JP2002511757A - Cell activation process and reagents therefor - Google Patents

Abstract

  (57) [Summary] Describes a cell activation process in which effector cells are transformed with DNA encoding a chimeric receptor containing two or more different cytoplasmic signaling components. At least one of the cytoplasmic signaling components comprises a tetra-span transmembrane protein, CD43, CD6, mannose, IL-7, IL-12 or a complement receptor, an integrin-related protein, or a gamma chain associated with a cytokine receptor. Derived from all or part. Activated cells may be useful in medicine, for example, in treating diseases such as cancer.

Description

DETAILED DESCRIPTION OF THE INVENTION                  Cell activation process and reagents therefor   The present invention relates to a process for activating cells, a DNA transport system for obtaining cell activation. It relates to the use of stems and activated cells in medicine.   Natural T-cell receptors are antigen-binding, transmembrane, and cytoplasmic components. It is a complex association of the polypeptide chains. When the antigen binds correctly to the receptor, T cells Activation and a series of intracellular events leading to, for example, destruction of antigen-presenting target cells Triggered. Antigen recognition can occur only when the antigen is presented in association with the MHC molecule You.   Manipulate T cells to be active on binding ligands other than the native MHC-presented antigen It would be highly desirable if MHC were no longer needed. This includes MHC Provide a means to prevent inter-individual variability related to presentation, and Therapies that allow targeting of surface antigens and thereby mediated lymphocytes, such as tumors The effectiveness of tumor treatment will be increased.   Chimeric receptors target T cells by targeting cells that express the antigen on their cell surface. It is designed to work. Such recombinant chimeric receptors include ‘T 結合, a binding domain from antibodies and intracellular signaling of T cell receptors Chimeras that include a chimeric domain [e.g., published international patent specification numbers, International Patent Publication No. 92/10591, International Publication No. 92/15322, International Publication No. 93/191 63 and WO 95/02686].   Recombinant chimeric receptors described in the art comprise a ligand binding component, It is composed of a transmembrane component and a cytoplasmic component. However, these recombinant forms When transfected with the chimeric receptor, T cells, for example, -Unless stimulated simultaneously by treatment with leukin 2 [IL-2], antigen will bind Nor does it provide acceptable levels of T cell activation. Because co-stimulation is needed, This method will be primarily suitable for ex vivo treatment of patients. This is redundant and complex Technique.   In International Patent Number, WO 97/23613, we have identified IL-2 as Current ex vivo activation was improved without the need for additional costimulators Provides an alternative to the ex vivo approach. The method described in that specification is: Successful in vivo repointing of T cells, especially in response to one type of extracellular interaction Advantageously provide orientation and activation.   In essence, the invention described in International Patent Specification No. 97/23613 Ming provides effector cells transformed with DNA encoding the chimeric receptor You. Chimeric receptors are not naturally associated and result in improved cell activation. Two or more different signals that are conveniently selected to cooperate Contains cytoplasmic components. DNA encoding such a recombinant chimeric receptor may be Transfect T cells or other effector cells ex vivo and / or ex vivo You may. Effector cells expressing one or more of these chimeric receptors will later Binding to target cells leads to processes involving chimeric receptor assembly and dimer formation Of effector cells or allosteric changes in chimeric receptors in Trigger another signaling- or receptor-triggered mechanism It is.   We have described in International Patent Specification Number WO 97/23613. To precisely trigger activation or reaction of effector cells There is provided a series of chimeric receptors that can be used to advantage. This is specific By utilizing a receptor having a co-stimulatory signaling domain Can be.   Thus, according to one aspect of the present invention, we consider that cytoplasmic components Is unbound, and at least one is derived from a transmembrane polypeptide. At least one of the cytoplasmic signaling components is a tetra-span transmembrane protein, CD 43, CD6, mannose, IL-7, IL-12, or complement receptor, integrin-related protein From all or part of white matter, or the gamma-chain associated with cytokine receptors A first cell characterized by a costimulatory signaling domain Coating one or more of the above recombinant chimeric receptors containing different cytoplasmic signaling components The first cell and the second marker provided by the DNA transport system containing the DNA to be loaded Cells associated with a specific cell made one type of extracellular interaction As a result, a method for activating a cell is provided.   The DNA encoding the chimeric receptor, when expressed, and with cells When two target cells interact extracellularly, through cytoplasmic signaling components Signals are converted to two or more different intracellular signaling messengers Are arranged as follows. As a result, the cells are activated and the biological response to the target cells Is triggered. As used herein, activation of a cell refers to one or more sigs. Activation of the null transmission pathway. This may be an increase in cell proliferation; Expression of cytokines by inflammatory response; stimulation of cytokine activity, differentiation or other Effector function; antibody secretion; poor diet; increased tumor invasion and / or adhesion May be indicated.   The cytoplasmic signaling component containing the costimulatory signaling domain is preferably They are chosen so that they can work in concert. They say "naturally `` Is not compatible '', as the term is used herein in its own right on a single polypeptide chain. Refers to the cytoplasmic signaling component that is not bound to any of the components. Preferably cytoplasmic signal Transmission components bind at one or more cytoskeletal components of the cells in which they are expressed And / or a component capable of causing a reaction. Especially useful Signaling components include those described below in connection with other aspects of the invention. It is.   In addition to the cytoplasmic signaling component, each recombinant chimeric receptor is preferably Or a binding component capable of recognizing cell surface molecules on target cells, and transmembrane Contains ingredients. The DNAs encoding these components can further comprise a once expressed chimeric receptor. Encodes a signal peptide to ensure that the receptor points to the cell surface membrane I do. All components may be encoded by a single DNA coding sequence.   Alternatively, each cytoplasmic signaling component is composed of two or more different DNA coding sequences. The columns may be coded. In this case, the respective DNA coding sequence is also a signal It may encode a peptide, a transmembrane component and / or a binding component. The binding component is Can be different, for example, by binding to the same molecule associated with the target cell. Thus, they can generally participate in the same type of extracellular event. One In a preferred embodiment, the binding components are the same.   In some of the applications described hereinafter, for example, if the binding component is an antibody or an anti- When the fragment is a body fragment, the DNA encoding the chimeric receptor has one sequence, for example, One of the binding components that binds to the signal peptide, transmembrane and cytoplasmic signaling components Part [Antibody, for example V_HComponent case], and the second sequence is the remainder of the binding component [Eg, V in a given embodiment_LIngredients], two different DNA It may include a code array.   To activate the desired cells, the DNA encoding the chimeric receptor is first Need to be transported to the cell. Thus, in accordance with the second aspect of the present invention, we Is a recombinant chimeric receptor in which DNA can perform one type of extracellular interaction. At least one of a carrier-associated DNA encoding a receptor, and a cytoplasmic component Is derived from a transmembrane polypeptide, wherein at least one of said cytoplasmic signaling components is Tetra-span transmembrane protein, CD9, CD43, CD6, mannose, IL-7, IL-12, also Or associated with complement receptors, integrin-related proteins, or cytokine receptors Is a costimulatory signaling domain derived from all or part of the γ chain Two or more different non-naturally associated extracellular signaling A DNA transport system comprising:   In this aspect of the invention, in particular, two or more different cytoplasmic signaling components The encoding chimeric receptor may be encoded by a single DNA coding sequence. like this In one preferred embodiment, the present invention relates to the following: of: i) a signal peptide component; ii) a binding component capable of recognizing a cell surface molecule on a target cell; iii) transmembrane components; iv) at least one of the cytoplasmic components is derived from a transmembrane polypeptide;   Two or more different cytoplasmic signaling components that do not match, and optionally v) at least one of said cytoplasmic signaling components is a tetra-span transmembrane protein;   CD9, CD43, CD6, mannose, IL-7, IL-12, or complement receptor, int   All or one of the gamma chains associated with phosphorus-related proteins or cytokine receptors   Having the feature of being a costimulatory signaling domain derived from the   One or more spacer regions for binding any two or more of the components i) to iv)   Encoding, encoding recombinant chimeric receptor, comprising carrier-associated DNA   Provide NA transport system.   The components of the recombinant chimeric receptor are signal transduction cytoplasmic components And thus the binding component recognizes cell surface molecules on the target cell. Activate one or more messenger systems as a result of knowledge Functionally combine.   Two or more components may be linked by one or more spacer regions. Space The protein region promotes the correct conformation of the component for biological activity. It may function to proceed. To combine the transmembrane component iii) with the binding component ii) It is particularly convenient to use a spacer region for   The spacer region is, for example, up to 300 amino acids, preferably 20 to 100 amino acids , And most preferably 25 to 50 amino acids.   The spacer may be all or part of the extracellular region of CD8, CD4, or CD28; or Naturally occurring, such as all or part of the constant region of an antibody including the hinge region It may be derived from all or part of the molecule. Functional part of intracellular signaling molecule All or part of the natural spacer component, such as ITAMS (immunoreceptor Spacers between rosin-based activation motifs) may also be used. Or The spacer may be a non-naturally occurring sequence.   The binding component ii) is any molecule capable of interacting with a cell surface molecule. And bombesin receptors expressed on lung tumor cells, Cells infected with the virus, such as baby antigens, polymorphic epithelial mucin, and CD33 inflammation Cells infected with bacteria: cells associated with a pathological condition, such as those associated with cancer cells Surface markers expressed on peptide hormones, adhesion molecules, and autoimmune diseases Inflammatory molecules, or T cell receptors, or antigens that cause autoimmunity You may choose to recognize.   Suitable binding components for use in the chimeric receptor of the present invention include cell surface related components. T cell receptor; CD4; CD8; CD28; cytokine receptor Condition, such as interleukin receptor, TNF receptor, or Ron receptor, eg, receptor for γ-IFN; colony stimulating factor, eg, GMCSF Receptor; eg, Fab, Fab ', F (ab') 2, single-chain F_V, F_V, And C_HAnd C_LDo V that may exist in relation to the main_HOr V_LComponent antibodies and antigen-binding fragments thereof, All or a part of The antibody or fragment can be mouse, human, chimeric or genetic. It may be a human antibody and a fragment that has been subjected to child manipulation. As used herein, genetic An engineered human antibody or fragment is a single antibody, for example, an otherwise human antibody. One or more CDRs from a mouse antibody buried within the framework and And antibodies or fragments having one or more framework residues. You. Such antibodies are well known, for example, International Patent Specification, International Publication No. 91/0. It may be prepared by a number of methods, as described in 9967.   Particularly useful binding moieties include Fab 'fragments, or especially single-chain F_VFragments are included.   Single-stranded F wherein the binding component comprises different binding chains_V, V_HOr V_LAntibodies other than components Is a transport system of the invention for encoding a second binding chain when is an antibody fragment Need to include a second different DNA coding sequence. In this case, the cytoplasmic sig The first DNA sequence, including the null transfer component and one strand of the antibody or fragment, contains the antibody binding The signal peptide and the antibody or fragment must be It is expressed together with a second DNA sequence encoding a second strand.   The transmembrane component iii) is the α, β, or ζ chain of the T cell receptor, CD28, CD8, CD4, Itokine receptor, e.g., interleukin receptor, TNF receptor, or Terferon receptor, or a colony stimulating factor receptor, e.g., all or May be from a wide range of sources, such as some. If desired, the transmembrane component Naturally a membrane associated with a costimulatory signaling domain as described herein Transmembrane domain and tetra-span transmembrane domain, CD43, CD 6, mannose, IL-7, IL-12 or complement receptor, integrin-related protein, Alternatively, it may be derived from a gamma chain associated with a cytokine receptor.   Binding and transmembrane components are bound directly or, preferably, by a spacer region May be. The spacer region may be one or more of the above regions. More than one If there are two regions, e.g. two regions, these are preferably different regions, e.g. For example, an antibody hinge region that binds to all or a part of the extracellular region of CD28.   Spacers and transmembrane components have their free thiol groups, thereby To provide the chimeric receptor with the ability to form multimers, especially dimers. Good. Receptors of this type, especially dimers, are particularly preferred, among which are CD28 components. , A ζ chain of a natural T cell receptor, and / or an antibody hinge sequence Is included.   The transmembrane component is the cell to which it binds, either directly or by a spacer It may or may not naturally bind to the quality component.   The cytoplasmic signaling component iv) may for example comprise one or more intracellular messages. Signal that results in activation of the marker system. Of cytoplasmic components Preferably, each activates a different messenger system. Directly Intracellular messenger systems that are activated, or indirectly, include, for example, NFAT and Pathways involving tyrosine kinase, PKC or MAP kinase, including cAMP-mediated pathway A G-protein or phospholipase-mediated pathway; a calcium-mediated pathway; One such as a pathway involving the synthesis of cytokines such as terleukins, eg IL-2 Including the above kinase pathway.   The at least one cytoplasmic signaling component iv) is, in particular, all of the above-mentioned receptors. Alternatively, it may be a costimulatory signaling domain from part. Generally, at least The signaling domain, or the signaling and transmembrane domains, To take advantage of convenient restriction sites when constructing the encoding DNA first, It may be used with the rest of the receptor, if desired.   Thus, for example, the costimulatory signaling domain may be, for example, the tetra- Span transmembrane protein [Lanza et al. Biol. Chem.266, 10638-10645 (1991)], particularly about amino acid positions 36 to 227; CD37 [Krasson (Classon, R.), J. et al. Exp. Med.172, 1007 (1990)], especially about amino acids 38- 281th place; CD53 [Angelisova P., Immunogenetics32, 281-285 (199 0)], especially about amino acids 37-219; CD63 [Metzelaar, M., J. Bio l.Chem.266, 3239-3245 (1991)], especially amino acids from about position 35 to about position 237: and CD82 [ Imai, T., J. et al. Immunol.149, 2879- (1992)], especially about amino acid position 35 CD43 [Pallant, A., P.N.A.S.86, 1328-1332 (1989)], especially amino acids about 262 to about 385 or about 240 to about 385; C D6 [Aluffo, A .; Exp. Med.174949-952 (1991)]. Amino acid about 401 to about 644, or about 374 to about 644; a mannose receptor [Ezecovi Ellekowitz, J. et al. Exp.Med.172, 1785-1794 (1990)], especially about 13 amino acids. Positions 94 to 1438, or about 1370 to about 1438; and the IL-7 receptor α-chain (CD127) [good Dewin (Goodwin, R.G.), Cell60, 941-951 (1990)], especially about amino acid position 245 About position 439, or about 220 to about 439; IL-12 receptor β chain [Preskey, D .; ), P.N.A.S.93, 14002-14007 (1996)], especially about amino acids 647-862, Is at about position 623 to about 862; a complement receptor such as CR1 (CD35) [Wong, W., P .; N.A.S.82, 7711-7715 (1985) 1, especially amino acids about 1955 to about 1998, or about Positions 1931 to about 1998, or CR3 and CR3-related proteins [Messlka, E., J. . Immunol.154, 6663- (1995)]; of CD47 including Form 1 and Form 2 Such integrin-related proteins [Lindberg et al. (Lindberg, F.), J. Cell. Biol.12 Three 485-496 (1993)]; or the gamma of the IL-2, IL-4, IL-7, IL-9, or IL-15 receptor Chain (CD132) [Takeshita, T., Science257, 379-382 (1992)] From about amino acids 262 to about 347, or from about 233 to about 347.   The amino acid ranges quoted above, as long as they retain the signaling function It is recognized that changes can be made if desired. Thus, for example, these Fragments within this range are convenient for constructing the DNA encoding the receptor. (E.g., taking into account convenient restriction sites as described above), and / or This prevents receptor features, for example to prevent possible glycosylation sites. It may be used when a change in sex and / or improvement is obtained. In this way, one In one example, the DNA encoding the CD9 fragment at amino acid residues 52 to 227 is replaced with an amino acid As a substitute for the DNA encoding the full length CD9 signaling domain at acid residues 36-227. May be used. Other signaling domain fragments are large The size depends on the properties of the DNA encoding the parent domain and / or the domain Depending on the amino acid sequence, especially any restriction sites and / or glycosylation sites Depending on the position of the position, it may be conveniently used according to the same principle. That Such sites can be visually inspected or by other conventional means. Can be easily identified.   One useful costimulatory signaling domain is the tetra-span transmembrane protein All or part, especially the domain from CD9.   In addition to the costimulatory signaling domains described, Other suitable cytoplasmic components iv) that may be present include, for example, ζ, η, or ε chains. Components derived from T cell receptors such as all or some; CD28; γ chain of Fc receptor; Or cytokine receptors such as interleukin, TNF and interfero Receptors, colony stimulating factor receptors such as GMCSF, tyrosine kinases such as Z Signal transduction components from AP-70, fyn, lyk, Itk and syk; adhesion molecules such as LFA -1 and LFA-2, B29, MB-1, CD3δ, CD3γ, CD5 or CD2. signal The transmitting cytoplasmic component is preferably an ITAM-containing cytoplasmic component.   Cytoplasmic signaling components are preferably selected such that they act in concert Is done. They may be in any direction relative to each other. Particularly useful ingredients In addition to the above costimulatory signaling domains, all of the signaling components of CD28 Or part of the T cell receptor ζ chain, or the γ chain of FcεR1 or FcγR111 included.   The signaling component may be naturally associated with the binding component and may be of other origin. May be derived from   Examples of suitable signal peptide components i) include immunoglobulin signal sequences It is.   The signal, binding, transmembrane and cytoplasmic components are preferably human From a column or based on a human sequence.   Homologs of individual components of the chimeric receptor may be used, and the present invention It is understood that it is expanded to. As used herein, the components of the chimeric receptor are encoded Homolog used for a particular nucleotide or amino acid sequence The term is used as long as the homologue always has substantially the same function as a particular component of the chimeric receptor. In which one or more nucleotides or amino acids are added, deleted, substituted, or U Otherwise, it represents the corresponding sequence that is chemically modified. Homologues are standard By biological and / or chemical techniques such as cDNA or gene cloning Or oligonucleotide-directed mutagenesis, oligonucleotide-directed synthetic techniques Method or enzymatic cleavage or enzymatic filling of gapped oligonucleotides It may be obtained by using mating.   Fragments of individual components in which one or more nucleotides have been deleted are chimeric This may also be used as long as it substantially retains the same function as the starting component of the receptor .   The DNA used in this and other aspects of the invention may be a standard molecular biology And / or using chemical techniques, eg, oligonucleotide-directed mutagenesis Oligonucleotide-directed synthesis techniques, enzymatic cleavage, or Easy entry by utilizing enzymatic compensation in the oligonucleotides It may be obtained from accessible DNA sources. Such techniques are described in Maniati et al. s) “Molecular Cloning”, Cold Spring Heart Bar Laboratories, New York, 1989 and in particular described in the Examples set forth below. You.   The carrier used in the DNA transport system of the present invention may be a target cell and / or a carrier. Are suitable for introducing DNA into target host cells ex vivo or in vivo. Or another carrier. An example of a suitable vector is a retrovirus , Adenovirus, adeno-associated virus, EBV, and HSV, and liposomes Includes non-viral vectors such as vectors and vectors based on DNA condensing agents It is. Alternatively, the carrier may be an antibody. Where appropriate, the vector may further comprise: Promoter / regulatory sequences and / or promoters from viruses such as the retrovirus LTR Or replication function, AAV repeat, SV40 and hCMV promoters and / or enhancers Splicing and polyadenylation signals; EBV and BK virus replication Functions may be included. TCR-α promoter, E-selectin promoter and CD Tissue-specific regulatory sequences such as the two promoters and locus control regions also It may be used in the same manner as inducible promoters such as disease induction promoters.   If two or more DNAs are used in the DNA transport system, As described above may be incorporated into the same or different carriers.   When used ex vivo, the DNA transport system of the present invention is well known in the art. Methods such as transfection, transduction, biolistics ics), protoplast fusion, calcium phosphate precipitated DNA transformation, electroporation, positive Obtained from the target host using onlipofection or targeted liposomes Effector cells. Next, the effector cells are transformed using standard techniques. And reintroduce it into the host.   In accordance with the present invention, a variety of target hosts, such as, for example, mammals, and especially humans, May be used.   Examples of suitable effector cells include lymphocytes, such as cytotoxic T-lymphocytes, Tumor infiltrating lymphocytes, natural killer cells, neutrophils, antibasophils, eosinophils, obesity Cells or T-helper cells; dendritic cells, B cells, hematopoietic stem cells, macrophages Or cells associated with the immune system, such as monocytes. Cytotoxic T lymphocytes It is particularly preferred to use. When using macrophages, costimulatory signaling The domain is preferably derived from all or part of an integrin-related protein, In particular, it is derived from all or part of mannose, complement receptor or related proteins. This In this case, it is convenient to use the γ chain of the Fc receptor as an additional cytoplasmic signaling component. Is good.   The DNA transport system of the present invention is particularly suitable for in vivo administration. One preferred In one embodiment, the delivery system directs DNA to effector cells for which a carrier is desired. In the form of targeted transport systems. Such targeted transport systems Specific examples of systems include targeted naked nuclear DNA, DNA-targeted retroviral systems And / or targeted liposomes complexed therewith, and protamine And targeted condensed DNA such as polylysine condensed DNA.   Targeting systems are well known in the art, for example, CD8; CD16; CD4; CD3; selectin, eg, E-selectin; CD5; CD7; CD34; activating antigen, eg, C Cell surface antigens expressed on target cells in vivo such as D69 and IL-2R And the use of antibodies or fragments thereof. Alternatively, express HIV gp160 Targeting target cells, e.g. CD4 Can be used.   Generally, antibody-targeted DNA, particularly antibody-targeted naked nuclear DNA, antibody-targeted condensed DNA and Particularly, it is preferable to use an antibody-targeted liposome. Liposomes that may be used Certain types of liposomes include, for example, liposomes using linkers that cleave at low pH. When combined with a chromosome, pH-sensitive liposomes are included. Fused with cell membrane Cationic liposomes that directly transport the recombinant chimeric receptor DNA of the invention to the cytoplasm Also, it may be used. The liposomes used in the present invention may also be, for example, polyether To increase circulating half-life such as Tylene glycol polymers May be bonded to a hydrophilic group. Attached to liposomes or other carriers Substituents suitable for are many examples in the art; see, for example, international patents. Specification number, International Publication No.88 / 04924, International Publication No. 90/09782, International Publication Publication No. 91/05545, International Publication No. 91/05546, International Publication No. 93/19738, National See International Publication No. 94/20073 and International Publication No. 94/22429. Antibody Or other targeting molecules can react with conventional readily available linking groups and antibodies. Sex substituents such as thiols or amines and DNA or DNA-containing materials The DNA, condensed DNA or liposomes may be attached using a reactive substituent.   Non-targeted transport systems may be used as well, in which case the targeted development of DNA The present is advantageous. Targeted expression of DNA can be performed, for example, by using the ζ promoter and the CD2 promoter. T-cell specific, such as promoter and locus control regions, and perforin promoter This may be performed using a promoter system.   The aspects of the invention described above utilize a single DNA sequence to encode a chimeric receptor. It is convenient to use. However, the present invention relates to two or more DNA Is understood to extend to the DNA transport system encoded by the sequence . Thus, in one example, in the same reading frame above, each component i) to iv) and optionally v), but the cytoplasmic signaling component iv) Differ from each other in that they are not the same, but with at least one signal The transduction component is a costimulatory signaling domain as described herein; The first and second different DNA coding sequences may be present in a transport system. Good. The two DNA coding sequences are such that at least one component on the first DNA is a second DN One or more signaling components, as long as they are different from the other signaling components on A Minutes may be coded. As mentioned above, the signaling components act in concert. Conveniently, the remaining components are the same as in the single DNA embodiment. May be any of the components described above. The result encoded by the first DNA The binding component iv) is preferably the same as the binding component encoded by the second DNA. You. The binding component encoded by the first DNA is encoded by the second DNA Be separated from the transmembrane component by a spacer region different from the binding component convenient.   The transport system may be used ex vivo, and in a further aspect, the book The invention relates to effector cells transfected by the DNA transport system of the invention. Provide vesicles. Effector cells previously described suitable for use ex vivo It may be any of the cells, preferably a T cell, and most preferably a cell It is a cytotoxic T cell.   The DNA transport system may be in the form of a pharmaceutical composition. This is therapeutic or diagnostic The composition may be in the form of a disintegrating composition or in any suitable dosage form suitable for administration. Preferably this is parenteral administration, such as injection or infusion, such as a single injection, or May be in a form suitable for continuous infusion. The composition is for injection or infusion In cases where this is the form of a suspension, solution or emulsion in an oily or aqueous medium, And such as suspending, preserving, stabilizing, and / or dispersing agents. Formulation substances may be included. Alternatively, the composition may be formulated with a suitable sterile liquid before use. In a dry dosage form.   If the composition is suitable for oral administration, the composition may contain, in addition to the active ingredient, a starch, e.g. For example, potato, corn, or wheat starch, or cellulose, Or a variety of starch derivatives such as microcrystalline cellulose, silica, lactose Additives such as sugar, magnesium carboxylate and / or calcium phosphate May be included. If the formulation is for oral administration, it is well tolerated by the patient's gastrointestinal tract. It is desirable that they be For this reason, the formulation contains a mucus-forming agent and a resin It may be desirable. Similarly, capsulate compositions that are insoluble in gastric juices. It may also be desirable to improve tolerance by encapsulating in the cell. Ma It may also be preferable to include the composition in a sustained release formulation.   The DNA transport system of the present invention is useful in medicine, and the present invention Administering to a subject an effective amount of a DNA transport system of the present invention. Expanded to the treatment of the subject. The exact amount to be used depends on the age and condition of the patient, Or the nature of the disorder, as well as conventional means, such as animal It may be determined by extrapolating data from experiments. In particular, Exbi When used in an animal model, the number of transfected effector cells Vivo transfection and re-directing of a wide range of effector cell numbers to cells May be established by input. Similarly, the amount of DNA required for use in vivo May be established using a wide range of DNA concentrations in animals.   The DNA transport system of the present invention is useful in treating many diseases or disorders. May be. Such diseases or disorders are infectious diseases, such as HIV infection; Diseases / autoimmune diseases such as rheumatoid arthritis, osteoarthritis, inflammatory bowel disease Disease: cancer; allergy / atopic disease, eg, asthma, eczema: congenital disease, eg, Cystic fibrosis, sickle cell anemia; skin disorders, such as psoriasis; neurological disorders, such as polyps Multiple sclerosis; transplantation, eg, organ transplant rejection, graft host rejection; metabolic / idiopathic disease It may include a disease, such as those described under the general heading Diabetes.   The DNA encoding the chimeric receptor described herein may also be specifically described herein. Is a feature of the present invention used in the transportation system of the present invention.   The DNA encoding the chimeric receptor of the present invention may be assembled from a known DNA sequence. , This specification and our international patent specification, WO 97/23613 and More specifically, the examples and figures in this specification and WO 97/23613 Expressed in effector cells using the methods and approaches generally described in You may.   The following examples illustrate the invention.1) Construction of chimera receptor gene   1 to 21 show the chimeric receptor constructs of the present invention [a to f in each figure].   The components of each of these chimeric receptor constructs were cloned by PCR. Or assembled by standard techniques (PCR protocol, Innis et al. (I. nnis), 1990, Academic Press Inc). Each component is described in our International Patent Statement Book number, cassette similar to that described in Example 1 of WO 97/23613 In-frame subcloning with pBIuescript SK + (Stratagene) Flanked by two to four amino acid bonds forming a restriction site It is.   Thus, for example, in FIG. 1a, the p67 scFv / h.CD28 / CD9-FcRγ chimeric receptor Is specific for human CD33 bound to an extracellular spacer containing the human IgG1 hinge Single engineered human antibody P67.6 single chain Fv, and human FcεR1 through part of human CD9 And a portion of the extracellular region of human CD28 bound to the intracellular domain of the gamma chain of   The single chain Fv is linked to the heavy chain of the engineered human antibody by a (Gly4 Ser) 5 linker. Contains the leader sequence and the variable component of the engineered human antibody light chain attached to the variable component . The extracellular spacer component comprises residues 234 to 243 of the human IgG1 hinge and human CD28. Includes residues 118-134. This corresponds to residues 52-227 of human CD9 (Boucheks et al. eix), 1991, J.M. Biol. Chem.266 117-122) through residue 2 of the gamma chain of human FcεR1 7-68 (Kuster et al., 1989, J. Biol. Chem.255, 6448-6452) It is.   As shown in the rest of FIG. 1 (b-f) and in FIGS. A similar construction may be made using the obtained components.   Thus, a single-chain Fv, such as the P67scFv just described, or HCTMO1scFv as described in Example 1 of 97/23613 is for example a human IgG1 hinge And an extracellular spacer containing part of the extracellular region of human CD28 just described. And may then bind to the various transmembrane and intracellular components indicated. Respectively In the figure, the size of each costimulatory domain is 36 to 227 for CD9 and 38 to 281 for CD37. Shown. In these figures, the specific CD28, ζ and FcRγ components used are If not shown, as above for FcRγ or for CD28 and ζ As described in Example 1 of WO 97/23613 Alternatively, components of the same or similar size may be used. That of these last ingredients Each was constructed in accordance with the invention in the same manner as described for the receptor described in the Examples. , May be incorporated into the receptor.2) Analysis of chimeric receptor constructs expressed in Jurkat cells   Transfection was performed using the following method, described in particular for the construct in FIG. And the expression and action of the receptor of the present invention in the affected cells.P67scFv / h.CD28 / CD9-FcR   The full length chimeric receptor construct was transferred from Hind III to the expression vector p on the EcoRI restriction fragment. EE6hCMV / ne (Bebbington, 1991), MethodsTwo 136-145] to pBluescr Subcloned from ipt.   The plasmid was linearized and biaded using two 1 second pulses at 1000 V and 3 μF. Jurkat E6.1 cells (ECACC) by electroporation using Orad gene pulsar Transfected. The cell line expressing the chimeric receptor was transformed with the drug G418 (2 mg / ml). Selection was performed in the containing medium. After about 4 weeks, determine whether the cells express scFv on their surface. And whether to produce IL-2.scFv Analysis of surface expression   About 5 × 10 cells^FiveStained with a saturating concentration of fluorescein-conjugated antigen (2 μg / ml) Was. Fluorescence was measured with a FACScan cytometer (Becton Dickinson). Was analyzed.Stimulation of antigen-expressing cells   Jurkat Transfectant 1 × 10^FivePieces into a 96-well plate (Falcon 1) control target cells that express or do not express antigen in^FivePieces With 37 ℃ / 5% CO_TwoFor overnight. After 18-20 hours, centrifuge the cells The supernatant was assayed for human IL-2 (Quanticaine kit, R & D system). Thames).Solid phase antigen stimulation   Jurkat Transfectant 1 × 10^FiveOf the cells, and In a 96-well plate (Nunc) at 37 ° C./5% CO_Two And incubated. After 18-20 hours, the cells are centrifuged and the supernatant is directed against human IL-2. Assay (Quanticaine kit, R & D Systems).3) result   FIG. 22 shows transfected cells measured using fluorescein-conjugated human CD33. Surface expression of P67scFv / h.CD28 / CD9-FcRγ chimeric receptor on Jurkat cells Show.   FIG. 23 shows that no target cells or antigen-negative cells [N.EE6 (control plasmid Expressing mouse myeloma NSO)] and antigen-positive target cells [HL60 and N.CD33 ( P67scFv / h.CD2 when stimulated by mouse myeloma NSO expressing human CD33) Antigen specificity by Jurkat cells transfected with 8 / CD29-FcRγ chimeric receptor IL-2 production is shown relative to untransfected Jurkat cells. The figure shows the anti In the presence of primordial (CD33) positive cells, transfected cells expressing the chimeric receptor Jurkat cells were stimulated to produce IL-2. The same cells take the antigen It did not produce IL-2 in the presence of unpresented cells. About each case As a result, untransfected Jurkat control cells did not respond, Did not produce a possible amount.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C07K 14/705 C07K 14/715 14/715 14/735 14/735 16/28 16/28 19/00 19 / 00 C12N 15/00 A C12N 5/10 5/00 B (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU , MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, L) S, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH , CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, GW, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ , TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Ware, Andrew, Nail, Charles Berkshire, England, Twiford, Willow Drive 7

Claims (1)

[Claims] 1. a cytoplasmic component not naturally bound and at least one transmembrane polypeptide     And at least one of the cytoplasmic signaling components is derived from Tetra Spa     Transmembrane protein, CD43, CD6, mannose, IL-7, IL-12 or complement receptor     Gamma chain associated with, integrin-related protein, or cytokine receptor     Characterized in that it is a costimulatory signaling domain derived from     One or more cells comprising two or more different cytoplasmic signaling components.     By a DNA transport system containing DNA encoding the above recombinant chimeric receptor     A fraction associated with a first cell and a second target cell, characterized by being provided.     Activate cells as a result of one type of extracellular interaction with the offspring     how to. 2. The method of claim 1, wherein the cytoplasmic signaling component is capable of acting cooperatively.     the method of. 3. The binding component can recognize cell surface molecules on the target cell,     Signal peptide, binding component and / or membrane of one or more chimeric receptors     3. The method according to claim 1 or 2, wherein the passage component is coded. 4. Signal peptide, transmembrane and cytoplasmic signaling components and binding components     4. All or part of is encoded by a single DNA coding sequence.     The method described in. 5. Each DNA sequence contains additional signal peptide, transmembrane components, and binding components.     The cytoplasmic signaling components that encode all or part of the     4. The method of claim 3, wherein the method is encoded by a DNA coding sequence. 6. DNA encodes a portion of the binding component, and additional different DNA coding sequences     A method according to claim 4 or 5, wherein the remainder of the composition is encoded. 7. The binding component encoded by one DNA sequence is     Are also involved in the same type of extracellular binding event     7. A method according to claim 5 or claim 6, wherein 8. The method of claim 7, wherein each binding component binds to the same molecule associated with the target cell.     The described method. 9. The method of claim 8, wherein each binding component is the same. 10. One or more of the recombinant chimeric receptors is a virus or cell surface component on a target cell.     10. The method according to any one of the preceding claims, wherein the child can be recognized. 11． Recombinant chimeric recipients in which DNA can perform one type of extracellular interaction     The carrier-associated DNA encoding the receptor, and at least the cytoplasmic component     One is derived from a transmembrane polypeptide and has at least a cytoplasmic signaling component.     One of them is a tetra-span transmembrane protein, CD43, CD6, mannose, IL-7, I     L-12 or complement receptor, integrin-related protein, or cytokine receptor     Costimulatory signaling domain from all or part of the gamma chain associated with the body     Two or more different cytoplasms that are not naturally associated     A DNA transport system comprising a signaling component. 12． Each of the receptors has one or more different cytoplasmic signals that do not naturally bind     A transmembrane polypeptide comprising a transduction component and at least one of the cytoplasmic components     Wherein at least one of the cytoplasmic signaling components is derived from Tetra Spa     Transmembrane protein, CD43, CD6, mannose, IL-7, IL-12 or complement receptor,     All gamma chains associated with integrin-related proteins or cytokine receptors     Or a co-stimulatory signaling domain derived from a part thereof     Two or more, each of which can perform the same type of extracellular interaction     The above-described recombinant chimeric receptor is encoded by a DNA, and the DN comprises a carrier-associated DNA.     A transport system. 13. DNA reads in the reading frame:   i) a signal peptide component;   ii) a binding component capable of recognizing a cell surface molecule on a target cell;   iii) transmembrane components;   iv) naturally at least one of the cytoplasmic components is derived from a transmembrane polypeptide;     Two or more different uncoupled cytoplasmic signaling components, and optionally;   v) at least one of the cytoplasmic signaling components is a tetra-span transmembrane protein;     , CD43, CD6, mannose, IL-7, IL-12 or complement receptor,     All or all of the gamma chains associated with gulin-related proteins or cytokine receptors     Is a costimulatory signaling domain derived from a part thereof,     One or more spacer regions that bind any two or more of the components of i) to iv)     The DNA transport system according to claim 11, which encodes: 14. DNA, 1) in reading frame:   i) a signal peptide component;   ii) one or a portion of the binding components;   iii) transmembrane components;   iv) naturally at least one of the cytoplasmic components is derived from a transmembrane polypeptide;     Two or more cytoplasmic signaling components that are not bound, and optionally;   v) at least one of the cytoplasmic signaling components is a tetra-span transmembrane protein;     , CD43, CD6, mannose, IL-7, IL-12 or complement receptor, integrin     All or part of the related protein or gamma chain related to cytokine receptor     Wherein i) to iv are co-stimulatory signaling domains derived from     ) Encodes one or more spacer regions that bind any two or more of the components     The first DNA that binds, and 2) the portion of the binding component     Signal peptide synthesis in the reading frame so that the offspring can be recognized.     And the further components of the binding component ii) encoded by the first DNA     12. The DNA transport system according to claim 11, comprising a second different DNA to be loaded. 15． DNA has the following in each reading frame:   i) a signal peptide component;   ii) a binding component capable of recognizing a cell surface molecule on a target cell;   iii) transmembrane components;   iv) naturally at least one of the cytoplasmic components is derived from a transmembrane polypeptide;     One or more different uncoupled cytoplasmic signaling components, and optionally;   v) at least one of the first DNAs is not encoded by the second DNA;     If coding for the signaling component iv), the cytoplasmic signaling component     At least one of the proteins is a tetra-span transmembrane protein, CD43, CD6, mannose     Protein, IL-7, IL-12 or complement receptor, integrin-related protein, or     Co-stimulatory sig derived from all or part of the gamma chain associated with the Itokine receptor     Any one of the above components i) to iv), which is a null transduction domain     A first DNA and one or more spacer regions encoding one or more     13. The DNA transport system according to claim 12, comprising a second and a different DNA. 16． DNA, 1) each of which in the reading frame:   i) a signal peptide component;   ii) part of the binding component;   iii) transmembrane components;   iv) naturally at least one of the cytoplasmic components is derived from a transmembrane polypeptide;     One or more different uncoupled cytoplasmic signaling components, and optionally;   v) at least one signal wherein the first DNA is not encoded by the second DNA     If coding for the transmission component iv), the cytoplasmic signaling component     One of them is tetra-span transmembrane protein, CD43, CD6, mannose, IL-7     , IL-12 or complement receptor, integrin-related protein, or cytochrome     Signaling from all or part of the gamma chain associated with the receptor     Characterized in that it is a domain, any two or more of the components i) to iv)     First and second different encoding one or more spacer regions to bind     DNA, and 2) the binding component portion provided by the first and third DNAs.     In addition, the binding component portions provided by the second and fourth DNA     In order to be able to recognize cell surface molecules on the target cells,     In the reading frame, the signal peptide component and the first and second     Encodes a further part of the binding component ii) each encoded by DNA     13. The DNA transport system according to claim 12, comprising different third and fourth DNAs. 17． Each signal peptide component is an immunoglobulin signal sequence,     17. The DNA transport system according to claim 13. 18． The binding component encoded by the first DNA is encoded by the second DNA     18. The DNA transport system according to claims 15 to 17, which is the same as the binding component. 19. The DNA transfer device according to claim 13, wherein the binding component is an antibody or an antigen-binding fragment thereof.     Feeding system. 20. The antibody or a fragment thereof is an engineered human antibody or an antigen-binding fragment thereof,     20. The DNA transport system according to claim 19. twenty one. 21. The DNA transport system according to claims 18 to 20, wherein the binding component is a single-chain Fv fragment. twenty two. 21. The DNA transport system according to claims 18 to 20, wherein the binding component is a Fab 'fragment. twenty three. Transmembrane components are T-cell receptor α, β, or ζ chains, CD28, CD8, CD4,     Tokain receptor, colony stimulating factor receptor, tetra-span transmembrane protein,     CD43, CD6, mannose, IL-7, IL-12 or complement receptor, integrin     All or part of the gamma chain associated with the     The DNA transport system according to any one of claims 13 to 22, which is derived from the DNA transport system. twenty four. 24.The DNA transport of claim 23, wherein the transmembrane component is derived from all or a portion of CD28.     system. twenty five. Co-stimulatory signaling domain is CD9, CD37, CD53, CD82, CD43, CD6, CD127     IL-12 receptor β-chain, CD35, CD3, and CR3-related proteins, CD47, or IL-2     From all or part of the gamma chain of the IL-4, IL-7, IL-9 or IL-15 receptor     The DNA transport system according to any one of claims 11 to 24, wherein: 26. The DNA transfer of claim 25, wherein the costimulatory domain is derived from all or a portion of CD9.     Feeding system. 27. At least one of the cytoplasmic signaling components is also a T cell receptor T, η,     Chain, CD28, γ chain of Fc receptor, cytokine receptor, colony stimulating factor     Child receptor, tyrosine kinase, or adhesion molecule B29, MB-1, CD3δ, CD3γ, C     From all or part of the cytoplasmic domain of D5 or CD2     The DNA transport system according to any one of claims 13 to 26. 28. The cytoplasmic signaling component is CD28, the T cell receptor ζ chain and / or FcεR1     Or the DNA transport according to claim 27, which is derived from all or a part of FcγR111.     system. 29. The cytoplasmic signaling components are in any direction relative to each other.     29. The DNA transport system according to any of 28. 30. DNA encoding components i) to iv) further comprises a binding component ii) and a transmembrane component iii).     The method of claim 13, further encoding one or more spacer regions that bind     The DNA transport system according to any one of the above. 31. Two or more different spacer regions link the binding component ii) and the transmembrane component iii).     31. The DNA transport system of claim 30, which is compatible. 32. The spacer region is selected to provide one or more free thiol groups;     32. The DNA transport system according to claim 30 or claim 31. 33. The spacer region is due to all or part of the extracellular region of CD8, CD4, or CD28.     33. The DNA transport system of claims 30-32. 34. The spacer region is all or part of the constant region of the antibody, or     32. The DNA transport system according to 32. 35. Antibody hinge with spacer region attached to all or part of the extracellular region of CD28     The DNA transport system according to claims 30 to 32, which is derived from all or a part of the region.     . 36. The carrier of claim 11 to 35, wherein the carrier is a viral vector or a non-viral vector.     The DNA transport system according to any one of the above. 37. The DNA transfer according to claim 36, wherein the non-viral vector is a liposome vector.     Feeding system. 38. 38. The DNA transport system according to claim 37, wherein the carrier is a targeted non-viral vector.     Stem. 39. The DNA of claim 38, wherein the targeted vector is an antibody-targeted liposome.     Transport system. 40. The DNA of claim 38, wherein the targeted vector is an antibody-targeted condensed DNA.     Transport system. 41. If the targeted vector is antibody-targeted protamine or polylysine condensed DNA     41. The DNA transport system according to claim 40, wherein 42. The DNA transport of claim 38, wherein the targeted vector is an antibody-targeted naked nuclear DNA.     system. 43. The DNA of claim 39, wherein the antibody is a whole antibody or an antigen-binding fragment thereof.     Transport system. 44. The antibody of claim 43, wherein the antibody is an engineered human antibody or an antigen-binding fragment thereof.     DNA transport system. 45. Transfected with the DNA transport system according to any one of claims 1 to 444     Effector cells. 46. Lymphocytes, dendritic cells, B cells, hematopoietic stem cells, macrophages, monocytes, or     46. The effector cell according to claim 45, which is an NK cell. 47. 47. The effector cell according to claim 46, which is a cytotoxic T lymphocyte. 48. Infectious disease, inflammatory disease, cancer, allergy / atopic disease, congenital disease, skin disease     Disease, neurological disease, transplantation and metabolic / idiopathic disease     48. The DNA transport system according to any one of claims 11 to 47. 49. Rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, asthma, eczema, cystic fiber     Disease, sickle cell anemia, psoriasis, multiple sclerosis, organ or tissue transplant rejection, transfer     49. The method of claim 48 for use in treating plant versus host rejection, or diabetes.     DNA transport system. 50. The DN according to any one of claims 11 to 44, together with one or more formulated substances.     A pharmaceutical composition comprising an A delivery system. 51. An agent wherein the formulation material is a suspending, preserving, stabilizing and / or dispersing agent.     A pharmaceutical composition according to claim 50. 52. Recombination for use in a transportation system according to any one of claims 11 to 44.     DNA encoding the chimeric chimeric receptor.