EP0737076A1 - PROCEDE D'IMMUNO-REGULATION SPECIFIQUE D'UN ANTIGENE PAR LA CHAINE $g(a) DES LYMPHOCYTES T - Google Patents

PROCEDE D'IMMUNO-REGULATION SPECIFIQUE D'UN ANTIGENE PAR LA CHAINE $g(a) DES LYMPHOCYTES T

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Publication number
EP0737076A1
EP0737076A1 EP95905413A EP95905413A EP0737076A1 EP 0737076 A1 EP0737076 A1 EP 0737076A1 EP 95905413 A EP95905413 A EP 95905413A EP 95905413 A EP95905413 A EP 95905413A EP 0737076 A1 EP0737076 A1 EP 0737076A1
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EP
European Patent Office
Prior art keywords
tcrα
antigen
tcrα chain
cell
immune response
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95905413A
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German (de)
English (en)
Inventor
Douglas Green
Arun Fotedar
Reid Bissonnette
Toshifumi Mikayama
Yasuyuki Ishii
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kirin Brewery Co Ltd
La Jolla Institute for Allergy and Immunology
Original Assignee
Kirin Brewery Co Ltd
La Jolla Institute for Allergy and Immunology
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Application filed by Kirin Brewery Co Ltd, La Jolla Institute for Allergy and Immunology filed Critical Kirin Brewery Co Ltd
Publication of EP0737076A1 publication Critical patent/EP0737076A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus

Definitions

  • This invention relates to methods for regulating the immune system in an antigen-specific manner.
  • T cell receptor alpha chains which are capable of binding to an antigen of interest are utilized in protocols designed for the suppression or augmentation of the immune response to the particular antigen.
  • the therapeutic protocols described herein may be used in the treatment of allergy, autoimmunity, graft rejection and cancer.
  • T cells respond to antigen stimulation by producing lymphokines which "help" or activate various other cell types in the immune system.
  • lymphokines which "help" or activate various other cell types in the immune system.
  • certain T cells can become cytotoxic effector cells.
  • the B cell response primarily consists of their secretory products, antibodies, which directly bind to antigens.
  • Helper T cells T H can be distinguished from cytotoxic T cells and B cells by their cell surface expression of a glycoprotein maker termed CD4.
  • T H1 Type 1 helper cells
  • T ⁇ type 2 helper cells
  • T H1 Based on the profile of lymphokine production, T H1 appear to be involved in promoting the proliferation of other T cells, whereas T m factors specifically regulate B cell proliferation, antibody synthesis, and antibody class switching.
  • T H1 may regulate each other since ⁇ -interferon produced by T H , inhibits the proliferation and function of T l H M 2-
  • MHC major histocompatibility complex
  • Class II gene products are mostly expressed on cells in various hematopoietic lineages and they are involved in cell-cell interactions in the immune response. Both Class I and Class II proteins have been shown to also function as receptors for antigens on the surface of antigen-presenting cells.
  • Another level of complexity in the interaction between a T cell and an antigen is that it occurs only if the haplotype (the combination of all alleles within the complex) of the MHC is the same between that of the antigen-presenting cells and the responding T cells.
  • haplotype the combination of all alleles within the complex
  • T cells could also negatively influence the course of an immune response.
  • This concept of immune regulation was initially met with skepticism, it eventually became accepted by the majority of immunologists as it provided a conceptual framework for the maintenance of homeostasis in the immune system after an antigen had been eliminated by a given immune response and a continued response was no longer necessary. Since then, antigen-specific suppressor T cells (Ts) have been reported in a wide variety of experimental systems (Green et al., 1983, Ann. Rev. Immunol. 1:439-463; Dorf and Benacerraf, 1984, Ann. Rev. Immunol. 2:127-158).
  • TsF T suppressor factors
  • Ts surface phenotype first showed that they expressed CD8 (Lyt-2), a marker shared by T cells with cytotoxic potential.
  • CD8 Lyt-2
  • an antiserum was reported that appeared to react with a structure specifically expressed by Ts in mice (Murphy et al., 1976, J. Exp. Med. 144:699; Tada et al., 1976, J. Exp. Med. 144:713).
  • Mapping studies of the gene encoding the antigen localized it to the I region between I-E ⁇ and I-E ⁇ within the murine MHC. This locus was called l-J.
  • T and B cell responses for antigen is a function of the unique receptors expressed by these cells.
  • Progress in the study of the B cell receptor advanced rapidly when it was found that B cells secreted their receptors in the form of antibodies.
  • Plasmacytomas are naturally-occurring tumors of antibody-producing cells that are monoclonal in origin. These tumors provided a continuous source of homogeneous proteins which were used in the initial purification and characterization of the structure of the antibody molecule (Potter, 1972, Physiol. Res.52:631-710). It has now been proven that antibodies are identical to their membrane-bound counterparts except that the cell surface form contains a domain for transmembrane anchoring (Tonegawa, 1983, Nature 21)2:575-581).
  • TCR is a heterodimer composed of two disulphide-linked glycoproteins known as ⁇ and ⁇ (Marrack and Kappler, 1987, Science 23_8_: 1073- 1079).
  • cDNA complementary DNA
  • CD3 is a complex of polypeptides which are non-covalently linked to the TCR and which may be involved in transmembrane signalling events leading to T cell activation triggered by TCR occupancy (Clevers et al., 1988, Ann. Rev. Immunol. 6:629). Direct stimulation of CD3 with antibodies has been shown to mimic the normal pathways of T cell activation (Meuer et al., 1983, J. Exp. Med. 158:988). The transport of CD3 to the T cell surface requires its association with complete heterodimeric TCR complexes intracellulary.
  • TCR TCR-binding protein
  • ⁇ ⁇ and ⁇ receptors of T cells are highly homologous to antibody molecules in primary sequence, gene organization and modes of DNA rearrangement (Davis and Bjorkman, 1988, Nature 3_3_4:395-402).
  • the T cell antigen receptors are distinct from antibodies in two major aspects: TCR are only found at cell surfaces and they recognize antigens only in the context of MHC-encoded molecules.
  • TCR may, in some occasions, be shed or released from cells (Guy et al., 1989, Science 244: 1477-1480; Fairchild et al., 1990, J. Immunol. 145:2001-2009).
  • secreted molecules are complete TCR, partial fragments, or other molecules with TCR cross-reactive epitopes.
  • the notion that functionally active TCR ⁇ chains could be released from T cells independently of the remaining TCR components was controversial and met with skepticism.
  • TCR ⁇ is expressed on the surface of immature thymocytes in the absence of TCR ⁇ or CD3 components (Kishi et al., 1991, EMBO J. ⁇ _:93-100).
  • a truncated TCR ⁇ chain gene has been constructed, including only VDJ and the C ⁇ , domain, that is secreted despite the expectation that such a molecule should be degraded (Gascoigne, 1990, J. Biol. Chem.265:9296- 9301).
  • TCR might be released in small quantities, possibly in a complex with other unidentified molecules and/or in a post-translationally truncated form.
  • Antisense oligonucleotides corresponding to TCR V ⁇ and V ⁇ were found to specifically inhibit cell surface TCR-CD3 expression, but only antisense for V ⁇ and not V ⁇ (or control oligonucleotides) inhibited the production of the soluble regulatory activity of A 1.1 (Zheng et al., 1989, Proc. Natl. Acad. Sci. USA 86: 3758-3762).
  • TCR ⁇ has been reported to be rapidly degraded in a nonlysosomal compartment before entering the Golgi apparatus (Wileman, et al, J. Cell. Biol, l ⁇ ):973-986, 1990; Lippincott-Schwartz, etal, Cell,5A:209-22Q, 1988; Baniyash, et al, J. Biol.
  • the corresponding lipid-linked TCR polypeptides were released from the membrane in soluble form by treatment of the cells with phosphatidylinositol-specific phospholipase C, and the solubilized TCR ⁇ heterodimers were shown to react specifically with an anti-clonotypic monoclonal antibody.
  • the yield of released TCR polypeptides was too low to apply this molecule for clinical use.
  • the third approach was to engineer hybrid proteins of TCR with immunoglobin constant region (Gregoire, et al, Proc. Natl. Acad. Sci., USA, £8:8077-8081, 1991; Weber, et al, Nature, 356:793-796.
  • TCR TCR in E. coli
  • a fusion protein of V ⁇ and V ⁇ polypeptide Soo Hoo, et al, Proc. Natl. Acad. Sci. USA, £2:4759-4763, 1992.
  • 1% of protein could be recovered as refolded protein.
  • the yield of refolded protein is as much as typical soluble proteins such as cytokines, which will make it possible to provide homogeneous TCR ⁇ molecule for clinical use.
  • E. coli In order to express the animal proteins in E. coli, various systems have been developed by many investigators. However, a number of difficulties are frequently encountered when expressing heterologous genes in this organism. For example, the significant differences between E. coli and animal genes, both in their patterns of codon usage and in their translation initiation signals, may interfere with the efficient translation of animal mRNA on bacterial ribosomes (Orormo, et al, Nucl. Acids Res. 10:2971-2996, 1982). Alternatively, heterologous proteins synthesized in E. coli may fail to accumulate to significant levels due to the activity of the host cell proteases (Gottesman., Annu. Rev. Genet. , 21: 163- 198, 1989).
  • the physical characteristics of therapeutical ly useful proteins can cause problems, since some secreted molecules or membrane as Cyprus - ciated molecules such as TCR require glycosylation and disulfied-crosslinking for both stability and solubility. Since such stabilizing processes are not available in the bacterial cytoplasm, heterologous proteins produced within E. coli often form insoluble aggregates known as "inclusion bodies" (Schein, et al, Bio/Technology, 7: 1141-1149, 1989).
  • the present invention provides methods to express truncated form of TCR ⁇ polypeptide in inclusion body in E. coli and to refold and purify biologically active TCR ⁇ . 2,. SUMMARY OF THE INVENTION
  • the present invention relates to methods which utilize the TCR ⁇ chain for modulating an immune response in an antigen-specific manner.
  • TCR ⁇ chains that demonstrate the following two important characteristics, which can be evaluated in vitro, are selected for production and use in the practice of the invention: TCR ⁇ chains used in the method of the invention must be capable of binding to the antigen of interest, and in the presence of an accessory component described herein, modulate the specific immune response generated against that antigen, L ⁇ ., by suppressing or augmenting the antigen-specific immune response.
  • the TCR ⁇ chains which demonstrate such properties may be used advantageously in protocols described for the down-regulation or up-regulation of the antigen-specific immune response in vivo in human or animal subjects or in vitro.
  • an effective dose of TCR ⁇ chain specific for the responsible antigen which, in the presence of the accessory component, suppresses the antigen-specific immune response can be administered in vivo.
  • body fluids of an immunosuppressed patient can be tested for the presence of soluble TCR ⁇ chains that exhibit immunosuppressive effects.
  • Augmentation of the patient's immune response for the antigen may be achieved by removal or neutralization of the soluble TCR ⁇ chains using antibodies specific for the TCR ⁇ chain, or antisense oligonucleotides that inhibit the expression of the TCR ⁇ chain.
  • PFC assays which can be used to evaluate the TCR ⁇ chains used in the invention are described herein.
  • a number of immunoaffinity techniques may be used to evaluate antigen binding
  • a plaque forming cell (PFC) assay described in detail infra, (hereinafter referred to as the "PFC assay") may be used to evaluate the regulatory function of the TCR ⁇ chain tested.
  • PFC assay plaque forming cell
  • the TCR ⁇ chain to be tested is added, in the presence of an accessory component, to a spleen cell culture containing the antigen of interest coupled to an immunogenic, lysable carrier, such as xenogeneic red blood cells.
  • the immunoregulatory effect of the TCR ⁇ chain is evaluated by assessing the immune response which is generated over the course of a few days, as indicated by the generation of plaque forming cells in the culture. That is, the immune response generates cells that produce complement-fixing antibodies against the carrier (e.g.. red blood cells), and these cells can be detected via an assay in which the cells are mixed with complement and the carrier (s ⁇ red blood cells) and formed into a monolayer. Lysis of the carrier results in the formation of one clear plaque, corresponding to the presence of one plaque forming cell (PFC). Inhibition of the generation of PFC in the culture indicates suppression of the immune response, mediated by the TCR ⁇ chain specific for the coupled antigen.
  • the carrier e.g. red blood cells
  • the accessory component used in the assay is prepared from stimulated T cell supernatants depleted of soluble factors, such as TCR ⁇ chains, that directly bind to the antigen used to stimulate the T cells.
  • the accessory component in and of itself, has no effect on an immune response unless the TCR ⁇ chain is present.
  • the invention is based, in part, on the discovery of a soluble TCR ⁇ chain which is constitutively secreted by a T cell hybridoma. As demonstrated in the working examples, this secreted TCR ⁇ chain is capable of directly binding to its antigen and, in the presence of accessory component, suppresses the immune response which would normally be generated against the antigen.
  • the invention is not limited to the use of naturally secreted TCR ⁇ chains, since any TCR ⁇ chain gene can be cloned, expressed and the gene product tested for its suitability in the practice of the invention using the techniques and methods described herein.
  • the assays described herein may be used to evaluate other molecules, &g., antibodies, other TCR components, which demonstrate an immunoregulatory function in an antigen-specific manner.
  • a new fusion gene expression system based on the use of rat calmodulin as fusion partner is provided.
  • the system can be preferably used for the high expression and purification of TCR ⁇ protein having biological activities.
  • the expression of rat calmodulin in E. coli has been successful by employing an expression vector containing the E. coli trp promoter and trpA terminator (Matsuki, et al, Biotech, Appl. Biochem., 12:284-291,
  • the rat calmodulin cDNA was modified so as to delete the 5'-nontranslated sequence and to incorporate a consensus sequence for the E. coli ribosome-binding site.
  • Several codons for the N-terminal amino acids were selected to fit the E. coli consensus nucleotide sequence around the translation initiation codon.
  • soluble rate calmodulin accounted for over 30% of total cellular proteins.
  • About 100 mg of recombinant calmodulin of 90% purity was obtained from 1 liter of culture by using phenyl-Sepharose column chromatography.
  • additional sequence encoding protease cleavage site, Lys-Val-Pro-Arg-Gly SEQ ID
  • FIGURE IA and B A T cell hybridoma, 3-1-V, produces an accessory component which mediates immunoregulatory activity in the presence of antigen-specific TCR ⁇ chain from Al.l cells.
  • FIGURE 2 The complete nucleoi ie sequence of the TCR ⁇ gene isolated from Al.l cells. The constant region of the gene is underscored (SEQ ID NO: 14).
  • FIGURE 3 Gene transfer of TCR ⁇ from Al.l cells to 175.2 cells (175.2-Al.l ⁇ ) transfers the ability to produce an antigen-specific regulatory activity.
  • A Expression of CD3 on 175.2 cells before and after the transfer of Al.l TCR ⁇ .
  • B relevant antigen
  • C carrier
  • FIGURE 4 Peptides used in testing the regulatory activity of TCR ⁇ chain from hybridoma Al.l (SEQ ID NOS: 16, 17, 18, 19, 20, 21, 22, 23 and 24).
  • FIGURE 5 The immunoregulatory activity produced by Al.l cells is neutralized by an antibody to TCR ⁇ chain.
  • FIGURE 7A and B Gene transfer of TCR ⁇ from BB19 cells to 175.2 cells does not transfer the ability to produce an antigen-specific immunoregulatory activity as shown by an anti-SRBC PFC assay.
  • FIGURE 8 Gene transfer of TCR ⁇ from Al.l cells to B9 cells (B9-A1.1 ⁇ ) transfers the ability to produce an antigen-specific regulatory activity.
  • FIGURE 10A and B Expression of the Al.l TCR ⁇ in cells lacking TCR ⁇ is sufficient for production of the antigen-specific regulatory activity.
  • FIGURE 1 IA. Antigen-specific binding activity in supematants of Al.l and other cell lines expressing Al.l TCR ⁇ .
  • FIGURE 1 IB. Competition of antigen-specific binding activity in supematants of Al.l and other cell lines expressing Al.l TCR ⁇ by peptides.
  • FIGURE 12 SDS-PAGE of in YittQ translated Al.l TCR ⁇ and ⁇ polypeptides.
  • FIGURE 13A and B The regulatory activity of Al.l TCR ⁇ gene product translated in vitro is bound by anti-TCR ⁇ and not anti-TCR ⁇ .
  • FIGURE 14 The complete nucleotide sequence and deduced amino acid sequence of Al.l TCR ⁇ cDNA is shown (SEQ ID NOS: 25 and 26).
  • FIGURE 15 The complete nucleotide and deduced amino acid sequence of 3B3-derived TCR ⁇ cDNA is shown (SEQ ID NOS: 27 and 28).
  • FIGURE 16 The expression plasmid pST811 which carries a trp promoter and a trpA terminator is shown.
  • FIGURE 17 The expression plasmid pST811-A1.1 TCR ⁇ S5 is shown.
  • FIGURE 18 The expression plasmid pTCAL7 which carries rat calmodulin cDNA and a trp promoter is shown.
  • FIGURE 19 The expression plasmid pCFl which carries rat calmodulin and a trp promoter and contains additional cloning sites from pTCAL7 is shown.
  • FIGURE 20 The expression plasmid pCFl-3B3TCR ⁇ is shown.
  • FIGURE 21 SDS-PAGE of E. coli produced calmodulin-TCR ⁇ from two expression plasmids is shown.
  • FIGURE 22 SDS-PAGE of E coli expressed Al.l TCR ⁇ S5 protein.
  • FIGURE 23 SDS-PAGE of E. coli expressed 3B3TCR ⁇ (calmodulin-TCR ⁇ fusion protein).
  • FIGURE 24a The immunosuppressive activity of recombinant Al.l TCR ⁇ S5 was dose dependent.
  • FIGURE 24b The immunosuppressive activity of recombinant Al.l TCR ⁇ S3 was dose dependent.
  • FIGURE 25 Immunosuppressive activity of the TCR ⁇ chain was observed when poly- 18 or EYKEYAEYAEYAEYA (SEQ ID NO: 2) was used.
  • the present invention involves the use of antigen-binding ⁇ chains of the T cell antigen receptors in the regulation of antigen-specific immune responses.
  • a TCR ⁇ chain is evaluated for its ability to bind antigen and to modulate the immune response specific for that antigen.
  • In vitro assays are described herein which can be used for this purpose.
  • TCR ⁇ chains which demonstrate appropriate activity can be produced in quantity, for example, using recombinant DNA and/or chemical synthetic methods and may be used to down- regulate or up-regulate the immune response to a specific antigen. For example, hypersensitivity reactions, autoimmune responses and graft rejection responses may be suppressed using TCR ⁇ chains which are specific for the corresponding antigens, and which induce antigen-specific suppression.
  • immunity to an antigen may be augmented by the removal of such ⁇ chains, or by inhibiting production of such ⁇ chains in a subject to specifically enhance the immune response to a particular antigen.
  • TCR ⁇ chains that augment the immune response to an antigen may be identified and utilized.
  • the invention is based, in part, on the discovery of a secreted form of TCR ⁇ chain which directly binds to antigen and suppresses the immune response generated against that antigen.
  • a CD4 + helper T cell hybridoma, Al.l is described, specific for a synthetic polypeptide antigen, poly 18, plus I-A d which contitutively releases a secreted form of its TCR ⁇ chain that binds to antigen, and in the presence of appropriate accessory component, inhibits the immune response to the antigen.
  • the present invention relates to TCR ⁇ chains (not the complete T cell surface antigen receptor of ⁇ and ⁇ ) possessing both antigen-binding and immunoregulatory activities.
  • An antigen- binding TCR ⁇ protein with antigen-specific regulatory activity may be produced in a variety of ways. For example, expression of TCR ⁇ chain protein may be achieved by recombinant DNA technology and/or chemical synthetic techniques based on known amino acid sequences. Alternatively, the TCR ⁇ chain may be purified directly from culture supematants of continuous T cell lines that release this activity.
  • TCR ALPHA CHAINS Regardless of the method used to produce such TCR ⁇ chains, the antigen binding capability and immunoregulatory activity of the molecule should be evaluated. For example, the ability of the TCR ⁇ chain to directly bind to an antigen of interest may be evaluated by modified immunoassay techniques including, but not limited to ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, Western blots, or radioimmunoassays in which the TCR ⁇ chain is substituted for the antibody normally used in these assay systems.
  • modified immunoassay techniques including, but not limited to ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, Western blots, or radioimmunoassays in which the TCR ⁇ chain is substituted for the antibody normally used in these assay systems.
  • the immunoregulatory capability of the antigen-binding TCR ⁇ chain may be evaluated using any assay system which allows the detection of an immune response in an antigen-specific fashion.
  • the PFC assay as described and exemplified herein may be utilized to identify TCR ⁇ chains that suppress immune responses directed toward a particular antigen.
  • a highly immunogenic carrier such as sheep red blood cells
  • SRBC SRBC
  • PFC plaque forming cells
  • the number of PFC generated per culture is assessed by mixing the cultured spleen cells with SRBC (or appropriate lysable carrier) and complement, and culturing the mixture as a monolayer. Cells surrounded by a clear plaque (e.g.. of lysed red cells) are counted as PFCs. Inhibition of PFC generation in the spleen cell culture, Lg., a reduction in the number of PFC/culture, indicates suppression of the immune response.
  • the PFC assay may be conducted as follows: the antigen of interest is coupled to SRBC (Ag-SRBC) and added to spleen cells from unimmunized mice.
  • the immunoregulatory effect of a TCR ⁇ chain specific for the antigen is assessed by adding the TCR ⁇ chain to be tested to the culture in the presence of an accessory component described below (i.e.. the accessory component should be added to the culture prior to or simultaneously with the TCR ⁇ chain to be tested).
  • Control cultures receive the TCR ⁇ chain in the absence of accessory component or vice versa, or may involve the use of an irrelevant antigen.
  • the number of PFC/culture is assessed for each condition. An inhibition of PFC generation in the test cultures, as compared to that observed in the controls, indicates that the TCR ⁇ chain tested suppresses, in an antigen-specific manner, the immune response which is normally generated in the culture system.
  • the accessory component used in the test system comprises the supernatant of stimulated T cells depleted of any soluble factors, including TCR ⁇ chains, that directly bind to the antigen which was used to stimulate the T cells, so that the accessory component in and of itself does not suppress immune responses.
  • the accessory component is produced from T cells stimulated in vivo with the carrier/indicator used in the antigen-specific PFC assay. For example, the following procedure may be used to prepare accessory component for use in the PFC assay system described above in which Ag-SRBC is the target.
  • Spleen cells derived from SRBC- immunized mice are depleted of B cells, and the enriched T cells are cultured or used to generate T cell hybridomas that can be used as a reproducible, continuous supply of culture supernatant.
  • the supematants of the T cell cultures are tested for their ability to inhibit an anti-SRBC immune response using a PFC assay in which SRBC are added to cultured spleen cells in the presence or absence of the T cell supematants.
  • the T cell culture supematants which are found to inhibit the anti-SRBC response are then adsorbed with SRBC to remove any soluble factors, such as soluble TCR ⁇ chains, which bind directly to the SRBC.
  • T cell hybridomas may be generated that produce the accessory component without the need for an adso ⁇ tion step; for example, hybridoma 3-1-V described in Section 8, infra (See FIG. 1) constitutively produces the accessory component in culture supematants.
  • the accessory component contains one or more factors that, while not inhibitory on its own, allows an antigen-specific factor, L£., the TCR ⁇ chain, to suppress an immune response in an antigen-specific fashion.
  • L£. the antigen-specific factor
  • An example of such an assay system is set forth in Section 6.1.5 infra.
  • TCR ⁇ chains which augment an immune response may be identified in a similar way.
  • an accessory component prepared from T cell hybridomas, or from T cell culture supematants that demonstrate increased PFC generation prior to adso ⁇ tion and no activity after adso ⁇ tion could be used in PFC assays designed to identify TCR ⁇ chains that augment the immune response against the Ag-SRBC in an antigen-specific manner.
  • the foregoing assay systems utilize unprimed spleen cell cultures to assess immune responses, and carrier-primed spleen cell cultures to prepare accessory component, they may be limited to the use of animal-derived sources for the cultured spleen cells (e.g.. mice, rats, rabbits, and non-human primates). However, this does not preclude their use for testing the immuno ⁇ regulatory activity of human TCR ⁇ chains. Indeed a number of human immune functions can be tested in animal-based assay systems; £,£., human antibody effector functions, such as complement mediated lysis and antibody dependent cellular cytotoxicity can be demonstrated using animal serum and animal effector cells, respectively.
  • animal-derived sources for the cultured spleen cells e.g. mice, rats, rabbits, and non-human primates.
  • human immune functions can be tested in animal-based assay systems; £,£., human antibody effector functions, such as complement mediated lysis and antibody dependent cellular cytotoxicity can be demonstrated using animal serum and animal effector cells, respectively.
  • the PFC assay described above may be modified using human cell cultures in place of the animal spleen cell cultures.
  • the effect of a TCR ⁇ chain on the immune response to a particular antigen can be evaluated using the reverse hemolytic PFC assay described by Thomas et al.,
  • Pokeweed mitogen stimulated T cell supematants may be used as a source of accessory component in this assay system.
  • T CELLS Antigen-specific T cells which can serve as the source of the TCR ⁇ chains and/or the source of genetic material used to produce the TCR ⁇ chains used in the methods of the invention may be generated and selected by a number of in vitro techniques that are well-known in the art.
  • a source of T cells may be peripheral blood, lymph nodes, spleens, and other lymphoid organs as well as tissue sites into which T cells have infiltrated such as tumor nodules.
  • the T cell fraction may be separated from other cell types by density gradient centrifugation or cell sorting methods using antibodies to T cell surface markers such as CD2, CD3, CD4, CD8, etc.
  • T cell subsets of interest may apply the above-mentioned techniques using antibodies to more specific markers such as anti-CD4 and anti-CD8 in selecting for helper and cytotoxic/suppressor T cells, respectively or to markers expressed on T cell subsets such as memory cells.
  • Antigen-specific T cell lines may be generated in vitro by repetitive stimulation with optimal concentrations of specific antigens in the presence of appropriate irradiated antigen-presenting cells and cytokines.
  • Antigen-presenting cells should be obtained from autologous or MHC- matched sources and they may be macrophages, dendritic cells, Langerhans cells, EBV- transformed B cells or unseparated peripheral blood mononuclear cells.
  • Cytokines may include various interleukins such as interleukin 1, 2, 4, and 6 in natural or recombinant forms. For one such technique, see, for example, Takata et al., 1990, J. Immunol. 145: 2846-2853.
  • Clonal populations of antigen-specific T cells may be derived by T cell cloning using limiting dilution cloning methods in the presence of irradiated feeder cells, antigen and cytokines.
  • T cell hybridomas may be generated by fusion of the antigen-specific T cells with fusion partner tumor lines such as BW5147 or BW1100 followed by HAT selection and recloning.
  • Antigen-specific T cells have also been cloned and propagated by the use of monoclonal antibodies to CD3.
  • T cell clones and T cell hybridomas can be generated using cells obtained directly from in vivo sources followed by testing and selection for antigen-specificity or antigen-specific T cell lines can be secured prior to the cloning and fusion events.
  • T cell clones can be maintained long-term in culture by repetitive stimulation with antigen or anti-CD3 every 7-14 days followed by expansion with cytokines while T cell hybridomas can be grown in the appropriate culture media without periodic antigen stimulation.
  • the antigen-specificity of monoclonal T cell populations can be assessed in i vitro assays measuring the proliferation and/or lymphokine production of these cells in response to antigen. Phenotype of the T cells may be confirmed by staining with antibodies to various T cell markers.
  • Such antigen-specific T cells may secrete TCR ⁇ chains constitutively or they may require activation signals for the release of their ⁇ chains.
  • the antigen-specific T cells may be used as the source of genetic material required to produce the TCR ⁇ chain by recombinant DNA and/or chemical synthetic techniques. Using this approach, certain antigen-specific T cells which may not secrete naturally-occurring TCR ⁇ chains can serve as a source of genetic material for the TCR ⁇ chain to be used in accordance with the invention.
  • Messenger RNA (mRNA) for the preparation of cDNA may be obtained from cell sources that produce the desired ⁇ chain, whereas genomic sequences for TCR ⁇ may be obtained from any cell source. Any of the T cells generated as described in Section 5.1.2. supra, may be utilized either as the source of the coding sequences for the TCR ⁇ chain, and/or to prepare cDNA or genomic libraries. Additionally, parts of lymphoid organs (££., spleens, lymph nodes, thymus glands, and peripheral blood lymphocytes) may be ground and used as the source for extracting DNA or RNA. Alternatively, T cell lines can be used as a convenient source of DNA or RNA. Genetically engineered microorganisms or cell lines containing TCR ⁇ coding sequences may be used as a convenient source of DNA for this pu ⁇ ose.
  • Either cDNA or genomic libraries may be prepared from the DNA fragments generated using techniques well known in the art.
  • the fragments which encode TCR ⁇ may be identified by screening such libraries with a nucleotide probe homologous to a portion of the TCR ⁇ sequence.
  • the TCR ⁇ gene or mRNA transcript which can be used to synthesize TCR ⁇ cDNA or to identify appropriate TCR ⁇ sequences in cDNA libraries prepared from such T cells or genomic clones.
  • oligonucleotides specific for the variable region of the desired TCR ⁇ chain could be constructed, but these would have to be designed on a case by case basis, depending on the sequence of the variable region. Oligonucleotide probes designed based on the constant region offer an advantage in this regard, since they can be used to "fish out" any TCR ⁇ chain gene or coding sequence.
  • oligonucleotide probes derived from specific TCR ⁇ sequences could be used as primers in PCR (polymerase chain reactions) methodologies to generate cDNA or genomic copies of TCR ⁇ sequences which can be directly cloned.
  • PCR polymerase chain reactions
  • expression cloning methods may be utilized to substantially reduce the screening effort. Recently, a one step procedure for cloning and expressing antibody genes has been reported (McCafferty et al., 1990,
  • TCR ⁇ chain genes may likewise be cloned directly into a vector at a site adjacent to the coat protein gene of a bacteriophage such as ⁇ or fd.
  • the phage carrying a TCR ⁇ gene expresses the fusion protein on its surface so that columns containing the antigen or a TCR ⁇ -specific antibody can be used to select and isolate phage particles with binding activity.
  • Transient gene expression systems may also be utilized to identify the correct TCR ⁇ gene. For example, the COS cell system (s&, Gerard & Gluzman, 1986, Mol. Cell.
  • Biol.6(12) 4570-4577 may be used; however, the expression of the TCR ⁇ chain should be detected in extracts of COS cells which had been co- transfected with the CD3 ⁇ chain gene (Bonifacino, et al., 1990, Cell 63: 503-513).
  • nucleotide coding sequences which encode analogous amino acid sequences for any known antigen-specific TCR ⁇ chain gene may be used in the practice of the present invention for the cloning and expression of TCR ⁇ .
  • Such alterations include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product.
  • the gene product may contain deletions, additions or substitutions of amino acid residues within the sequence, which result in a silent change thus producing a bioactive product.
  • Such amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues involved.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.
  • the TCR ⁇ chain sequence may be modified to obtain a gene product having improved properties for use in vivo, such as improved stability and half-life.
  • a hybrid gene can be constructed by ligating the TCR ⁇ chain gene, or its variable region, to the constant region of a human immunoglobulin gene such as IgG.
  • a technique which can be applied see Capon et al., 1989, Nature 337: 525-531.
  • the nucleotide sequence coding for TCR ⁇ is inserted into an appropriate expression vector, Lg., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Modified versions of the TCR ⁇ coding sequence could be engineered to enhance stability, production, purification or yield of the expressed product.
  • the expression of a fusion protein or a cleavable fusion protein comprising TCR ⁇ and a heterologous protein may be engineered.
  • Such a fusion protein may be readily isolated by affinity chromatography; ⁇ JJ. by immobilization on a column specific for the heterologous protein.
  • the TCR ⁇ chain can be released from the chromatographic column by treatment with an appropriate enzyme or agent that disrapts the cleavage site (e.g.. see Booth et al., 1988, Immunol. Lett. 19:65-70; and Gardella et al., 1990, J. Biol. Chem. 265:15854-15859).
  • an appropriate enzyme or agent that disrapts the cleavage site e.g. see Booth et al., 1988, Immunol. Lett. 19:65-70; and Gardella et al., 1990, J. Biol. Chem. 265:15854-15859.
  • a variety of host-expression vector systems may be utilized to express the TCR ⁇ coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing a TCR ⁇ coding sequence; yeast transformed with recombinant yeast expression vectors containing the TCR ⁇ coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g.. Ti plasmid) containing a TCR ⁇ coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g...).
  • microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing a TCR ⁇ coding sequence; yeast transformed with recombinant yeast expression vectors
  • baculovirus containing a TCR ⁇ coding sequence
  • animal cell systems infected with recombinant virus expression vectors e ⁇ g., retroviruses, adenovirus, vaccinia virus
  • bacterial expression systems may be advantageously utilized for high yield TCR ⁇ production.
  • glycosylation may be important for in vivo applications, even though it is not required for immunoregulatory activity; e.g.. the glycosylated product may demonstrate an increased half-life in vivo.
  • expression systems that provide for translational and post-translational modifications may be used; £,£., mammalian, insect, yeast or plant expression systems.
  • any of a number of suitable transcription and translation elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g.. Bitter et al., 1987, Methods in Enzymology 153:516-544).
  • inducible promoters such as pL of bacteriophage ⁇ , plac, pt ⁇ , ptac (pt ⁇ -lac hybrid promoter) and the like may be used.
  • promoters derived from the genome of mammalian cells e.g..
  • metallothionein promoter or from mammalian viruses (e.g.. the retrovirus long terminal repeat; the adenoviras late promoter; the vaccinia virus 7.5K promoter) may be used.
  • mammalian viruses e.g. the retrovirus long terminal repeat; the adenoviras late promoter; the vaccinia virus 7.5K promoter
  • Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the inserted TCR ⁇ coding sequence.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the TCR ⁇ expressed. For example, when large quantities of TCR ⁇ are to be produced, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Those which are engineered to contain a cleavage site to aid in recovering TCR ⁇ are preferred.
  • Such vectors include but are not limited to the £ coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the TCR ⁇ coding sequence may be ligated into the vector in frame with the lac Z coding region so that a hybrid
  • TCR ⁇ -lac Z protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like.
  • yeast a number of vectors containing constitutive or inducible promoters may be used.
  • Current Protocols in Molecular Biology Vol. 2, 1988, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et al., 1987, Expression and Secretion Vectors for Yeast, m Methods in Enzymology, Eds.
  • vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome.
  • the expression of a TCR ⁇ coding sequence may be driven by any of a number of promoters.
  • viral promoters such as the 35 S RNA and 19S RNA promoters ofCaMV (Brisson etal., 1984, Nature 310:511-514), or the coat protein promoter to TMV (Takamatsu et al., 1987, EMBO J. 3:1311) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J.
  • TCR ⁇ is an insect system.
  • Autographa califomica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the TCR ⁇ coding sequence may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • TCR ⁇ coding sequence Successful insertion of the TCR ⁇ coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (Lg., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (Tig., see Smith et al., 1983, J. Viol. 46:584; Smith, U.S. Patent No. 4,215,051).
  • Eukaryotic systems and preferably mammalian expression systems, allow for proper post- translational modifications of expressed mammalian proteins to occur.
  • Eukaryotic cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, phosphorylation, and, advantageously secretion of the gene product should be used as host cells for the expression of TCR ⁇ .
  • Mammalian cell lines are preferred. Such host cell lines may include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, -293, and WI38.
  • T cell hosts including but not limited to T cell tumor cell lines, T cell hybridomas, T cells which produce accessory component, or T cells which produce immunoregulatory factors may be utilized.
  • Mammalian cell systems which utilize recombinant viruses or viral elements to direct expression may be engineered.
  • the TCR ⁇ coding sequence may be ligated to an adenoviras transcription/translation control complex, e.g.. the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenoviras genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g.. region El or E3) will result in a recombinant viras that is viable and capable of expressing the TCR ⁇ chain in infected hosts (e.g...
  • the vaccinia virus 7.5K promoter may be used, (g.g., see, Mackett et al., 1982, Proc. Natl. Acad. Sci. USA 79: 7415-7419; Mackett et al.,
  • vectors based on bovine papilloma viras which have the ability to replicate as extrachromosomal elements (Sarver, et al., 1981, Mol. Cell. Biol. 1: 486). Shortly after entry of this DNA into mouse cells, the plasmid replicates to about 100 to 200 copies per cell. Transcription of the inserted cDNA does not require integration of the plasmid into the host's chromosome, thereby yielding a high level of expression.
  • These vectors can be used for stable expression by including a selectable marker in the plasmid, such as the n__2 gene.
  • the retroviral genome can be modified for use as a vector capable of introducing and directing the expression of the TCR ⁇ chain gene in host cells (Cone & Mulligan, 1984, Proc. Natl. Acad. Sci. USA 81:6349-6353). High level expression may also be achieved using inducible promoters, including, but not limited to, the metallothionine IIA promoter and heat shock promoters.
  • TCR ⁇ cDNA controlled by appropriate expression control elements (e.g.. promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g. promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • a number of selection systems may be used, including but not limited to the he ⁇ es simplex viras thymidine kinase (Wigler, et al.,
  • t ⁇ B which allows cells to utilize indole in place of tryptophan
  • hisD which allows cells to utilize histinol in place of histidine
  • ODC ornithine decarboxylase
  • TCR ⁇ protein product by genetically-engineered cells can be assessed immunologically, for example by Western blots, immunoassays such as radioimmuno- precipitation, enzyme-linked immunoassays and the like.
  • the ultimate test of the success of the expression systerr.. involves the production of biologically active TCR ⁇ gene product.
  • the host cell secretes the gene product
  • the cell free media obtained from the cultured transfectant host cell may be assayed for TCR ⁇ or its immunoregulatory activity.
  • cell lysates may be assayed for such activity.
  • a number of assays can be used to assess TCR ⁇ activity, including but not limited to assays measuring the ability of the expressed TCR ⁇ to bind antigen, and assays to evaluate its immunologic function, such as the PFC assays described in Section 5.1.1. supra and exemplified in Section 6.1.5, infm-
  • the clone may be expanded and used to produce large amounts of the protein which may be purified using techniques well-known in the art including, but not limited to immunoaffinity purification, chromatographic methods including high performance liquid chromatography and the like. Where the protein is secreted by the cultured cells, TCR ⁇ may be readily recovered from the culture medium.
  • TCR ⁇ from crude culture media of T cells may be adapted for purification of the cloned, expressed product.
  • TCR ⁇ from Al.l cells used in the examples, infra
  • TCR ⁇ from Al.l cells can be purified from the crude culture media of T cells by ammonium sulfate precipitation followed by affinity chromatography (Zheng et al., 1988, J. Immunol. 140:1351-1358; Bissonnette et al., 1991, J. Immunol. 146:2898-2907).
  • Purified monoclonal antibodies specific for a commonly shared determinant on all murine TCR ⁇ chains or an antigen or a fragment containing a specific antigenic epitope thereof can be coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia) and used for affinity chromatography.
  • the biological activity of the protein purified in this manner from crude culture media has been shown to be enriched 3, 000- fold.
  • antibodies made to products of different V ⁇ gene families may also be used if it is known which specific V ⁇ gene segment encodes the ⁇ chain protein in question.
  • antibodies may be raised to the variable region of a specific TCR ⁇ chain and used in the purification of the ⁇ chain from a mixture of other irrelevant TCR ⁇ chains. In this case, a specific ⁇ chain may be isolated even from the crude media of bulk culture T cells if sufficient quantity of the protein is present.
  • TCR ⁇ coding sequence is engineered to encode a cleavable fusion protein
  • the purification of TCR ⁇ may be readily accomplished using affinity purification techniques.
  • a protease factor Xa cleavage recognition sequence can be engineered between the carboxyl terminus of TCR ⁇ and a maltose binding protein.
  • the resulting fusion protein can be readily purified using a column conjugated with amylose to which the maltose binding protein binds.
  • the TCR ⁇ fusion protein is then eluted from the column with maltose containing buffer followed by treatment with Factor Xa.
  • the cleaved TCR ⁇ chain is further purified by passage through a second amylose column to remove the maltose binding protein (New England Biolabs,
  • any cleavage site or enzyme cleavage substrate may be engineered between the TCR ⁇ sequence and a second peptide or protein that has a binding partner which could be used for purification, e.g.. any antigen for which an immunoaffinity column can be prepared.
  • TCR ⁇ chain gene Once a specific TCR ⁇ chain gene has been molecularly cloned and its DNA sequence determined, its protein product may be produced by a number of methods in addition to those described supra. For example, solid phas chemical synthetic techniques can be used to produce a TCR ⁇ chain in whole or in part based on an amino acid sequence deduced from the DNA sequence (see Creighton, 1983, Proteins Structures and Molecular Principles, W.H. Freeman and Co., N.Y. pp. 50-60). This approach is particularly useful in generating small portions of proteins that correspond to the active site of a molecule.
  • variable region in the amino-terminal end of the protein encoded by the V and J gene segments is important to antigen-binding. Therefore, synthetic peptides cc sponding to the variable region of the ⁇ chain may be produced.
  • a larger peptide containing a specific portion of an ⁇ chain constant region may also be synthesized if, for example, that region is known to be important for its interaction with accessory factors in achieving a full immunoregulatory response.
  • TCR ⁇ chain based on its cloned DNA sequence is by transcription and translation of its gene in an in vitro cell free system.
  • the A 1.1 TCR ⁇ chain gene is in vitro transcribed and translated and its product is shown to be a protein of about 32,000 dalton molecular weight by SDS-PAGE. This protein corresponds to an unglycosylated TCR ⁇ polypeptide chain.
  • the advantage of this approach is to provide a method for definitively demonstrating the contribution of a specific TCR ⁇ chain in a specific immunological reaction in the absence of the synthesis of other proteins.
  • Molecular mimicry of protein conformation by antibody combining sites provides another method for producing a protein with binding specificity similar to that of a specific TCR ⁇ chain.
  • anti-idiotypic antibodies or monoclonal antibodies directed to the same antigenic determinant as the specific TCR ⁇ chain may possess a binding site that is structurally identical to the TCR ⁇ chain variable domain.
  • Such antibodies which demonstrate a TCR ⁇ chain immunoregulatory function as evaluated in the PFC assays described herein may be used in place of TCR ⁇ chain. The use of such antibodies may be preferred under certain circumstances, for example, where it can be shown that the antibody has a longer in vivo half life than a native ⁇ chain.
  • monoclonal antibodies which bind to and neutralize the TCR ⁇ chain may be desired.
  • These antibodies may also be used in diagnostic assays in vitro, e.g.. radioimmunoassays, ELISAs, to detect circulating TCR ⁇ chains in humans.
  • Such monoclonal antibodies can be readily produced in large quantities using techniques well known in the art.
  • various host animals including but not limited to mice, rabbits, hamsters, rats, and non-human primates, may be immunized with the desired antigen or an anti-TCR ⁇ chain antibody in order to generate antibodies that mimic the desired antigen or an anti-TCR ⁇ chain antibody in order to generate antibodies that mimic the desired antigen or an anti-TCR ⁇ chain antibody in order to generate antibodies that mimic the desired antigen or an anti-TCR ⁇ chain antibody in order to generate antibodies that mimic the
  • TCR ⁇ chain as measured by their ability to competitively inhibit the antigen-specific binding of the TCR ⁇ chain to its antigen, and their ability to regulate an immune response specific for the antigen as evaluated in the PFC assays described herein.
  • the host animal would be immunized with the TCR ⁇ chain itself.
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corvnebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • Monoclonal antibodies may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein, (Nature, 1975, 256:495- 497), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today, 4:72; Cote et al., 1983, Proc. Natl. Acad. Sci. 80:2026-2030) and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • Antibody fragments which contain the specific desired binding sites may be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • techniques described for the construction of Fab expression libraries Huse et al., 1989, Science, 246: 1275-1281 to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity can be used.
  • TCR ⁇ chain The antigen-specific immunoregulatory activity of a TCR ⁇ chain provides for a wide variety of uses in vivo in human or animal subjects and in vitro. Any TCR ⁇ chain, or fragments and derivatives thereof, which are capable of binding to the antigen and which exhibit immunoregulatory activities as assayed in vitro may be used in the practice of the method of the invention. Where the factors of the accessory component are present in a subject's serum, the
  • TCR ⁇ chain may be administered as the sole active agent. However, the TCR ⁇ chain could be administered in conjunction with biologically active factors found in the accessory component, growth factors, or inhibitors.
  • the TCR ⁇ chains which are capable of binding to the antigen and which suppress the immune response that would normally be generated against the antigen may be especially useful in the down-regulation of antigen-specific immune responses such as hypersensitivity reactions, transplantation rejections, and autoimmune disorders. Alternatively, the removal or neutralization of such TCR ⁇ chains, or the factors which associate with such
  • TCR ⁇ chains may be useful as a means of augmentating an immune response against diseases such as cancer and immunodeficiency.
  • TCR ⁇ chains specific for the antigen which augment the immune response could be utilized to augment such antigen-specific responses in vivo.
  • the antigen-binding TCR ⁇ chains that demonstrate antigen-specific immunosuppression may be used in the treatment of conditions in which immune reactions are deleterious and suppression of such responses in an antigen-specific manner is desirable.
  • disorders which may be treated in accordance with the invention include but are not limited to hypersensitivity (types I- IV), autoimmune disease as well as graft rejection responses after organ and tissue transplantations.
  • Hypersensitivity reactions are commonly classified into four groups. Type I reactions are immediate-type hypersensitivity which result from mast cell degranulation triggered by antigen- specific IgE. Examples of type I diseases include most common allergies caused by substances such as plant pollens, mold spores, insect parts, animal danders, bee and snake venom, industrial dusts, house dusts, food products, chemicals and drags.
  • Type II reactions are caused by the action of specific antibodies, usually IgG and IgM, on target cells leading to cellular destruction.
  • Type II diseases include transfusion reactions, erythroblastosis fetalis, autoimmune hemolytic anemia, myasthenia gravis and Grave's disease.
  • Type III reactions are caused by antigen-antibody complex formations and the subsequent activation of antibody effector mechanisms.
  • type III diseases include immune complex glomerulonephritis, Goodpasture's syndrome and certain forms of arthritis.
  • Type IV reactions are cell-mediated reactions involving T cells, macrophages, fibroblasts and other cell types. These are also referred to as delayed-type hypersensitivity. Allergic contact dermatitis is a typical example of this category.
  • Autoimmune disorders refer to a group of diseases that are caused by reactions of the immune system to self antigens leading to tissue destruction. These responses may be mediated by antibodies, auto-reactive T cells or both. Many of these conditions overlap with those described under hypersensitivity above. Some important autoimmune diseases include diabetes, autoimmune thyroiditis, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosis, and myasthenia gravis.
  • organs and tissues such as kidneys, hearts, livers, skin, pancreatic islets and bone marrow.
  • graft rejections can still occur.
  • an antigen-specific TCR ⁇ may be used to specifically suppress an immune response mediated by T cells, antibodies or both while retaining all other normal immune functions.
  • EAE experimental allergic encephalomyelitis
  • T H have been shown to play a critical role in the pathogenesis of the disease (Wraith et al., 1989, Cell 57:709-715). The number of antigenic determinants recognized by auto-reactive
  • T cells in a given mouse strain are limited. Furthermore, the V ⁇ and V ⁇ gene segments used for the construction of autoimmune TCR is equally restricted so that the majority of the T cell response to the small number of encephalitogenic epitopes has an identical TCR. Antibodies to TCR determinants have been successfully used.to deplete antigen-specific T cells in vivo leading to protection from disease. (Owhashi and Heber-Katz, 1988, J. Exp. Med. 168:2153-2164).
  • a TCR ⁇ chain gene may be isolated from such auto-reactive T cells, expressed in an appropriate host cell and tested for its ability to suppress the antigen- specific immune responses in vitro and in vivo.
  • TCR ⁇ chains for this pu ⁇ ose is particularly important in light of the recent findings that in certain human diseases such as multiple sclerosis and myasthenia gravis, autoimmune T cells have been detected and they appear to similarly have a restricted usage of certain V ⁇ and V ⁇ alleles (Oksenberg et al., 1989, Proc. Natl. Acad. Sci. USA 86:988-992).
  • the foregoing conditions may be treated by administering to the patient an effective dose of TCR ⁇ chain specific for the relevant antigen which suppresses the immune response generated against that antigen.
  • the TCR ⁇ chains selected for use may be evaluated by an immunoregulatory assay in vitro, such as the PFC assays described herein.
  • the TCR ⁇ chain may be administered in a variety of ways, including but not limited to injection, infusion, parenterally, and orally.
  • TCR ⁇ and its related derivatives, analogs e.g.. peptides derived from the variable region, may be used as the sole active agent, or with other compounds.
  • Such compositions may be administered with a physiologically acceptable carrier, including phosphate buffered saline, saline and sterilized water.
  • liposomes may be used to deliver the TCR ⁇ .
  • the liposome may be conjugated to antibodies that recognize and bind to cell specific antigens, thereby providing a means for "targeting" the TCR ⁇ compositions.
  • An effective dose is the amount required to suppress the immune response which would have been generated against the relevant antigen in vivo.
  • the amount of TCR ⁇ employed will vary with the manner of administration, the use of other active compounds, and the like. Generally a dose which will result in circulating serum levels of 0.1 ⁇ g to 100 ⁇ g/ml may be utilized.
  • the most effective concentration for suppressing antigen-specific responses may be determined in vitro by adding various concentrations of TCR ⁇ to an in vitro assay such as the PFC assays described in Section 6.1.5., infra, and monitoring the level of inhibition achieved.
  • Certain diseases are the result of deficient or defective immune responses.
  • An impaired immune response may be due to the absence or aberrant function of certain compartments of the immune system, or the presence of factors that specifically down-regulate these responses.
  • a patient's ove ⁇ roduction of an antigen-specific TCR ⁇ which suppresses the immune response directed toward a particular antigen may be involved.
  • the systemic removal or neutralization of TCR ⁇ may be able to rescue the responding cells from such suppression and thereby enhance their efficacy against the antigens they recognize.
  • the PFC assay described herein can be utilized to assay the patient's body fluids, such as serum for the presence of circulating or soluble TCR ⁇ chains that exert an antigen-specific immunosuppressive effect.
  • Such patients may then be treated using antibodies for the TCR ⁇ chain to remove or neutralize the circulating suppressive molecules.
  • T cell-mediated suppression can explain the continual growth of a tumor in the face of a demonstrable tumor-specific immune response.
  • Ts and TsF have been reported to uncover the underlying anti- tumor responses (North, 1982, J. Exp. Med. 55:106-107; Hellstrom et al., 1978, J. Exp. Med.49- 799-804; Nepom et al., 1977, Biochim Biophy. Acta; 121-148).
  • Antigen-specific suppressor factors may be released directly by tumor cells or by Ts which are activated upon recognizing certain suppressogenic epitopes of tumor antigens (Sercarz and Krzych, 1991, Immunol. Today
  • a monoclonal antibody originally raised to a TsF may also react with a tumor- specific T suppressor factor produced by a T cell hybridoma (Steele et al., 1985, J. Immunol. 134: 2767-2778).
  • TsF tumor-specific T suppressor factor produced by a T cell hybridoma
  • TCR ⁇ chains therefore, with appropriate specificity for tumor antigens may participate in the dampening of tumor immunity, in which case, the removal or neutralization of such TCR ⁇ will likely be advantageous to the restoration and augmentation of tumor-specific responses.
  • TCR ⁇ chains in a patient may be inhibited by the administration in vivo of antibodies specific for the ⁇ chain (see Section 5.1.4, supra, which neutralize its activity, i.e.. either its ability to bind antigen and/or its resulting antigen-specific immunosuppressive effect. While antibodies to the constant or variable region of TCR ⁇ may be used, those which bind the variable region may be preferred since only the TCR ⁇ chains specific for that particular antigen will be neutralized so that the immune response is augmented in an antigen-specific fashion.
  • sera of cancer patients with detectable levels of soluble tumor antigen- specific TCR ⁇ chain may be adsorbed by gx vivo passage through columns containing an antibody to ⁇ chain or the antigen or a peptide thereof; g., plasmaphoresis.
  • an antibody may be administered in a variety of ways, including but not limited to injection, infusion, parenterally and orally.
  • the antibody may be administered in any physiologically acceptable carrier, including phosphate buffered saline, saline and sterilized water.
  • the amount employed of the subject antibody will vary with the manner of administration, the employment of other active compounds, and the like, generally being in a saturating dose which will result in the binding of most if not all of the free systemic TCR ⁇ chains.
  • the amount of antibody or antigen coupled to the column will be that which is sufficent for removing most if not all free TCR ⁇ chain in patients' sera.
  • Antisense oligonucleotides may be used to interfere with the expression and systemic release of a specific immunosuppressive TCR ⁇ chain, and thereby selectively enhance an antigen-specific immune response.
  • complementary oligonucleotides which exhibit catalytic activity Lg., a ribozyme approach may be used. See generally, ⁇ g., PCT International
  • V ⁇ antisense or ribozyme oligonucleotides for each TCR ⁇ chain is preferred over the use of the complete TCR ⁇ since this will only inhibit a specific TCR ⁇ of interest.
  • nuclease resistant antisense V ⁇ oligodeoxynucleotides complementary to the mRNA of any known TCR ⁇ chain sequence may be synthesized. Following their uptake into the antigen-specific T cells, these agents can hybridize to their complementary mRNA sequences through base pairing, block translation and disrupt the production of the encoded protein products (for review of such techniques, see Green et al., 1986, Ann. Rev. Biochem. 55:569-597).
  • TCR ⁇ chains specific for the antigen of interest and which augment the antigen specific immune response may be identified as described in Section
  • Therapeutically effective doses of such TCR ⁇ chains may be administered to a patient to augment the patient's immune response against that particular antigen.
  • the invention provides a substantially pure fusion polypeptide R,-[X,]- R 2 , wherein R t is a carrier peptide, R 2 is a polypeptide encoded by a structural gene, and X, is a proteolytic enzyme recognition sequence.
  • the "carrier peptide” is located at the amino terminal end of the fusion peptide sequence.
  • the carrier peptide of the fusion polypeptide of the invention may function to transport the fusion peptide to inclusion bodies, the periplasm, the outer membrane or, preferably, the extemal environment.
  • the carrier peptide is believed to function to transport the fusion polypeptide across the endoplasmic reticulum.
  • Carrier peptides of the invention include, but are not limited to, the calmodulin polypeptide. Categories of carrier peptide which can be utilized according to the invention include pre-pro peptides and outer membrane peptides which may include a proteolytic enzyme recognition site. Acceptable carrier peptides also include the amino terminal pro-region of hormones. Other carrier peptides with similar properties described herein are known to those skilled in the art, or can be readily ascertained without undue experimentation.
  • a carrier peptide is included in an expression vector, which is specifically located adjacent to the N-terminal end of the carrier protein. While the vector used in the example of the present invention uses the calmodulin nucleotide sequence, other sequences which provide the means for transport of the fusion protein to the endoplasmic reticulum (for eukaryotes) and into the extemal environment or into inclusion bodies (for prokaryotes), will be equally effective in the invention. Such sequences as described above are known to those of skill in the art.
  • the carboxy-terminal end of the carrier peptide of the invention contains a proteolytic enzyme recognition site so that polypeptide encoded by the structural gene can be easily separated from the fusion polypeptide. Differences in the cleavage recognition site are possible in that different enzymes exist for the proteolytic specificity.
  • the cleavage site is the sequence, Lys- Val-Pro-Arg-Gly (SEQ ID NO: 1), which is recognized by thrombin. This recognition site allows for an unexpectedly high level of active protein encoded by the structural gene to be produced.
  • Other cleavage sites, such as that recognized by Factor Xa protease will be known to those of skill in the art.
  • the fusion polypeptide of the invention includes a polypeptide encoded by a structural gene, preferably at the carboxy terminus of the fusion polypetide. Any structural gene is expressed in conjunction with the carrier peptide and cleavage site. The structural gene is operably linked with the carrier and cleavage site in an expression vector so that the fusion polypeptide is expressed as a single unit.
  • An example of a stractural gene that can be used to produce a fusion polypeptide of the invention encodes the truncated form of TCR ⁇ , which includes only the extracellular membrane domain of TCR ⁇ .
  • the invention provides a substantially pure polypeptide.
  • substantially pure refers to a polypeptide which is substantially free of other proteins, lipids, carbohydrates or other materials with which it may be naturally associated.
  • One skilled in the art can purify the polypeptide using standard techniques for protein purification, such as affinity chromatography using a monoclonal antibody which binds an epitope of the polypeptide.
  • the substantially pure polypeptide will yield a single major band on a polyacrylamide gel.
  • the purity of the polypeptide can also be determined by amino-terminal amino acid sequence analysis.
  • the polypeptide includes functional fragments of the polypeptide, as long as the activity of the polypeptide remains. Smaller peptides containing the biological activity of polypeptide are included in the invention.
  • the invention also provides polynucleotides encoding the fusion polypeptide.
  • These polynucleotides include DNA, cDNA, and RNA sequences. It is understood that all polynucleotides encoding all or a portion of the fusion polypeptide are also included herein, as long as they encode a polypeptide of which the cleavage product has biological activity.
  • Such polynucleotides include naturally occurring, synthetic, and intentionally manipulated poly ⁇ nucleotides. For example, the polynucleotide may be subjected to site-directed mutagenesis.
  • the polynucleotide sequence also includes antisense sequences and sequences that are degenerate as a result of genetic code.
  • DNA sequences of the invention can be obtained by several methods as described above. For example, the DNA can be isolated using hybridization procedures which are well known in the art. These include, but are not limited to : 1) hybridization of probes to genomic or cDNA libraries to detect shared nucleotide sequences; 2) antibody screening of expression libraries to detect shared stractural features; and 3) synthesis by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • DNA sequences are frequently the method of choice when the entire sequence of amino acid residues of the desired polypeptide product is known.
  • the direct synthesis of DNA sequences is not possible and the method of choice is the synthesis of cDNA sequences.
  • the standard procedures for isolating cDNA sequences of interest is the formation of plasmid- or phage-carrying cDNA libraries which are derived from reverse transcription of mRNA which is abundant in donor cells that have a high level of genetic expression. When used in combination with polymerase chain reaction technology, even rare expression products can be cloned.
  • the production of labeled single or double-stranded DNA or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA/DNA hybridization procedures which are carried out on cloned copies of the cDNA which have been denatured into a single-stranded form (Jay etal, Nucl. Acid Res. 11:2325, 1983).
  • DNA sequences encoding the fusion polypeptide of the invention can be expressed in vitro by DNA transfer into a suitable host cell.
  • "Host cells” are cells in which a vector can be propagated and its DNA expressed. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell” is used.
  • Preferred host cells of the invention include E. coli, S. aureus and P. aeruginosa, although other Gram-negative and Gram-positive organisms known in the art can be utilized as long as the expression vectors contain an origin of replication to permit expression in the host.
  • the polynucleotide sequences may be inserted into a recombinant expression vector.
  • recombinant expression vector refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or inco ⁇ oration of the genetic sequences for TCR ⁇ , for example, and a carrier peptide and cleavage site.
  • Such expression vectors contain a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host.
  • the expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells.
  • Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg et al, Gene 56:125, 1987), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J. Biol. Chem. 261:3521 ,
  • the DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g., T7, metallothionein I, or polyhedrin promoters).
  • a promoter e.g., T7, metallothionein I, or polyhedrin promoters.
  • the expression of the fusion peptide of the invention can be placed under control of E. coli chromosomal DNA comprising a lactose or lac operon which mediates lactose utilization by elaborating the enzyme beta-galactosidase.
  • the lac control system can be induced by IPTG.
  • a plasmid can be constructed to contain the lac Iq repressor gene, permitting repression of the lac promoter until IPTG is added.
  • Other promoter systems known in the art include beta lactamase, lambda promoters, the protein A promoter, and the tryptophan promoter systems. While these are the most commonly, used, other microbial promoters can be utilized as well.
  • the vector contains a replicon site and control sequences which are derived from species compatible with the host cell.
  • the vector may carry specific gene(s) which are capable of providing phenotypic selection in transformed cells.
  • the beta-lactamase gene confers ampicillin resistance to those transformed cells containing the vector with the beta- lactamase gene.
  • Polynucleotide sequences encoding the fusion polypeptide of the invention can be expressed in either prokaryotes or eukaryotes.
  • Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art.
  • Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to inco ⁇ orate DNA sequences of the invention.
  • the host cell of the invention may naturally encode an enzyme which recognizes the cleavage site of the fusion protein.
  • the host cell in which expression of the fusion polypeptide is desired does not inherently possess an enzyme which recognizes the cleavage site, the genetic sequence encoding such enzyme can be cotransfected to the host cell along with the polynucleotide sequence for the fusion protein.
  • Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art.
  • the host is prokaryotic, such as E. coli
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl 2 method by procedures well known in the art.
  • CaCl 2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.
  • Eukaryotic cells can also be cotransfected with DNA sequences encoding the fusion polypeptide of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the he ⁇ es simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein.
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
  • SV40 simian virus 40
  • bovine papilloma virus bovine papilloma virus
  • Techniques for the isolation and purification of either microbially or eukaryotically expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies.
  • preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies.
  • C57B1/10 and C57B1/6 animals were purchased from Jackson Laboratories (Bar Harbor, ME).
  • CD3e Monoclonal antibodies with specificity for CD3e (145-2C11, hamster IgG) (Leo et al., 1987, Proc. Natl. Acad. Sci. USA £4:1374-1378), and TCR C ⁇ (H28-710.16, hamster IgG) (Becker et al., 1989, Cell 5_8:911-921) were purified by protein A affinity chromatography (Protein A Superose, Pharmacia). Fluorescent staining and FACS analysis of surface CD3 (Zheng et al., 1989, Proc. Natl. Acad. Sci. USA £6:3758-3762) and antibody-affinity chromatography with
  • H28-710 (Bissonette et al., 1991, J. Immunol. 146:28-98-2907) were performed as previously described.
  • SRBC were purchased from Morse Biologicals (Edmonton, AB) or from Colorado Serum Co. (Denver, CO).
  • the nonrandom synthetic polypeptide, poly- 18, and peptides based on its stracture (listed in FIG. 4) were generated as previously described (Fotedar et al., 1985, J. Immunol. H£:3028-3033).
  • Total cellular RNA was isolated from IO 9 cells by the conventional guanidium-isothiocyanate and cesium chloride method. Poly A + RNA was recovered by oligo-dT cellulose affinity chromatography. The first strand synthesis was generated using an oligo-dT primer and reverse transcriptase and the second strand using DNA polymerase I and RNase H. The methylated blunt ended double-stranded (ds) cDNA was ligated to EcoRI linkers. Subsequent to EcoRI digestion the dscDNA was size selected on agarose gels, purified by spermine precipitation, and cloned into ⁇ gtlO.
  • the ⁇ DNA was in vitro packaged (Gigapack Gold, Stratagene, La Jolla, CA). Approximately 200,000 plaques were screened by in situ hybridization using " 32 P radiolabelled C ⁇ and C ⁇ probes. The C ⁇ probes and C ⁇ probes were used to screen a cDNA library (made from a beef insulin specific T cell hybridoma). Insert DNA from the positive clones was ligated into M13mpl 8 and M13mpl9 for standard dideoxy sequencing.
  • All of the retroviral vectors used in the examples described herein are derivatives of the N2 vector (Keller et al., 1985, Nature H£:149-154).
  • the Al.l TCR ⁇ and ⁇ cDNAs were completely sequenced.
  • the Al.l ⁇ cDNA uses the V ⁇ , 2 (Arden et al., 1985, Nature 11 ⁇ :783-787) and J ⁇ TA65 and the Al.l ⁇ cDNA uses V ⁇ 6 (Barth et al., 1985, Nature 316:517- 523), D ⁇ 2 (Sui et al., 1984, Nature 111:344-349), and J ⁇ 2.7 (Gascoigne et al., 1984, Nature
  • the recipient T cell hybridomas were infected by the supematants of the producer lines as directed (Keller et al., 1985, Nature U£: 149-154) and selected in G418 (0.8 -1.0 mg/ml) for 10 days. In the case of 175.2 cells expressing Al.l ⁇ , further selection was performed by fluorescent staining with anti-CD3, followed by cell sorting using a FACStar Plus (Becton-Dickinson). The expression of the transduced TCR gene was determined by FACS analysis using either an anti-V ⁇ 6 monoclonal antibody (Payne et al., 1988, Proc. Natl. Acad. Sci.
  • RNAs from control and infected recipient T cell hybridomas were used for PCR.
  • Primers specific for the V ⁇ , and C ⁇ gene segments were used for PCR.
  • the amplified products were hybridized with a 5'end-labeled antisense oligonucleotide specific for the junctional region of Al.l ⁇ cDNA.
  • Suppressive activity was assessed by adding filter- sterilized hybridoma supernatant with or without an "accessory component” (10-15%) to the cultures.
  • This accessory component was prepared from cultures of murine T cells from animals immunized to SRBC, followed by adso ⁇ tion of the supernatant with SRBC, as described supra. in Section 5.1.1. (see also Zheng et al., 1988, J. Immunol.140:1351-1358; Bissonette et al., 1991,
  • culture supernatant of a T cell hybridoma, 3-1-V (described in Section 8, infra., may be used as accessory supernatant (FIG. IA and B).
  • the cultures were incubated at 37'C in humidified 92% air/8% C0 2 and anti-SRBC PFC assessed 5 days later.
  • neither the T cell hybridoma supematants nor the accessory supernatant significantly affected the immune response when added alone.
  • EYK(EYA) 4 EYK SEQ ID NO: 3
  • EYKEYAEYAAYAEYAEYK SEQ ID NO: 4
  • antigen-binding activity was assessed in cell supematants by a modified
  • the cell line 175.2 expresses TCR ⁇ and the CD3 components, but lacks a functional TCR ⁇ gene (Glaichenhaus et al., 1991, J. Immunol.14$: 2095). 175.2 cells were infected with a retroviras expressing A 1.1 -TCR ⁇ (See Section 6.1.4, supra, and the cells were selected in G418, then further selected by cell sorting of CD3 + cells. The expression of CD3 on the selected cells (FIG. 3A), confirmed that the TCR ⁇ chain was expressed in the selected cells (175.2-Al.l ⁇ ).
  • FIGS. 6A and B show that transfection of a TCR ⁇ gene from T cell hybridoma BB19 specific for an epitope of poly 18 which is distinct from that recognized by A 1.1 induced CD3 expression on the cell surface of 2 subclones of 175.2 (AF5 and AF6).
  • the transfectants did not produce any immunoregulatory activity in their supernatant (FIG. 7A and B). Therefore, not all poly 18-specific T cells secrete a TCR ⁇ chain with immunoregulatory activity.
  • B9 another cell line, B9, was infected with retroviral vectors carrying the TCR ⁇ or ⁇ of Al.l. Like Al.l, B9 expresses both TCR ⁇ and ⁇ , and produces IL2 in response to the antigen, poly 18, presented with I-A d (Fotedar et al., 1985, J. Immunol. 111:3028-3033). As shown in Figure 8, supematants from Al.l, but not B9, displayed antigen- specific regulatory activity, and B9 cells expressing the Al.l TCR ⁇ chain (B9-Al.l ⁇ ) also produced this activity, while those expressing the Al .1 TCR ⁇ chain (B9-A1.1 ⁇ ) did not. The latter was not due to a blocking effect of TCR ⁇ , since B9 cells expressing both the TCR ⁇ and ⁇ from Al.l (B9-Al.l ⁇ ) produced the regulatory activity.
  • the Al.l TCR ⁇ gene was transduced using retroviral vectors into another poly 18-specific cell line, B 1.1. Following selection with G418, the B 1.1 -A 1.1 ⁇ lines were found to produce the antigen-specific immunoregulatory activity, although the original cell line (Bl.l) does not.
  • B9, Bl.l two TCR ⁇ +
  • TCR ⁇ gene To further address whether or not expression of Al.l TCR ⁇ , in the absence of TCR ⁇ , can lead to production of the antigen-specific regulatory activity, Al.l TCR ⁇ or Al.l
  • TCR ⁇ was transferred into BWl 100 cells. Since BWl 100 cells lack intact TCR ⁇ and ⁇ (White et al., 1989, J. Immunol.141:1822-1825), any effect of TCR ⁇ gene transfer should be directly attributable to TCR ⁇ . As shown in FIG. 10A and B, supematants from BWl 100-Al .1 ⁇ , but not
  • BWl lOO-Al.l ⁇ displayed immunoregulatory activity. As with the other gene transfer experiments, this activity showed the antigenic specificity of Al .1.
  • TCR ⁇ chain versus that of other cells.
  • supematants from Al.l, and cell lines expressing Al.l TCR ⁇ contain an antigen-binding component as detected in a modified ELISA assay (See Section 6.1.6., supra.. This antigen binding was effectively competed by the unlabeled peptide, but not by two inappropriate peptides (FIG. 1 IB), one of which differs from the antigenic peptide by only a single residue. This substitution has been previously shown to destroy the antigenicity of the peptide for the Al .1 TCR (in an antigen presentation assay) (Boyer et al., 1990, Eur. J. Immunol.
  • the CD4 + T cell hybridoma, Al.l constitutively releases an immunoregulatory activity specific for the synthetic antigen poly 18 and related peptides (Zheng et al., 1988, J. Immunol.14 :1351-1358; Bissonette et al., 1991, J.
  • This example also conslusively demonstrates that the TCR ⁇ chain is released from the cell in a form that is independent of the CD3/TCR complex, and which modulates an antigen-specific immune response.
  • the transfer of the Al.l TCR ⁇ gene into BWl 100, which completely lacks TCR ⁇ nevertheless resulted in constitutive production of the antigen-specific regulatory activity (FIG. 10).
  • the results described herein also indicate that it is the direct recognition of antigen by the Al .1 TCR ⁇ chain (FIG. 11) that gives this molecule activity in the PFC assay, and that other T cells release TCR ⁇ chains that fail to directly bind to the epitope and therefore do not display such activity.
  • TCR ⁇ mediates the immune response. It is possible, for example, that the complex of TCR ⁇ and antigen is immunogenic, resulting in regulatory immune responses to the TCR.
  • TCR ⁇ the complex of TCR ⁇ and antigen is immunogenic, resulting in regulatory immune responses to the TCR.
  • immunization with specific T cells Lider et al., 1988, Science 212: 181-183; Sun et al., 1988, Nature 112:843-845) or peptides corresponding to regions in the TCR variable region (Vandenbark et al., 1989, Nature 241:541-544; Howell et al., 1989, Science 246:668-670) can result in dramatic immunoregulatory effects in vivo.
  • the regulatory effects associated with the Al.l TCR ⁇ chain may represent a form of such TCR "vaccination" in__i_ -
  • an unidentified molecule associates with the antigen-binding TCR ⁇ chain and this second molecule imparts biological function to the system.
  • Iwata, et al. Iwata et al., 1989, J. Immunol. 141:3917-3924
  • Iwata et al. have described a soluble complex of a molecule with glycosylation-inhibitory activity and a molecule bearing TCR determinants, released into supematants of some T cell hybridomas.
  • DNA oligonucleotide probes were designed based on the known sequences of the C ⁇ and C ⁇ genes in mice. The probes were synthesized and used to screen a cDNA library prepared from poly 18-specific Al.l hybridoma cells. Full-length TCR ⁇ and TCR ⁇ cDNAs from Al.l were characterized and cloned into a Bluescript vector (Stratagene, La Jolla, CA). RNA for both C ⁇ and C ⁇ was transcribed in vitro using a eukaryotic in vitro transcription system (BRL, Gaithersburg, MD). The RNA was then translated in vitro using a rabbit reticulocyte lysate system (BRL, Gaithersburg, MD). For autoradiography, 3S S-Met (New England Nuclear, Boston, MA) was included in the translation. For bioassays, the material was translated in the absence of radionucleotides.
  • the in vitro translated material was then enriched by affinity chromatography with monoclonal anti-TCR ⁇ or anti-TCR ⁇ antibodies. Labelled material was analyzed by SDS-PAGE, treated with Enhance, and exposed to X-ray film. Biological activity was assayed as in the system described in Section 6.1.5, supra.
  • the TCR ⁇ protein was found to have biological activity in the PFC assay, and this activity was completely bound (and eluted) from the anti-TCR ⁇ antibody (FIG. 13A and B).
  • FIG. 13 A the immunoregulatory activity from the in vitro translated TCR ⁇ was found in filtrates of the anti-TCR ⁇ antibody column and in eluates of the anti-TCR ⁇ antibody column. Titration of the active filtrate (anti-TCR ⁇ ) and eluate (anti-TCR ⁇ ) showed the activities to be similar.
  • T experiments detailed above coupled with the studies described in Section 6, supra. demonstrate that recombinant TCR ⁇ has biological function.
  • a TCR ⁇ chain gene encoding such a biologically active factor can be expressed in various expression systems to yield a product with biological activity; i ⁇ ., a TCR ⁇ chain that can specifically suppress an immune response directed against its target antigen.
  • BWl 100 and T cell hybridomas were maintained in RPMI-1640 plus 10% FCS.
  • Monoclonal antibody directed to CD4 (GK1.5) (Dialynas et al., 1983, Immunol. Rev. 74:29) was obtained from American Type Culture Collection (Rockville, MD). Rabbit and guinea pig complement were obtained from SciCan (Edmonton, Alberta, Canada) and from GIBCO (Grand Island, NY), respectively. Both complement samples were first screened for low background activity before use. Magnetic beads coated with anti-rat IgG antibodies were purchased from Dynal.
  • Spleen cells from C57B1/6 mice immunized to SRBC were obtained and treated with an antibody to CD4 (Gkl.5) in the presence of complement.
  • the CD4 depleted cells were subsequently reacted with magnetic beads coated with anti-rat IgG antibodies (Dynalbeads) for the removal of all IgG + cells.
  • the remaining T cells were centrifuged on lympholyte M (Cedarlane Laboratories, PA) and viable T cells were fused with B W 1100 in a 1 : 1 ratio in the presence of
  • Hybridomas were selected in the presence of hypoxanthine, thymidine, aminopterin and ouabin. Mouse red blood cells were used as filler cells. Supematants of the wells that scored positive for growth were tested for ability to substitute for accessory supernatant in combination with Al.l supernatant in the PFC assay as described in detail in 6.1.5., supra. Cultures with activity were split into subcultures and the sublines were retested for activity. The sublines with activity were again split and those with activity were cloned at 0.4 cells/well. Clones were rescreened for activity.
  • SRBC-imm unized murine spleen cells depleted of CD4 + T cells and IgG + B cells were fused with
  • a cDNA library can be prepared from T cells using techniques well known in the art. Since the nucleotide sequences encoding the single constant region gene for TCR ⁇ (C ⁇ ) in human and mice are known (Willson, etal, Immunol. Rev., 101:149-172. 1988), DNA probes homologous to C ⁇ can be synthesized by standard methods and used to screen such libraries to identify TCR ⁇ cDNA.
  • oligonucleotide probes derived from specific TCR ⁇ sequences could be used as primers in PCR (polymerase chain reaction) method (Mullis, et al, Methods in Enzymol, Hl ⁇ 335-350, 1987) to generate cDNA of TCR ⁇ sequences which can be directory cloned
  • helper T cell hybridoma Al.l
  • TCR ⁇ and ⁇ molecules specific for a synthetic polypeptide designated poly- 18 (poly (Glu-Tyr-Lys-(Glu-Tyr-Ala) 5 )) and, in the presence of specific antigen and 1-Ad, releases lymphokines.
  • This T cell hybridoma also constitutively produces a poly- 18- specific soluble factor involved in antigen-specific suppression. It has been shown that the factor produced by A 1.1 displayed the same antigenic fine specificity exhibited by the TCR on the A 1.1 cell (Zheng., et al, J.
  • TCR ⁇ cDNA of Al.l cells was cloned from cDNA library using C ⁇ probes.
  • mRNA was isolated from IO 9 cells by the conventional guanidine-isothiocyanate and cesium chloride method, and recovered by oligo-dT cellulose affinity chromatography.
  • the first strand cDNA was synthesized using an oligo-dT primer and reverse transcriptase and the second strand using DNA polymerase 1 and RNase H.
  • the methylated blunt ended double-strand cDNA was ligated to
  • GIF glycosylation inhibiting factor
  • This T cell hybridoma constitutively produces immunosuppressive factor, GIF, and PLA 2 binding GIF upon stimulation with homologous antigen and antigen presenting cells.
  • antigen binding GIF specifically suppress the immune response to the antigen in vivo, and that the antigen binding GIF may be encoded, at least in part, by TCR ⁇ expressing on the cell (Iwata, et al, J. Immunol, 141:3270-3277, 1988; Iwata, et al, J. Immunol, 141:3917-3924, 1989; Mori, et al, Int. Immunol, 1:833-842, 1993).
  • TCR ⁇ cDNA of 3B3 cells was cloned by PCR following the method described by Mullis, et al, Nucl. Acids. Res., &3895-3950, 1980).
  • mRNA was isolated from 5 X IO 7 3B3 cells by using Fast TrackTM mRNA isolation kit (Invitrogen).
  • cDNA was generated by using cDNA synthesis system (Pharmacia). After their generation, cDNAs were ligated at the 5'-end and the 3'-end by using T4 ligase (Takara) to construct circular DNA.
  • Oligonucleotide primers encoding murine C ⁇ DNA were synthesized by DNA/RNA synthesizer (Applied Biosystems) using phosphoramidite method (Beaucage, et al, Tetrahedron Lett., 22:1859-1862, 1981).
  • PCR was carried out by Taql DNA polymerase (Takara) in the presence of template cDNA, primers and dNTPs in a thermo cycler.
  • the conditions of PCR were that the denaturation step was 94 ⁇ C, 1 min; the annealing step was 54 ⁇ C, 1 min; and the elongation step was 72°C, 2 min; for 35 cycles.
  • Amplified cDNA was subcloned into pCRlOOO vector of TA cloning systemTM (Invitrogen). DNA sequences of the inserts were confirmed by dideoxy sequencing technique (Sanger, etal, Proc. Nat'lAcad. Sci. USA, 24:5463-5467, 1977).
  • TCR ⁇ cDNA Three different TCR ⁇ cDNA were cloned and sequenced. Two of them were identified to be originated from the fusion partner cell of 3B3 hybridoma, BW5147 (Chien, etal, Nature, . 12:31-35. 1984; Kumar et al, J. Exp. Med, 170:2183-2188, 1989). The other TCR ⁇ cDNA was confirmed not to be expressed in BW5147 by using several PCR primers encoding the different portion of this TCR ⁇ gene, which indicated that this TCR ⁇ originated from PLA 2 -specific T cells. Two of independent clones encoding this TCR ⁇ cDNA were isolated and their DNA sequences were confirmed to be identical.
  • TCR ⁇ cDNA The DNA sequence of this 3B3 derived TCR ⁇ cDNA is shown in Figure 15.
  • This TCR ⁇ cDNA encodes 268 amino acids open reading frame and the first 20 amino acids were identified to be a signal peptide (McEUigott, et al, J. Immunol, 140:4123-4131. 1988). 10. Expression of recombinant TCR ⁇ in E.coli - direct expression
  • Al.l TCR ⁇ cDNA which encodes amino acid 26 to 240 in extracellular region, and includes a Cla ⁇ restriction site
  • the denaturation step in each PCR cycle was set at 94 °C for 1 min, and elongation was at 72 ⁇ C for 2 min.
  • the DNA fragment was digested with Clal and BamHl, and cloned into the expression plasmid pST811 vector carrying a t ⁇ promoter and a t ⁇ A terminator ( Figure 16, Japanese patent, Kokaikoho 63269983) at the unique Clal and B amUl sites.
  • the new plasmid, called pST811-A1.1 TCR ⁇ S5 ( Figure 17) was transformed into competent RR1 E. coli host cells.
  • Selection for plasmid containing cells was on the basis of the antibiotic (ampicillin) resistance marker gene carried on the pST811 vector.
  • the DNA sequence of the synthetic oligonucleotides and the entire TCR ⁇ gene was confirmed by DNA sequencing of plasmid DNA.
  • RRl E. coli carrying plasmid pST81 l-Al.lTCR ⁇ S5 or pST81 l-Al.lTCR ⁇ S3 were cultured in 50 ml of Luria broth containing 50 ⁇ g/ml of ampicillin, and grown ovemight at 37 ⁇ C.
  • the inoculum culture was aseptically transferred to 1 liter of M9 broth which was composed of 0.8% glucose, 0.4% casamino acid, 10 mg/liter thiamine and 50 mg/liter ampicillin, and culture for 3 hours at 37°C. At the end of this initial incubation, 40 mg of indoleacrylic acid was added and the culture was incubated for an additional 5 hours at 37°C.
  • Matsuki, et al. has developed a rat calmodulin expression plasmid, pTCAL7, which carries rat calmodulin cDNA and t ⁇ promoter (Matsuki, et al, Biotech. Appl. Biochem., 12:284-291. 1990) (FIGURE 18).
  • pTCAL7 rat calmodulin expression plasmid
  • FIGURE 18 In order to express fusion proteins, several cloning sites were generated at the 3'- end of calmodulin cDNA, which also contains a thrombin cleavage sequence.
  • calmodulin cDNA inserted into pTCAL7 was amplified by PCR using two primers: one encoded 5'-terminus of calmodulin cDNA containing Clal site, the oth_- one provided the sequence of 3'- terminus of calmodulin cDNA, thrombin cleavage site and both BamHl, Xbal, Notl and BgRl sites.
  • the DNA fragment of 3B3-derived TCR ⁇ extracellular region which encodes amino acid 21 to 241, was amplified from pCR1000-3B3TCR ⁇ plasmid by PCR using two primers containing Xbal site for 5'-terminus, stop codon and Notl site for 3'-terminus respectively.
  • the sequences of those primers were:
  • the amplified DNA fragment was ligated with Xbal and Notl digested pCFl plasmid.
  • the new plasmid, called pCFl-3B3TCR ⁇ ( Figure 20) was transformed into competent W3110 E.coli cells, and the DNA sequence was confirmed.
  • a 1.1 -derived TCR ⁇ cDNA which encodes amino acid 26 to 240 was also inserted into pCFl by the method described above by using two primers; 5'-GATCTAGACAGAGCCCAGAATCCCTCAGTG-3' (SEQ ID NO: 12) S'-AAGCGGCCGCTTATTGAAAGT ⁇ AGGTTCATATC-S * (SEQ ID NO: 13)
  • the supematant fraction was added slowly, with stirring, to 40 ml of an appropriate mixture such that the final concentration of components in the mixture were 2.5 M urea, 5 mM sodium acetate, 0.01 mM EDTA 50 mM Tris- HC1 pH8.5, 1 mM glutathione (reduced form) and 0.1 mM glutathione (oxidized form).
  • the sample solution was added an appropriate mixture such that the final concentration of components in the mixture were 150 mM NaCl, 1 mM CaCl 2 and 5 mM MgCl 2 .
  • This mixture was applied at 4"C to a phenyl sepharose 6 fast flow low sub column (Pharmacia, 3 X 6 cm) equilibrated with 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM CaCl 2 and 5 mM MgCl 2 and ran at a flow rate of 0.5 ml per min.
  • calmodulin-TCR ⁇ fusion protein was eluted with 50 mM Tris-HCl pH8.0 containing 4 mM EDTA at a flow rate of 0.5 ml per min. Aliquots of fractions was analyzed by SDS- polyacrylamide gel electrophoresis, which indicated that the fusion protein was highly enriched ( Figure 23).
  • the elution fraction was dialyzed against 50 mM Tris-HCl buffer pH8.0 containing 150 mM
  • Each culture received 50 ⁇ l 1% SRBC coupled with poly- 18 (EYK(EYA) 5 ) or a substituted polypeptide.
  • Suppressive activity was assessed by adding recombinant TCR ⁇ with or without an "accessory component" (10-15%) to the cultures.
  • This accessory component was prepared from cultures of murine T cells from animals immunized to SRBC, followed by abso ⁇ tion of the supematant with SRBC. The cultures were incubated at 37°C in humidified 92% air/8% C02 and anti-SRBC PFC (plaque forming cells) assessed 5 days later.
  • Al.l cell cultured supematant was used as a positive control.
  • the recombinant TCR ⁇ protein showed suppressive activity at a final concentration of 4 X 10"'° M only with poly- 18 or EYKEYAEYAEYAEYA ( Figure 25).
  • the figure represents the data from four experiments, in which each of the peptides shown on the left (or saline) were added into coded tubes. The coded samples were then used for coupling to SRBC for the assay culture in the presence of accessory supematant. No suppression was observed in any case in the absence of accessory supematant. The codes for each experiment were different.
  • DNP dinitrophenyl derivatives of bee venom PLA 2 (Sigma) were prepared by standard procedure.
  • Balb/C mice were immunized by an i.p. injection of 10 ⁇ g of DNP-PLA 2 absorbed to 2 mg of alum.
  • Recombinant 3B3 TCR ⁇ was injected i.p. on day -1, 0, 2, 4, 6 at a dose of 5 ⁇ g/injection, and control mice received PBS alone.
  • serum was obtained from each animal and anti DNP-lgGl and anti DNP-lgE were measured by ELISA (Iwata, etal, J. Immunol, 141:3270-3277. 1988).
  • Anti-DNP-IgGl and anti-DNP-IgE were significantly suppressed (Table 1).
  • DNP-ovalbumin was used as an antigen and the activity of recombinant 3B3 TCR ⁇ was assessed.
  • anti- DNP antibody response to DNP-OVA was not affected by the treatment of immunized mice with the recombinant TCR ⁇ .
  • Anti-DNP IgE( ⁇ g/ml) a Anti-DNP IgGl( ⁇ g/ml)*
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE:
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE:
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE:
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE:
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE:
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE:
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE:
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE:
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE:
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE:
  • ATCAATGTGC CGAAAACCAT GGAATCTGGA ACGTTCATCA CTGACAAAAC TGTGCTGGAC 660
  • MOLECULE TYPE protein
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE:
  • MOLECULE TYPE protein

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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
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  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Saccharide Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract

Des procédés permettent de moduler des réponses immunitaires de manière spécifique d'un antigène. Ces procédés consistent à utiliser des chaînes α solubles de récepteurs des lymphocytes T qui peuvent se lier à un tel antigène et qui, en présence d'un composant accessoire, répriment la réponse immunitaire de manière spécifique à un antigène. On décrit l'utilisation des chaînes α des récepteurs des lymphocytes T qui témoignent d'une telle activité dans des protocoles thérapeutiques destinés à traiter des troubles dus à une hyperimmunité ou à une immunodéficience.
EP95905413A 1993-12-13 1994-12-13 PROCEDE D'IMMUNO-REGULATION SPECIFIQUE D'UN ANTIGENE PAR LA CHAINE $g(a) DES LYMPHOCYTES T Withdrawn EP0737076A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US16549693A 1993-12-13 1993-12-13
US165496 1993-12-13
PCT/US1994/014542 WO1995016462A1 (fr) 1993-12-13 1994-12-13 PROCEDE D'IMMUNO-REGULATION SPECIFIQUE D'UN ANTIGENE PAR LA CHAINE α DES LYMPHOCYTES T

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EP0737076A1 true EP0737076A1 (fr) 1996-10-16

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EP (1) EP0737076A1 (fr)
JP (1) JPH08149981A (fr)
CN (1) CN1145589A (fr)
AU (1) AU1403495A (fr)
CA (1) CA2179018A1 (fr)
WO (1) WO1995016462A1 (fr)

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Publication number Priority date Publication date Assignee Title
AU2651997A (en) * 1996-05-10 1997-12-05 Kirin Beer Kabushiki Kaisha T-cell receptor alpha-chain constant-region peptides, processes for producing the peptides, and use thereof
EP1029918A4 (fr) 1997-09-26 2003-01-02 Kyowa Hakko Kogyo Kk Recepteur de lymphocyte t tueur reconnaissant le vih
GB0223399D0 (en) * 2002-10-09 2002-11-13 Avidex Ltd Receptors
JP2009508517A (ja) * 2005-09-22 2009-03-05 コーエン，イルン，アール Ｔ細胞受容体定常ドメインの免疫原性断片及びそれに由来するペプチド
FR2919065B1 (fr) 2007-07-19 2009-10-02 Biomerieux Sa Procede de dosage de l'apolipoproteine ai pour le diagnostic in vitro du cancer colorectal
FR2919063B1 (fr) 2007-07-19 2009-10-02 Biomerieux Sa Procede de dosage du leucocyte elastase inhibitor pour le diagnostic in vitro du cancer colorectal.
JP5715817B2 (ja) 2007-07-19 2015-05-13 ビオメリューＢｉｏｍｅｒｉｅｕｘ 結腸直腸癌のインビトロ診断のための肝臓脂肪酸結合タンパク質、ｃｅａおよびｃａ１９−９のアッセイのための方法
FR2919060B1 (fr) * 2007-07-19 2012-11-30 Biomerieux Sa Procede de dosage de l'ezrine pour le diagnostic in vitro du cancer colorectal.
CA2729219A1 (fr) * 2008-06-23 2010-01-21 Perkinelmer Health Sciences, Inc. Substrats de kinases
US9850278B2 (en) 2013-04-25 2017-12-26 Carmel-Haifa University Economic Corp. Synthetic anti-inflammatory peptides and use thereof
WO2021147891A1 (fr) * 2020-01-21 2021-07-29 苏州克睿基因生物科技有限公司 Cellule effectrice immunitaire modifiée et son utilisation

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JP3875730B2 (ja) * 1993-02-22 2007-01-31 サノフィ・アベンティス株式会社 自己免疫疾患の予防治療剤

Non-Patent Citations (1)

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Title
See references of WO9516462A1 *

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AU1403495A (en) 1995-07-03
WO1995016462A1 (fr) 1995-06-22
CA2179018A1 (fr) 1995-06-22
JPH08149981A (ja) 1996-06-11
CN1145589A (zh) 1997-03-19

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