individual activation back T cells with antigenic specificity (consulting) as U.S. Patent number 5,057,423.Aspect some, particularly when using allogeneic or heterogenous cell, can use fewer purpose cell of the present invention, scope is 10 ⁶/ kg (every patient 10 ⁶-10 ¹¹).In one embodiment of the invention, use about 1 * 10 to the patient ⁸Individual T cell.Can repeatedly use the T cell composition with the dosage in these scopes.T cell for the patient who receives treatment after the activation can be from body or allogenic.As described herein, if desired, treatment can also comprise use mitogen (as PHA) or lymphokine, cytokine and/or chemokine (as GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1 α, etc.) to strengthen inducing to immunne response.

Aspect some, the T cell of using is being used the back is kept them in vivo between 2 weeks and 1 year polyclone at least of the present invention.In other embodiments, the T cell of using was kept polyclone in 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 weeks after using.In also having some embodiments, the T cell of using is at least 5,5.5,6,6.5,7,7.5,8,8.5,9,9.5,10,10.5,11,11.5,12 months or the polyclone of keeping more of a specified duration after using.

Can carry out in any mode easily by using of reagent compositions, comprise the aerosol inhalation, inject, ingest, inculcate, implant or transplant.Can give that the patient is subcutaneous, in the intracutaneous, intramuscular, intravenously (i.v.) injection, tumour or intraperitoneal use the present composition.Preferably inject and use T cell composition of the present invention by i.v..Can directly be expelled in tumour or the lymphoglandula activating back T cell composition.

In also having an embodiment, can in controlled release system, deliver pharmaceutical composition.In one embodiment, can use pump (to consult Langer, Science 249:1527-1533,1990; Sefton, CRC Crit.Ref.Biomed.Eng.14:201,1987; People such as Buchwald, Surgery88:507,1980; People such as Saudek, N.Engl.J.Med.321:574,1989).In another embodiment, can use polymeric material (consult Medical Applications ofControlled Release, 1974, Langer and Wise compile, CRC press, Berkeley village, Florida State; Controlled Drug Bioavailability, Drug Product Design andPerformance, 1984, Smolen and Ball compile, Wiley, New York; Ranger and Peppas, J.Macromol.Sci.Rev.Macromol.Chem.23:61,1983; Also can consult people such as Levy, Science 228:190,1985; People such as During, Ann.Neurol.25:351,1989; People such as Howard, J.Neurosurg.71:105,1989).In also having an embodiment, controlled release system can be placed the treatment target near, thereby only need the small portion of body dose (to consult Release as Medical Applications of Controlled, 1984, Langer and Wise compile, CRC press, the Berkeley village, the Florida State, the 2nd volume, 115-138 page or leaf).

Can also use many matrix to use T cell composition of the present invention.Matrix has been used many years (consult the Engineering as Priciples of Tissue, Lanza, Langer and Chick compile, 1997) in the organizational engineering field.The present invention uses these matrix in uncharted field, support, keep or regulate and control immunity system as artificial lymphoid organ, normally passes through modulating T cell.Therefore, the present invention can use those substrate compositions and the preparation that has proved effectiveness in organizational engineering.Therefore, the matrix type that can be used for the present composition, apparatus and method can comprise biology and synthetic two class matrix in fact without limits.In a specific examples, 889,5,913,998,5,902,745,5,843,069,5,787,900 or 5,626,561 composition and the devices that propose have been used by U.S. Patent number 5,980.The characteristic that matrix had is usually with to have biocompatibility when being applied to mammalian hosts relevant.Can constitute matrix by natural or synthetic two class materials.In hope permanent structure maybe can be eliminated structure and stay in the animal body in the situation such as implant, matrix is can right and wrong biodegradable; Or it is biodegradable.Matrix can be taked the form of cavernous body, implant, pipe, telfa pad, fiber, tubular fibre, freeze-dried component, gel, powder, porous composition, liposome, cell, nanoparticle.In addition, matrix can be designed to be able to continue to discharge institute's inoculating cell or institute's founder cell factor or other active agent.In certain embodiments, matrix of the present invention is soft resilient, and can be described as such as inorganic salt, aqueous fluids and dissolved gases reagent and comprise the permeable semi-solid supports of material such as oxygen.

Matrix is used as the example of biocompatible substance in this article.Yet, the invention is not restricted to matrix, thereby Anywhere in the appearance of term matrix, these terms should be understood to comprise device and other material, its permissive cell extends or cell crosses, it is biocompatible to be, can allow that also polymer directly passes crossing (because material self is a semi-permeable membranes, or unites specific semi-permeable material and use together) of material.

Be hereby expressly incorporated by reference at this all reference as cite herein of quoting.In addition, all digital scopes used herein clearly comprise all round valuess in the scope, and the selection of concrete numerical value is considered to draw according to concrete purposes in the scope.In addition, explanation and the following example is provided without limitation by way of example.

Embodiment 1

The T cytositimulation

In some experiment described herein, adopted to be called XCELLERATE I ^TMMethod.In brief, in the method, prepare XCELLERATED T cell by the single blood sampling composition of peripheral blood lymphocytes (PBMC) art product.After the clinical collection of patient, clean the single blood sampling composition of PBMC art product, then with the DYNABEADS of " bag by "

M-450Epoxy is incubation together.During this period, phagocytic cell is such as the monocytic cell ingests bead.Behind the incubation, handle cell and bead to remove any monokaryon/phagocytic cell that adheres on bead and the bead with MaxSepMagnetic Separator (magnetic separator).After this eliminated monocytic step, taking-up contained and adds up to 5 * 10 ⁸Individual CD3 ⁺The volume of T cell is with 1.5 * 10 ⁹Individual DYNABEADS

M-450CD3/CD28TCell Expander assembles together, to start XCELLERATE ^TMProcess (about 3: 1 beads: the T cell).Then with cell and DYNABEADS

The mixture of M-450CD3/CD28T Cell Expander is in 37 ℃, 5%CO ₂About 8 days of incubation is used for to generate XCELLERATED T cell inculcating for the first time.Eliminated the freezing preservation of monocytic PBMC until the second time or further cellular products amplification (after about 21 days) with remaining, at that time with its thawing, cleaned, taking-up contains total 5 * 10 then ⁸Individual CD3 ⁺The volume of T cell is with 1.5 * 10 ⁹Individual DYNABEADS

M-450CD3/CD28T Cell Expander assembles together to start the XCELLERATE method and is used for inculcating for the second time.Be 37 ℃, 5%CO ₂Incubation is in about 8 days stage, CD3 ⁺The T cell obtains activating and amplification.Obtain anti-CD3mAb (clone BC3 by Fred Hutchinson DKFZ (Seattle, the State of Washington); XR-CD3), by Diaclone (

, France) obtain anti-CD28mAb (clone B-T3; XR-CD28).

Be called XCELLERATE II ^TMImprove one's methods and adopt method mentioned above and make some modifications, wherein separately do not eliminate monocytic step, and in some method, before first contact bead with cell freezing and carry out further concentrating and stimulating.In a version of this method, by the circulation blood acquisition T cell of single blood sampling composition art by donor or patient.The component of single blood sampling composition art product generally includes lymphocyte, monocyte, granulocyte, B cell, other karyocyte (white corpuscle), red corpuscle and thrombocyte.Typical single blood sampling composition art product contains 1-2 * 10 ¹⁰Individual karyocyte.Clean cell to remove plasma proteins and thrombocyte with no calcium, no magnesium phosphate buffered saline (PBS).Cleaning step is following carrying out, and with cell centrifugation, removes supernatant liquor, replaces with PBS then.Use semi-automatic " flowing through " whizzer (COBE 2991 systems, Gambro BCT, thunder gram Wood, the state of Colorado) to finish this process.When handling, in closed system, keep cell.

Can not comprise monocyte (enrichment activated cell) and continue with stimulation and further handle cell by eliminating in conjunction with cell.Perhaps, can and handle the cell freezing after cleaning, preservation after a while, this paper proves that this can improve propagation intensity and eliminate granulocyte.In an example, for frozen cell, the 35ml cell suspending liquid is placed 250ml Cryocyte with the 35ml refrigerating fulid ^TMIn the freezer bag (Baxter).The 35ml cell suspending liquid contains 3.5 * 10 usually in PBS ⁹To 5.0 * 10 ⁹Individual cell.Add isopyknic refrigerating fulid (being dissolved in 20%DMSO and 8% human serum albumin of PBS).Final concentration of cells is 50 * 10 ⁶Individual cell/ml.The scope of Cryocyte bag receiving volume can be 30-70ml, and the scope of cell concn can be 10-200 * 10 ⁶Individual cell/ml.In case packed cell and the refrigerating fulid of having expired of Cryocyte places the fast freezer of control with bag, and reduces to-80 ℃ with 1 ℃/min frozen cell.To freeze cell then places the liquid nitrogen saved system until needs.

Thaw by liquid nitrogen saved system taking-up cell and in 37 ℃.In order to remove DMSO, cleaning the cell after melting with no calcium, no magnesium PBS in the COBE2991 system.Make the stream of cells after the cleaning cross 80 tm screen web filters then.

With the cell about 0.5 * 10 after melting ⁹Individual CD3 ⁺Cell places 1 liter of Lifecell bag of plastics, and in the bag 100ml being housed does not have calcium, no magnesium PBS.PBS contains 1% to 5% human serum.Equally with 1.5 * 10 ⁹ Individual 3 * 28 beads (DYNABEADS M-450CD3/CD28T Cell Expander) place the bag (3: 1DYNABEADS M-450CD3/CD28T Cell Expander: CD3 that cell is housed ⁺Cell).Mixed bead and cell about 30 minutes in room temperature at 1rpm (putting upside down rotation).The bag that pearl and cell are housed is placed on the MaxSep Magnetic Separator (Nexell Therapeutics, Irving, California).Between bag and MaxSep, place plastics spacer (approximately 6mm is thick).(, can remove spacer in order to improve magneticstrength.) any cell of adhering on bead and the bead is retained on the magnet, and PBS and unconjugated cell are taken away.

With cell culture medium (1 liter contains X-Vivo 15, Bio Whittaker; And the heat-inactivated PHS of 50ml, 20ml 1M Hepes, 10ml 200mM L-glutaminate, contain or do not contain about 100,000I.U.IL-2) bonded concentrating cells on 3 * 28 beads and the bead is rinsed in 3 liters of Lifecell culture bag.With 3 * 28 beads with after just selecting cell transfer in the Lifecell bag, add substratum 1000ml is housed in bag.Be equipped with cell the bag place incubator (37 ℃, 5%CO ₂), and make cell amplification, as required with passage.

By having measured t cell activation and propagation in cultivation the 3rd day and the 8th day harvested cell.T cell activation is by evaluating in the expression of cultivating the 3rd day measurement cell size, cell surface marker expression level, particularly CD25 and CD154.The 8th angel's cell (approximately 150ml/min) under gravity flows through the MaxSep magnet to remove magnetic-particle, use COBE device mentioned above to clean and concentrating cells, and be resuspended in to being suitable for intravenously and use in the good electrolytic solution of balance, such as Plasma-Lyte A (Baxter-Healthcare).This moment is freezing in suitable refrigerating fulid with cell equally.

As indicated above, XCELLERATE I ^TMRefer to and above similar condition, just before stimulation, do not stimulate and concentrate and carried out the monocyte elimination.

Eliminated monocytic PBMC with 3 * 28 coupling beads (DYNABEADS M-450CD3/CD28T Cell Expander) stimulation from 4 donors.The IL-2 in the supernatant liquor, IL-4, TNF-α and IFN-γ concentration have been measured by ELISA.Also IL-4, TNF-α and IFN-T concentration have been measured behind the inoculating cell (stimulating once more) once more with new DYNABEADSM-450CD3/CD28T Cell Expander at the 12nd day.

Shown in table 1, table 2 and table 3, at XCELLERATE ^TMEach fate of stimulating course has been measured IFN-γ, IL-4 and TNF-α concentration by ELISA once more.

Table 1:XCELLERATE ^TMThe T cell of method was at the 3rd day and XCELLERATE ^TMThe T cell that activates and stimulate once more generates at the 3rd day gamma-interferon

	XCELLERATE TMThe 2nd day [IFN-γ] ng/ml of method	Stimulate the 2nd day [IFN-γ] ng/ml once more
			Mean value	13.61	31.59
Scope	7.99-27.11	10.8-95.5
			Standard deviation	5.64	22.98
Intermediate value	11.95	26.4
			N	24	24

Anti-CD3 and anti-CD28 (DYNABEADSCD3/CD28T Cell Expander) stimulation with coupling DYNABEADS M-450Epoxy have been eliminated cytophagous PMBC (XCELLERATE from 3 donors ^TM).The 2nd day by the IFN-γ concentration in the ELISA mensuration supernatant liquor.The 12nd day,, and after 2 days, measure IFN-γ concentration with the anti-CD3 of new coupling DYNABEADS M-450Epoxy and anti-CD28 inoculating cell (stimulating once more) once more.

Table 2:XCELLERATE ^TMThe T cell of method was at the 2nd day and XCELLERATE ^TMThe T cell that activates and stimulate once more generates at the 2nd day IL-4

	XCELLERATE TMThe 2nd day [IL-4] pg/ml of method	Stimulate the 2nd day [IL-4] pg/ml once more
			Mean value	310	274
Scope	170-460	50-500
			Standard deviation	143	224
Intermediate value	297	268
			N	3	3

Anti-CD3 and anti-CD28 stimulation with coupling DYNABEADS M-450Epoxy have been eliminated cytophagous PMBC (XCELLERATE from 3 donors ^TM).IL-4 concentration in the supernatant liquor is measured by ELISA in the 2nd day and the 4th day.The 12nd day,, and after 2 days, measure IL-4 concentration with the anti-CD3 of new coupling DYNABEADSM-450Epoxy and anti-CD28 inoculating cell (stimulating once more) once more.

Table 3:XCELLERATE ^TMThe T cell of method was at the 2nd day and the 4th day and XCELLERATE ^TMThe T cell that activates and stimulate once more is in the TNF-α generation of the 2nd day and the 4th day

Anti-CD3 and anti-CD28 stimulation with coupling DYNABEADS M-450Epoxy have been eliminated cytophagous PMBC from 4 donors.TNF-α concentration in the supernatant liquor is measured by ELISA in the 2nd day and the 4th day.The 12nd day,, and after 2 and 4 days, measure TNF-α concentration with the anti-CD3 of new coupling DYNABEADS M-450Epoxy and anti-CD28 inoculating cell (stimulating once more) once more.

By the CDw137 (41BB) on the flow cytometry Xcellerated T cell, CD154 (CD40L) and CD25 expression level, and mean fluorecence mapped.CDw137 (41BB) expression level raises and reached the climax at the 4th day, reduces gradually then.After stimulating once more, CDw137 expresses rapidly and raises.CD154 expresses and raises gradually until about the 7th day, reduces then.Yet after stimulating once more, the CD154 level raises rapidly, reaches the level more much higher than primary stimulus.The CD25 level raises until about the 3rd day, reduces gradually then until the 8th day (the final time point of analysis).

Embodiment 2

T cell spectral pattern is analyzed

This embodiment has described the assay determination of use spectral pattern and has used XCELLERATE ^TMClone's property of expressed TCR in the T cell mass before method stimulates and afterwards.This paper has described the analysis of resetting V β gene.Those of skill in the art will be easy to recognize and can analyze V α, V γ and V δ tcr gene in a similar manner.

In essence as U.S. Patent number 5,837,447 and people (C.Ferrand, E.Robinet, Emmanuel Contassot, J-M Certoux, Annick Lim, P.Herve and P.Tiberghien such as C.Ferrand, Human Gene Therapy 11:1151-1164,2000) describedly carry out the spectral pattern analysis.In brief, initiator cell suspension is from PBMC, clone, the PBMC that has eliminated the CD8+ cell and/or XCELLERATED T cell.Use Trizol (Gibco-BRL) to separate total RNA, get 2 μ g and in standard cDNA building-up reactions, carry out reverse transcription with sexamer (PharmaciaBiotech) at random.

(people such as Puisieux, J.Immunol.153:2807-2818,1994 as discussed previously; People such as Pannetier, Immunol.Today 16:176-181,1995), with one of 24 kinds of TCR BV subtribe Auele Specific Primers each TCR BV section of C β primer amplification with identification TCR β chain two constant region C β 1 and C β 2.The coupling of C β primer 6-Fam fluorescence dye (Gibco-BRL).For quantitative analysis, with cDNA and internal standard (PTZ-δ CD3 plasmid) coamplification.

In thermal cycler in 25 μ l reaction with the aliquots containig of one of 24 kinds of TCRBV oligonucleotide with unmarked C β primer amplification cDNA building-up reactions.

The pcr amplification of TCRBV transcript length pattern.At thermal cycler (PTC-200; MJ Research, water is honest, the Massachusetts) in 25 μ l reaction with one of 24 kinds of TCRBV oligonucleotide and unmarked C _βThe aliquots containig of primer amplification cDNA building-up reactions.Each reaction contains 1x Taq polymerase buffer (Promega, Charbonniere, France), 1.5mM MgCl ₂, every kind of dNTP concentration 0.2 μ M, every kind of concentration of primer 0.5 μ M and 0.5U Taq polysaccharase (Promega).Use following program under state of saturation, to carry out PCR: pre-94 ℃ of 3min of sex change; 40 circulation sex change (94 ℃ of 25sec), annealing (60 ℃ of 45sec) and polymerization (72 ℃ of 45sec); Be final 72 ℃ of 5min of extension subsequently.Get some TCRBV/C _βThe PCR product carries out 2% agarose electrophoresis, comprises in the experiment that negative control (lacking cDNA) is to check amplification and possible pollution.To 24 kinds of TCRBV/C _β-40 cycle P CR products are got 2 microlitres for every kind and are carried out two round-robin prolongations (out of control) under the same conditions, except C in 10 μ l _βThe final concentration of fluorescent primer is 0.1 μ M.

The quantification that the TCRBV subtribe characterizes in the cell mass

Competitive δ CD3PCR.To each sample, DNA plasmid (having deleted the δ CD3 chain of 4bp) the amplification synthetic cDNA of the set amount by adding serial dilution, the scope of competition thing is 10 ¹¹-10 ⁷Individual copy (people such as Garderet, 1998).Normal concentration when best titration point is defined as the PCR product standard and natural cDNA are produced the signal of comparable intensity.In brief, use 1x Taq polymerase buffer (Promega), 1.5mM MgCl ₂, every kind of dNTP concentration 0.2 μ M, every kind of concentration of primer 0.5 μ M and 0.5U Taq polysaccharase (Promega), in 25 μ l reaction, carry out δ CD3PCR.Use following program under state of saturation, to carry out PCR: pre-94 ℃ of 3min of sex change; 40 round-robin sex change (94 ℃ of 1min), annealing (60 ℃ of 1min) and polymerization (72 ℃ of 45sec); Be final 72 ℃ of 5min of extension subsequently.Two round-robin dye to 2 microlitre first round PCR in prolonging under the same conditions, except the final concentration of 3 ' δ CD3 fluorescent primer in 10 μ l volumes is 0.1 μ M.Fluorescence PCR products is separated on sex change 6% acrylamide gel, and on the automated DNA sequenator of being furnished with Genescan 1.2.1 version (Applied Biosystems, Foster city, California) analysis software, analyze.

Quantitative TCRBV/C _βPCR.For the TCRBV subtribe that quantizes universal class characterizes, in PCR linear stage process (26-28 circulation) (be equivalent to 5 * 10 by cDNA ⁷The δ CD3RNA of individual copy) carries out 24 TCRBV/C _βReaction (15 μ l), condition is to described similar about 40 amplification cycles, except for every kind of TCRBV subtribe primer, C _βThe working concentration of fluorescent primer is 0.1 μ M.For TCRBV in the universal class characterizes, by the summation at all peaks of TCRBV subtribe is calculated the relative percentage of every kind of TCRBV subtribe divided by the summation of all TCRBV subtribes.Because the initial number of δ CD3 copy equates in all TCRBV PCR, so all samples can compare each other.

Electrophoresis and CDR3 clip size are analyzed.40 circulations and 26-28 round-robin pcr amplification reaction thing are mixed with equal-volume (being respectively 10 or 15 μ l) 20mM EDTA-deionized formamide, the Rox-1000 size criteria is used as molecular weight marker (Applied Biosystems).The mixed solution of 2.5 μ l volumes is added on the 24cm 6% acrylamide sequencing gel, and upward size and fluorescent strength determining is analyzed with Immunoscope software at automatic 373ADNA sequenator (Applied Biosystems).

Use the CDR3 length of TCRRNA of polymerase chain reaction (PCR) the product length reflection input of this technology, it depends on that the N Nucleotide of the use of connection (J) and diversity (D) gene fragment and exonuclease activity and terminal enzyme (DNA) is added on the balance of joining region.Detect peak corresponding to transcript in the frame.The appearance at advantage peak explanation exists few clone or clone T cell mass, does not have the T cell of specifying CDR3 length or V β subtribe and have peak or whole subtribe spectral pattern to illustrate respectively, perhaps has in the T cell that generation property tcr gene resets not have the TCR transcript.

As shown in Figure 2, use XCELLERATE ^TMMethod is kept the T cell aggregation, and the set deflection of seeing with the OKT3/IL-2 activated T cell time is compared.At XCELLERATE ^TMActivate and handle the T cell of analyzing before and afterwards from B-CLL patient.(particularly the figure of the 4th row, V β 4,9,15 and 22) as shown in Figure 3, B-CLL patient shows the T cell aggregation of deflection, promptly show to express the polyclone reduction of the T cell of numerous V 'beta ' family genes (comprising V β 4,9,11,13,14,15 and 22).XCELLERATE ^TMThe 12nd day T cell spectral pattern analysis of method shows that the polyclone of these T cells has recovered.

Fig. 4 use Gorochov analyze people such as (, Nat.Med.4:215-221,1998) G.Gorochov ascribe to add with each donor at XCELLERATE ^TMThe numerical value that " disturbance " aggregate level obtains is gathered in amplification before and afterwards.Fig. 4 a has reflected by 8 different B-CLL donor at small-scale XCELLERATE ^TMBefore the amplification and the numerical value that obtains afterwards, and Fig. 4 b has reflected by 5 different donors at clinical scale XCELLERATE ^TMBefore the amplification and the numerical value that obtains afterwards.It is exception that a donor is arranged, the set of its deflection for a long time deflection more that becomes, and the every other donor of initial deflection tends to normalizing (Gaussian distribution).Analyze in 13 duplicate samples 8 parts and return to normal level by high-caliber set disturbance.One of 13 duplicate samples show that disturbance reduces, and are not considered as normal level but reach, and undeflected 3 parts of donor samples run through amplification procedure and keep proper distribution during beginning.

Also checked that by the surface expression of analyzing various TCR V 'beta ' families TCR V β uses, described analysis is used standard technique and adopts different V 'beta ' family members are had specific antibody by flow cytometry.Shown in Fig. 5 a and 5b, measured per-cent (V β 1,2,5,8,14,17 and 21.3) at representative proteinic cd4 t cell of TCRV 'beta ' family of its surface expression and cd8 t cell.Analyzed by 2 isolating T cells of normal donor with by 2 isolating T cells of CLL donor.In the case of B-CLL sample, figure has reflected XCELLERATE ^TMBefore and pattern afterwards.According to these data, obvious every part of B-CLL sample has been demonstrated excessive and not enough that specific V 'beta ' family characterizes, particularly in CD8 group.For example, before activating and increasing, in the cd8 t cell group, CLL donor 1 has the expression V β 2 of very high per-cent and the T cell of V β 21.3, and CLL donor 2 has the T cell of the expression V β 14 of high per-cent.On the contrary, be these 2 donors equally, donor 1 shows V β 5,8,14 expressers of extremely low per-cent, and donor 2 shows V β 1,2,8 expressers of extremely low per-cent.Similar to the observations in the spectral pattern research, through XCELLERATE ^TMAfter the method amplification, these per-cents tend to more normal level, and overexpression person's per-cent reduces, and show that the per-cent of not enough V β raises.

In order to assess leukemia B cell had the frequency of specific T cell, with XCELLERATED ^TMThe T cell has carried out gamma-interferon (IFN γ) ELISPOT and has analyzed with mixing from body leukemia B cellular targets.As shown in table 4, at 1: 167 to 1: 2, can detect tumour-specific T cell in 500 the scope.XCELLERATE ^TMBefore frequency＜1: 10,000 (boundary of sensitivity) illustrate that the number of tumour-specific T cell has obtained selectivity expansion, perhaps, more likely be that tumour-specific T cell is anergic before activating and increasing, and XCELLERATE ^TMMethod has been recovered response.The frequency of the tumor response T cell of being reported has reflected the minimizing of IFN γ positive cell background frequency when lacking the CLL irritation cell.

Table 4:XCELLERATE ^TMThe frequency of amplification back tumor response T cell

Test	Scale	Donor	Tumor response T cell frequency (IFN γ) by the ELISPOT measurement
				CLL-3 CLL-4 CLL-5 CLL-6 CLL-7 CLL-8 CLL-18 CLL-20 CLL-23 CLL-30 CLL-31 CLL-32 CLL-33 CLL-34 CPDCLL-12 CPDCLL-13	The small-scale small-scale is wave wave on a small scale	OHSU-10 OHSU-11 OHSU-12 OHSU-17 OH-CL- 101B OH-CL- 103B RCLL-1 OH-CL- 105L OHSU-16 RCLL-7 RCLL-14 RCLL-14 RCLL-8 RCLL-7 RCLL-8 RCLL-7	1∶256 1∶556 1∶333 1∶681 1∶284 1∶850 1∶1667 1∶200 1∶2500 1∶1111 1∶1000 1∶909 1∶1111 1∶555 1∶167 1∶833

Mean number (N=16) 1:813

Table 4.To from 14 13 parts of frequencies of having assessed antineoplastic specificity T cell by ELISPOT in amplification and the 16 parts of Xcellerated T cells increasing on a large scale for 2 times on a small scale.Tissue is from 13 different donors.

Therefore, as shown here, the TCR that not only can recover to reduce express and give birth to therefrom to antigenic response, and can widen the width of immunne response by the stripped Xcelleration of individual T cell.XCELLERATE ^TMMethod can be used for keeping or recovering the polyclone of T cell.Xcellerated T cell with polyclone of increase of the present invention can be used as preventive measures or is used for the treatment of present illness, such as B-CLL.Make in this way the activation that generates and the T cell after the amplification thereby be used in the non-responsiveness individuality and recover immune response.

Embodiment 3

The XCELLERATE method has been improved the lymphocyte recovery in accepting the myelomatosis patient who transplants

This embodiment has described the data of the preliminary clinical trial of multiple myeloma patients, and indication XCELLERATED T cell has improved the recovery of accepting the myelomatosis patient of transplanting.

The leukapheresed cell of being gathered by the patient before having registered after the clinical trial, gathering stem cell carries out XCELLERATE II to it as described in example 1 above in essence and handles.After inculcating stem cell, inculcated XCELLERATED T cell in 3 days.As shown in Figure 6, XCELLERATE ^TMMethod has been improved the lymphocyte recovery in accepting the myelomatosis patient who transplants.In addition, CD4 and cd8 t cell have increased after XCELLERATED T cell is inculcated.

Therefore, this clinical data shows that XCELLERATED T cell has improved recovery in accepting the myelomatosis patient who transplants, and supports and T cell composition described herein can be beaten in the donor body so that the idea of extensive and effective immunoprotection to be provided.

Claims (15)

1. external method of recovering T cell mass polyclone, it comprises:

(a) provide a group cell, wherein its part comprises the T cell at least;

(b) described cell mass is exposed to first kind of reagent and second kind of reagent, wherein said first kind of reagent comprises anti-CD 3 antibodies, or its Fab and described second kind of reagent comprises anti--CD28 antibody, or its Fab;

Wherein described cellular exposure is enough to increase polyclone in the time of described first kind of reagent and second kind of reagent; Wherein the increase of polyclone comprises the conversion of at least a V 'beta ' family gene from monoclonicity or few clone's property to polyclone, and described V 'beta ' family gene is selected from by V β 4,9,11,13,14,15 and 22 groups of forming,

Recover the polyclone of T cell mass thus.

2. according to the process of claim 1 wherein that described first kind and second kind of reagent are attached to the surface.

3. the method for claim 2, wherein said first kind and second kind of reagent are by covalency absorption, directly absorption or be attached to described surface indirectly.

4. the T cell mass that has recovered polyclone according to the method for claim 1 is used for recovering application in the medicament of immunne response at the non-responsiveness individuality in preparation.

5. the application of claim 4, wherein said non-responsiveness individuality suffers from cancer.

6. the application of claim 5, wherein said cancer is selected from the group of being made up of following cancer: melanoma, non Hodgkin lymphoma, Hodgkin, nasopharyngeal carcinoma, leukemia, plasmoma, sarcoma, neurospongioma, thymoma, mammary cancer, prostate cancer, colorectal cancer, kidney, renal cell carcinoma, carcinoma of the pancreas, esophagus cancer, the cancer of the brain, lung cancer, ovarian cancer, cervical cancer, multiple myeloma, hepatocellular carcinoma, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CML), large granular lymphocyte leukemia (LGL), and lymphocytic leukemia (CLL).

7. the application of claim 5, wherein said cancer are B cell lymphocytic leukemias.

8. the application of claim 4, wherein said non-responsiveness individuality has infected virus.

9. the application of claim 8, wherein said virus is selected from the group of being made up of following virus: single strand RNA virus, single-stranded DNA viruses, human immunodeficiency virus (HIV), first type, B-mode or hepatitis C virus, hsv (HSV), human papillomavirus (HPV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV).

10. the application of claim 4, wherein said non-responsiveness individuality suffers from the congenital heredity disease, suffers from the chronic disease that influences kidney, liver or pancreas, or suffers from and old and feeble relevant immune deficiency.

11. the application of claim 4, wherein the non-responsiveness individuality suffers from autoimmune disease, or suffers from the hematologic disease relevant with hemocytopenia.

12. the application of claim 11, wherein said autoimmune disease is selected from the group of being made up of following disease: rheumatoid arthritis, multiple sclerosis, insulin-dependent diabetes mellitus, bronzed disease, celiac disease, chronic fatigue syndrome, inflammatory bowel, ulcerative colitis, regional ileitis, Fibromyalgia, systemic lupus erythematous, psoriasis, Sjogren syndrome, hyperthyroidism/Graves disease, hypothyroidism/Hashimoto thyroiditis, insulin-dependent diabetes mellitus (I type), and myasthenia gravis.

13. the application of claim 4, wherein said non-responsiveness individuality has passed through the processing of chemotherapy, cytotoxic reagent or inhibitive ability of immunity reagent.

14. the application of claim 11, wherein said hematologic disease is selected from the group of being made up of following disease: aplastic anemia, myelodysplastic syndromes, Fanconi anemia, idiopathic thrombocytopenic purpura and autoimmune hemolytic anemia.

15. a composition, it comprises T cell mass and the pharmaceutical excipient that has recovered polyclone according to the method for claim 1.

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