+

WO1994025489A1 - Moderation d'une reponse immunitaire specifique d'un allergene - Google Patents

Moderation d'une reponse immunitaire specifique d'un allergene Download PDF

Info

Publication number
WO1994025489A1
WO1994025489A1 PCT/CA1994/000228 CA9400228W WO9425489A1 WO 1994025489 A1 WO1994025489 A1 WO 1994025489A1 CA 9400228 W CA9400228 W CA 9400228W WO 9425489 A1 WO9425489 A1 WO 9425489A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
cells
tcr
ova
peptides
Prior art date
Application number
PCT/CA1994/000228
Other languages
English (en)
Inventor
Shyam S. Mohapatra
Alec H. Sehon
Original Assignee
University Of Manitoba
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by University Of Manitoba filed Critical University Of Manitoba
Priority to EP94914290A priority Critical patent/EP0697024A1/fr
Priority to AU66740/94A priority patent/AU6674094A/en
Priority to CA002161316A priority patent/CA2161316A1/fr
Publication of WO1994025489A1 publication Critical patent/WO1994025489A1/fr

Links

Classifications

    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to the discovery that the antibody (Ab) production in a mammal can be specifically suppressed to a given allergenic antigen (Ag) by treatment with a peptide(s) corresponding to the sequence of the junctional segment of the a and/or ⁇ chain of the Ag receptor of suppressor T (Ts) cells, which have been induced by the tolerogenic conjugates of the antigen in question with monomethoxypolyethylene glycol (mPEG) .
  • This method is useful for the treatment of a broad spectrum of immunological diseases, including diseases due to an aberration of the immunological system, as is the case in allergic and asthmatic disorders.
  • IgE-mediated allergic diseases such as hayfever and asthma
  • IgE-mediated allergic diseases are induced by a number of antigens present in a variety of environmental antigens such as the multideterminant proteins or glycoproteins in pollens in -20% of the genetically predisposed population (ref. l - a list of the references appears at the end of the specification) .
  • environmental antigens such as the multideterminant proteins or glycoproteins in pollens in -20% of the genetically predisposed population (ref. l - a list of the references appears at the end of the specification) .
  • allergens such as hayfever and asthma
  • APC antigen-presenting cells
  • MHC major histocompatibility complex
  • the binary peptide-MHC complexes interact with the corresponding specific T-cell receptors (TCR) of, respectively, (i) helper T (Th) cells or (ii) CTLs or suppresser T (Ts) cells, and the resulting triads determine the up- or down-regulation of the appropriate B cells (ref. 19) .
  • TCR T-cell receptors
  • the Th cell subpopulation may be further subdivided into three subsets, i.e., ThO, Thl and Th2 cells (ref. 20).
  • ThO Th2 cells
  • Th2 cells have been shown to produce IL-4, IL-5 and IL-6, and IL-4 has been shown to activate B cells leading to the production of IgE antibodies.
  • Thl cells produce IFN ⁇ which blocks the production of IL-4.
  • the mechanism(s) involved in the suppression of antibody responses by Ts cells is still not fully understood. It has been suggested that the suppression of antibody production is due to inactivation of a specific Th cells as a result of non-professional APC, i. e., a T cell (ref. 21).
  • the primary reaction of the IgE antibodies secreted from the Be cells is their binding to specific IgE receptors on the surface of mast cells, basophils and eosinophils.
  • the cell-fixed IgE antibodies react with and are crosslinked by the allergenic molecules, which leads to the release from these cells of chemical mediators of anaphylaxis.
  • these mediators act rapidly on the smooth muscles of different target organs resulting in the inflammatory manifestations characteristic of the hypersensitivity diseases of the immediate type.
  • T cell dependent antibody immune responses Induction of tolerance to T cell dependent antibody immune responses is considered to be of pivotal importance in devising appropriate therapeutic interventions for several immunologic diseases.
  • Several mechanisms, involving clonal deletion, clonal anergy, and suppressor T (Ts) cells have been postulated to be involved in the induction of immunological tolerance (ref. 23).
  • Ts suppressor T
  • TCRs T cell antigen receptors
  • the TCRs of helper (Th) , cytotoxic (Tc) and Ts cells consist of cell surface heterodimers of at ⁇ or ⁇ chains (refs. 25 to 30). Each of these chains is composed of regions encoded by separate gene segments: variable (V) , junctional (J) and constant (C) elements, in addition the j ⁇ chain contains a diversity (D) segment (ref. 31) .
  • V variable
  • J junctional
  • C constant
  • D diversity
  • TCRs The functional repertoire of TCRs is restricted because of the limited usage of TCR chains detected on T cells when primed with antigens (Ags) (ref. 32) .
  • Ags antigens
  • V ⁇ and/or V/3 genes appear to be selected for specific T cell responses as exemplified by different antigenic systems, such as cytochrome C (ref. 33), p-azobenzene arsonate
  • TCRs recognize the antigenic peptides in the context of a complex with MHC molecules of APCs and (ii) some antigenic peptides are associated with either the or ⁇ chain of the TCR, it was postulated that whereas one of these chains may be involved in Ag binding, the other is involved in binding to the appropriate MHC molecule (ref. 38) .
  • mice susceptible to MBP were found to utilize mainly the V ⁇ 8.2 gene (ref. 44) and their pretreatment with the V ⁇ 8.2 peptide 39-59, emulsified in adjuvant, induced cross-reactive and EAE protective T cells and Abs (refs. 45, 46) .
  • TCR genes In relation to allergic diseases, recently studies have implicated the pivotal role of TCR genes in the allergic immune response. For instance, the antigen receptors utilized in experimental Leishmaniasis (ref. 48) , and OVA-specific IgE responses in mice (ref. 49) were restricted. Moreover, analysis of T cell receptor genes of human T cell clones specific to dustmite allergens (ref. 50) and to those of grass pollen allergens (ref. 51) revealed restricted usage of TCR genes.
  • mice by the Ag(mPEG) n conjugates were transferable to normal mice by unfractionated spleen cells, or by slg", CD8 + splenic cells (ref. 59), or their crude freeze-thaw extract (FTE) , or by a suppressor factor (TsF) released from these cells by their interaction with an immunosorbent consisting of a moAb to TCR ⁇ . chain of T cells.
  • the tolerance induced in recipients of either Ts cells or their FTE or TsF lasted for extended periods (at least 90 days) (ref. 60) .
  • Ts cell clones specific to HIgG e.g., clone 23.32
  • OVA e.g., clone 17.2
  • the cloned Ts cells were shown to be Thy-1 + , CD4 " , CD5 * and CD8 + , and >95% of the cloned cells co-expressed CD3 and the a ⁇ TCR.
  • immunosorbents incorporating mAbs to the and ⁇ chain epitopes of TCR, it was demonstrated that whereas the FTE of clones 23.32 shared the epitopes of the a chain of TCR (ref. 61), the FTE of cells of clone 17.2 was serologically and structurally related to the ot/ ⁇ heterodimer of TCR.
  • the present invention provides a synthetic peptide having an amino acid sequence comprising at least a portion of the complementarity determining region 3 (CDR3) region of a T-cell receptor, particularly a human T-cell receptor, of an antigen, particularly an allergen, and which is capable of eliciting a T-cell response.
  • CDR3 complementarity determining region 3
  • the amino acid sequence is encoded by a nucleotide sequence comprising the J-gene of the ⁇ -chain of the TCR, or by a nucleotide sequence comprising the J-gene of the S-chain of the TCR.
  • a mixture of such peptides is particularly useful in immunotherapy, as described in more detail below, one peptide being encoded by a nucleotide sequence comprising the J-gene of the ⁇ -chain of TCR and another encoded by a nucleotide sequence comprising the J-gene of the /S-chain of the TCR.
  • the synthetic peptides provided herein may have about 8 to about 12 amino acids and be capable of binding to a class I MHC molecule or about 12 to about 27 amino acids and be capable of binding to a class II MHC molecule.
  • the present invention provides a method of immunotherapy, which comprises administering to a host, particularly a human, a peptide according to the invention or an immunosuppressive composition comprising at least one such peptide and a pharmaceutically-acceptable carrier.
  • a host particularly a human
  • an immunosuppressive composition comprising at least one such peptide and a pharmaceutically-acceptable carrier.
  • Such composition may further comprise an adjuvant.
  • the host may have previously exposed to the antigen or the material may be administered for prophylatic immunization of the host.
  • the immunization may be effected to suppress an allergic response in the host to the antigen or to protect the host from an allergic response to the antigen.
  • the immunization also may be effected to suppress an autoimmune response in the host to the antigen or to protect the host from an autoimmune response to the antigen.
  • the immunotherapeutic treatment may be effected herein in conjunction with the administration of therapeutic molecules prone to producing unwanted immunological responses.
  • the peptides of the present invention also are useful in screening a host for an immunogenic response to an antigen, particularly an allergen.
  • a method of diagnosing an allergic response of a host to an allergen which comprises screening a serum from the host with a plurality of the peptides according to the invention and corresponding to a plurality of allergenic antigens, and detecting reactivity of at least one of the peptides to the serum as a detection of an allergen to which the host has been exposed.
  • the at least one peptide which is detected in this manner then may be administered to the host as an immunotherapeutic treatment according to the method described above.
  • the present invention also includes a method of identifying the amino acid sequence of the peptides provided herein. Accordingly, in an additional aspect of the present invention, there is provided a method of identifying a peptide having an amino acid sequence comprising at least a portion of the CDR3 region of a T- cell receptor of an antigen and capable of eliciting a T- cell response, which comprises effecting induction of regulatory T-cells to a desired antigen, particularly an allergen, determining the nucleotide sequence of T-cell receptors of the regulatory T-cells, determining the portion of the nucleotide sequence of the T-cell receptors which codes for the CDR3 region of the T-cell receptors, and deducing the amino acid sequence of the determined portion of the nucleotide sequence as a determination of the amino acid sequence of the peptide.
  • the regulatory T-cells preferably are induced by administration to a host of a conjugate of a non- immunogenic substrate, particularly a polymer, and the desired antigen.
  • the polymeric substrate may be selected from carboxymethyl celluloses, monomethoxypolyethylene glycols (MPEGs) and polyvinyl alcohols.
  • the conjugate comprises an antigen - MPEG conjugate.
  • the regulatory T-cells induced by the antigen- substrate conjugates generally comprises CD8 + cells and the nucleotide sequence of T-cell receptors of such cells are determined, generally by conventional cloning procedures.
  • the determination of the CDR3 region of the T-cell receptors preferably comprises determining the individual nucleotide sequences for the - and /3-chains of the T- cell receptor and effecting sequence analysis of the individual sequences to determine the sequences of the J- gene of both a- and ⁇ -chains.
  • Figure 1 contains a schematic representation of anchored PCR protocols for amplification of 5' end of the a chain of Ts clones.
  • Total RNA was reverse-transcribed with a gene-specific primer, C ⁇ E 3 , to create a (-) strand of cDNA (hatched box) .
  • a poly- (A) tail was added, annealed with dT 17 adaptor primer and extended to generate the (+) strand.
  • a gene-specific internal oligo primer, C ⁇ .5RT, and the adaptor-primers were used for further amplification.
  • Figure 2 contains the nucleotide sequence (SEQ ID NO: 1) of the a transcript of 17.A2 (or 23.Al) of the cloned Ts cells #17.2 (or #23.32).
  • the asterisk (*) refers to the highly conserved residues in V H , V L , V/3 and V ⁇ regions.
  • Figure 3 contains a comparison of deduced amino acid sequences of the TCR a chain transcripts of cloned 17.A2 (SEQ ID NO: 2) and 23.Al (SEQ ID NO: 3) cDNAs with those of P14.A1 (SEQ ID-NO: 4) and human V ⁇ .3.1 (SEQ ID NO: 5) .
  • the asterisk (*) refer to the highly conserved residues in the V H , V L , V/3 and Va regions.
  • the symbol '-' designates the residues that are identical to those of cDNA 17.A2.
  • Figure 4 contains the nucleotide sequences (SEQ ID NOS: 6 and 7) and deduced amino acid sequences (SEQ ID NOS: 8 and 9) for the junctional region of the ⁇ . chains of TCRs in Ts clones 17.2 and 23.32, aligned with respect to their common V ⁇ .15 segment.
  • the shaded regions designate the residues of T cell clone #23.32 that differ from those of cloned T cells #17.2, the positions of which are identical.
  • Figure 5 contains the nucleotide sequences (SEQ ID NOS: 10 and 11) and deduced amino acid sequences (SEQ ID NOS: 12 and 13) for the junctional region of ⁇ chains of TCRs of Ts clones.
  • the alignment is based upon the common V/38.2 segment.
  • the Ts clone #17.2 utilized J/31.1
  • clone #23.32 utilized J-62.5. Spaces are introduced to align the highly conserved sequences among the J/3 regions.
  • the shaded regions designate the residues of clone #23.32 that differ from that of clone #17.2.
  • FIGS. 6A, 6B, 6C, 6D, 6E and 6F show down- regulation of OVA-specific antibody responses by TCR peptides.
  • Each group of five Balb/c mice were injected s.c. on day -8, day -6 and day -4 with PBS, or with 25 ⁇ g the Q.-CDR3 peptide or of the /3-CDR3 peptide, or with 1:1 mixture (25 ⁇ g each) of the ⁇ and ⁇ CDR3 peptides, in 0.2 ml PBS.
  • mice On days 1 and 28 all mice were immunized (i.p.) with 20 ⁇ g of aggregated OVA ( Figures 6A, B and C) or 20 ⁇ g of heat-treated HIgG ( Figures 6D, E and F) , and were bled 15 days after secondary immunization. IgGl antibody titers were determined by ELISA. Normal mouse serum was used as a negative control and gave an average O.D. readings of 0.05 at a 1:200 dilution. Titration curves for each individual mouse serum are shown.
  • mice treated with PBS ⁇ » A, ⁇ , ⁇ , and ⁇
  • mice o, ⁇ , V, D, and O
  • mice o, ⁇ , V, D, and O
  • FIG. 7 shows the immunological response of mice to repeated immunization of allergen.
  • Each group of six Balb/c mice received on day -5 and day -3 either PBS, or 25 ⁇ g each of Ja or of J ⁇ peptide, or mixture of 25 ⁇ g each of J ⁇ and J ⁇ in PBS.
  • IgG- L ELISA titers were determined on individual sera on days 43, 51, 77, 84, 107, 114, 159 and 216.
  • FIG 8 shows the immunological response of mice to allergen challenge.
  • Each group of six Balb/c mice received (intraperitoneally) on day 0, 10 ⁇ g of aggregated OVA, and then treated subcutaneously with three courses of PBS alone or, or ⁇ -CDR3 or 1:1 mixture of ⁇ -CDR3 and /3-CDR3 in PBS on days 15, 30 and 45. All mice were immunized (intraperitoneally) with aggregated OVA or heat-treated HIgG in 0.5 ml PBS, on days 52 and 80. Mice were bled 15 days after each immunization and IgGl titers were determined for individual serum by ELISA. Each bar represents mean titer +.SD.
  • Figures 9 shows the long lasting in vivo suppression of OVA-specific IgE antibody response by pretreatment with J ⁇ /J/3 peptides of the TCR of OVA-Ts cells #17.2.
  • mice On days 1, 28, 63, 93, 138 and 195 all mice were immunized (i.p.) with 20 ⁇ g of Fr-I OVA in PBS (as indicated by arrows) , and the PCA titers of OVA-specific IgE antibodies of pooled sera were determined, one week after immunizations, i.e., on days 35, 70, 100, 145 and 202 by 24-hr PCA-reaction in hooded rats. Each PCA titer represents the inverse of the highest dilution of the serum, still giving a clearly visible skin reaction (> 5mm in diameter) .
  • o PBS treatment; •: J ⁇ treatment; A : j ⁇ treatment; D: J ⁇ plus J ⁇ treatment.
  • FIG. 10 shows the antigen-specific abrogation of an established IgE-antibody response by TCR peptides.
  • Each group of four Balb/c mice received (i.p.) 10 ⁇ g of FR-I OVA on day 0, and was then treated s.c. with three courses of PBS alone or, J ⁇ or a mixture of J ⁇ and J ⁇ in PBS on days 15, 30 and 45. All mice were re-immunized (i.p.) with Fr-I OVA in 0.5 ml PBS, on days 52 and 80, and were bled 15 days after each immunization; the IgE titers of pooled sera were determined by the 2 -hr PCA reaction. Similar results were obtained in a second experiment.
  • Each course consisted of three s.c. injections of 25 ⁇ g of J ⁇ or a mixture of 25 ⁇ g of J ⁇ and J ⁇ in 0.2 ml PBS, administered on alternate days. Mice were injected with OVA on days 52 (secondary response) and 80 (tertiary response) . OVA specific IgE titers were determined on day 66 (A) and day 94 (B) . Black bars, PBS treatment; hatched bars, J ⁇ treatment; doubly crosshatched bars, J ⁇ +J/3 treatment.
  • the present invention relates to a procedure for the induction of tolerance to specific antigens by the -treatment of a host with a peptide segment of the TCR ⁇ and/or ⁇ chain(s) of Ts cells, particularly induced in vivo by tolerogenic Ag-mPEG conjugates.
  • the cDNAs encoding the ⁇ and ⁇ chains of TCRs of cloned Ts cells specific for (i) ovalbumin (OVA) and (ii) human monoclonal (myeloma) IgG (HIgG) were produced by polymerase chain reaction (PCR) . Analysis of the V ⁇ genes indicated that these clones utilized a new member of the V ⁇ l5 gene family, which was productively joined to J ⁇ genes that were different for the Ts cells of the two distinct specificities.
  • mice with a mixture of pentadecapeptides comprising the V ⁇ l5 chain including the J ⁇ region (residues 95 to 109) of the TCR of the OVA- specific 17.2 Ts cells, downregulated the immune response specific to OVA, but not to HIgG.
  • injection of mice with the peptide of this J ⁇ region alone suppressed >90% of the Ab response to OVA, whereas the J ⁇ region peptide downregulated Ab production by only 70%.
  • mice Treatment of mice with a single course of the synthetic peptide(s) corresponding to the CDR3 region(s) of TCR ⁇ and ⁇ chains of OVA Ts cells, led to the immunosuppression of these mice for >200 days with respect to their potential to mount an anti-OVA antibody response.
  • established OVA-specific antibody production was abrogated following peptide vaccination.
  • the immunosuppression induced by these TCR peptide(s) involved CD4 + T cells, which were necessary but not sufficient.
  • this invention provides a method for inducing the antigen specific downregulation of the immune response by administration of peptides designed on the basis of the amino acid sequences of ⁇ and ⁇ chains of TCR of Ts cells, induced by the corresponding Ag-mPEG conjugates.
  • the present invention may serve as a method directed toward the treatment of diseases and abnormalities which are characterized by the synthesis of unwanted antibodies to specific antigens, such as IgE antibodies responsible for common forms of IgE-mediated allergies.
  • This method is also useful for the treatment of a broad spectrum of immunological diseases including (a) diseases resulting in aberrant immune responses, such as autoimmune disorders, and (b) malignacies and organ transplant operations, requiring the injection of xenogenic and/or immunogenic, biologically and therapeutically active molecules, such as (i) xenogenic or engineered Abs, including human monoclonal antibodies
  • Immunotoxin (moAbs) , and immunoconjugates of different Abs or their fragments, (ii) ribosome-inactivating proteins(RIP) , which are used as conjugates with cell targetting Abs or Ag-binding fragment thereof, referred to as Immunotoxin
  • compositions suitable to be used for protecting allergic individuals from developing an allergic reaction or for ameliorating an existing allergic condition, or other immunotherapeutic treatment as described herein, may be prepared from the peptides disclosed herein.
  • Compositions may be prepared as injectables, as liquid solutions or emulsions.
  • the peptides may be mixed with pharmaceutically-acceptable excipients which are compatible with the peptides. Excipients may include, water, saline, dextrose, glycerol, ethanol, and combinations thereof.
  • the composition may further contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the effectiveness thereof.
  • compositions may be administered parenterally, by injection subcutaneously or intramuscularly. Alternatively, other modes of administration including suppositories and oral formulations may be desirable.
  • binders and carriers may include, for example, polyalkylene glyco-ls or triglycerides.
  • Oral formulations may include normally employed incipients, such as, for example, pharmaceutical grades of saccharine, cellulose, magnesium carbonate and the like.
  • compositions are administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically effective immunotherapeutic treatment of interest.
  • quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual's immune system to synthesize antibodies. Precise amounts of peptide required to be administered depends on the judgement of the practitioner. However, suitable dosage ranges are readily determinable by one skilled in the art and may be of the order of nanograms to micrograms of the peptides. Suitable regimens for initial administration and booster does are also variable, but may include an initial administration followed by subsequent administrations. The dosage of the composition may also depend on the route of administration and will vary according to the size of the host.
  • the peptides of the present invention are useful as antigens in immunoassays including enzyme-linked immunosorbent assays (ELISA) , RIAs and other non-enzyme linked antibody binding assays or procedures known in the art for the detection of allergen specific IgE antibodies.
  • ELISA assays the peptide is immobilized onto a selected surface, for example, a surface exhibiting a protein affinity, such as the wells of a polystyrene microtiter plate.
  • a nonspecific protein such as bovine serum albumin (BSA) or casein that is known to be antigenically neutral with regard to the test sample, may be bound to the selected surface.
  • BSA bovine serum albumin
  • casein that is known to be antigenically neutral with regard to the test sample.
  • BSA bovine serum albumin
  • a mixture of peptides may be used, either as an immunogen in a composition or as a diagnostic agent.
  • the immobilizing surface is then contacted with a sample, such as clinical or biological materials to be tested, in a manner conducive to immune complex
  • the sample is then allowed to incubate for from 2 to 4 hours, at temperatures, such as of the order of 25° to 37°C. Following incubation, the sample-contacted surface is washed to remove non- immunocomplexed material.
  • the washing procedure may include washing with a solution, such as PBS/Tween, or a borate buffer.
  • the occurrence, and even amount, of immunocomplex formation may be determined by subjecting the immunocomplex to a second antibody having specificity for the first antibody.
  • the second antibody would be an antibody having specificity for human IgE or IgG antibodies.
  • the second antibody may have an associated activity, such as an enzymatic activity that will generate, for example, a color development upon incubating with an appropriate chromogenic substrate. Quantification may then achieved by measuring the degree of color generation using, for example, a visible spectra spectrophotometer-.
  • Ts cell clones The generation of the HIgG-specific clone #23.32 and of the OVA-specific clone #17.2 were described previously (refs. 61, 63) .
  • RNA pellets were washed in 70% ethanol and then dissolved in TE (10 mM Tris, pH 8, 1 M EDTA) at 65°C. The RNA was precipitated in 0.2M sodium acetate and absolute ethanol, and redissolved in water.
  • RNA-PCR RNA-PCR.
  • the RNA was heated to 70°C for 10 minutes in the presence of oligo(dT) or gene specific primer, and was cooled down quickly on ice.
  • the single stranded (ss) cDNA was synthesized using 20 units of M-MLV (H " ) reverse transcriptase (Superscript, Gibco, BRL) (ref. 66) in 5 fold concentrated reaction buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl 2 ) , 20 mM DTT and 0.5 mM dNT mixture.
  • M-MLV H " reverse transcriptase
  • 5 fold concentrated reaction buffer 250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl 2
  • 20 mM DTT 0.5 mM dNT mixture.
  • PCR Taq DNA polymerase Gabco, BRL
  • oligo(dT) For the amplification of TCR ⁇ /3 chains the corresponding cDNAs were obtained employing oligo(dT as primer. Amplification of transcripts encoding the chain was achieved with the 5' end of V/38 and the 3' en of C/3 (C/3E 3 ) oligonucleotides (Table la - the variou Tables appear at the end of the disclosure. SEQ ID NOS 20 to 29) . The transcripts encoding ⁇ -chains were amplified using the 5' end of V ⁇ l5 and the 3' end of C (C ⁇ E 3 ) oligonucleotides. Each cycle consisted of 1 mi at 94°C, 2 min at 55°C and 1 min at 72°C. Anchor PCR.
  • gene specific internal primer selected from the 5' en of C ⁇ , i.e. C ⁇ 5RT, and adapter primers were used fo amplification and generation of double stranded (ds) DNA Cloning of PCR products.
  • PCR products were cloned usin the TA cloning system (In Vitrogen, CA) , which take advantage of the A-overhangs of amplified cDNAs that ar used to insert the PCR product into a spatially designe vector, namely pCR 1000, providing single 3'T-overhang at the insertion site.
  • the amplified PCR product obtained as described above were directly ligated ont the pCR 1000 vector, in a 1:2 ratio of vector to insert
  • the ligati ⁇ n products were transformed in E. col strain DH5 ⁇ F' laclq " as described elsewhere (ref. 69) Briefly, the frozen competent cells were thawed on ice for 30', incubated with plasmids for 30', then heat shocked for 2' at 42°C and then kept for 2' on ice.
  • the mixture was then grown in SOC medium (2% Bacto tryptone, 0.5% Bacto yeast extract, lOmM NaCl, 2.5 mM KC1, lOmM MgCl 2 , lOmM MgS0 4 and 20mM glucose) at 37°C for 1 hr and spread on LB agar plates containing kanamycin (50 ⁇ g/ml) and 25 ⁇ l of X-gal (40mg/ml) . The colonies were allowed to grow for 40 hrs. and the white colonies were picked up and analysed by plasmid isolation. At least 10 transformant colonies were analysed from each of two independent PCR amplifications to ensure the reproducibility of this procedure. Plasmid DNA isolation.
  • Plasmid DNA was isolated by a modification of the protocol of Birnboim and Doly (ref. 70) . Briefly, the bacterial cells were lysed in a solution of 1:2:1 ratios of TE, lysis buffer (1.25% SDS, 0.2N NaOH) , and 10M NH 4 OVAc. The supernatant was precipitated by absolute EtOH. RNA was removed by digestion with DNAse-free RNAse and the protein was removed by digestion with proteinase K. The DNA was then extracted with phenol, phenol/choloroform and chloroform and precipitated with NaOVAC and two fold EtOH.
  • Inserts were excised by digestion with Eco RI and Hind III restriction endonucleases and checked by electrophoresis in an 1% agarose gel.
  • the plasmids containing the inserts were chosen for further analysis.
  • DNA sequencing DNA sequencing was performed employing ds DNA cycle sequencing system (BRL) as per manufacturer's instruction. Polylinker of TA vector was flanked by T 7 promoter and M13 forward primer regions. Therefore, positive recombinants were sequenced directly using T 7 and M13 forward primers. Furthermore, internal gene specific primers of C ⁇ and C ⁇ regions were used to sequence the entire TCR a/ ⁇ genes. Each of the amplifications was repeated at least twice with different batches of mRNA of each of the Ts cell clones. At leas 5 clones from each batch of transformants were sequence to avoid PCR amplification and cloning induced artifacts Results Amplification, cloning and sequencing of TCR a chai genes of Ts clones.
  • the ss cDNAs were synthesized from mRNAs of Ts clones #17.2 and #23.3 which were then tailed with poly- (A) and amplified Transcripts of 540 bps were generated which were ge purified and cloned in pCRlOOO vector.
  • Several cDNA were isolated from the #17.2 and #23.32 Ts cells Individual cDNAs were sequenced using T 7 and M13 forwar primers. It is to be noted that each of th amplifications was repeated at least twice with differen batches of mRNA of the cloned Ts cells.
  • Figure 2 i illustrated the nucleotide sequence (SEQ ID NO: 1) of th V ⁇ gene utilized by cDNAs 17.A2 and 23.Al, which were derived, respectively, from mRNAs of Ts cell clones #17. and #23.32. Both cDNAs possessed identical nucleotid sequences.
  • a unique family of rearranged V ⁇ genes encode TCR o Ag(mPEG) n induced Ts cells.
  • FIG. 3 are shown the deduced amino aci sequences (SEQ ID NOS: 2 and 3) of the V ⁇ chains of 17.A and 23.Al Ts celis which have been aligned with respec to the corresponding sequence of a previously reporte partial and non-functional cDNA clone pl4A.l (SEQ ID NO: 4), which has been considered to represent V ⁇ l5 (ref. 34) .
  • V regions of the ⁇ chain of the TCR of #17.2 (or #23.32) Ts cells are homologous to the extent of 82% with the sequence of P14A.1 over a span of 81 amino acid residues.
  • P14A.1 did not yield a rearranged transcript because it lacked an open reading frame.
  • V ⁇ genes of the cloned Ts cells are distinct and may represent a new member of the V ⁇ 15 family; moreover, this novel family of murine V ⁇ genes was homologous to the extent of -71% with respect to the sequence of the human V ⁇ 3.1 gene (ref. 35) (SEQ ID NO: 5).
  • CDR3 in TCR a chain of Ts cell clonest
  • the J ⁇ genes utilized by Ts clones #17.2 and #23.32 are shown in Figure 4 (SEQ ID NOS: 6 to 9) . From these data it may be concluded that the diversity of the TCR ⁇ chains of these Ts clones was confined to a single V ⁇ and two J ⁇ genes.
  • the OVA-specific cloned Ts cells expressed V ⁇ l5/J ⁇ 17.A2, whereas the HIgG-specific Ts cells expressed V ⁇ l5/J ⁇ 23.Al.
  • the C ⁇ region of each of these Ts cells was sequenced using C ⁇ E- ⁇ and C ⁇ E 3 primers. Two bp changes at positions 684 (i.e. C --> T) and 768 (i.e.
  • the mRNA of Ts clone #23.32 and #17.2 were subjected to RNA-PCR.
  • the ss cDN were synthesized from total RNAs using oligo(dT) a primer.
  • the cDNAs were then amplified utilizing the 5 end of V/38 (a consensus primer common to V/38.1, 8.2 an 8.3) and 3' end of C/3 primers, C/3E 3 .
  • An 850 bp transcrip was generated from each of these Ts cells.
  • the amplifie products were cloned directly into pCRlOOO vector (I Vitrogen, CA) .
  • FIG. 5 is shown the alignment of junctiona regions of the V/3-D/3-J/3 sequences of the two cloned T cells (SEQ ID NOS: 10 to 13). Comparison of sequence with the data bank revealed that both Ts cell clone utilized V/38.2 (ref. 37) .
  • the OVA-specific Ts cell clon #17.2 expressed the V/38.2-D / 31.l-J/31.1 gene, whereas th HIgG specific clone #23.32 expressed the V/38.2-D/31.1 J/32.5 gene.
  • the D / 3 segments in each set were identica except for 1 or 2 N-region additions at the 3' end Alignment of V/38.2 segment and of the residues common t both J/31.1 and J/32.5 segments revealed that th nucleotide differences between the putative TCR-CDR loops of the ⁇ chains of OVA- and HIgG-specific Ts clone resided in only 9 residues within the junctional regio
  • the peptide with the amino acid sequence GPNTEVFFGKGTRLTV (SEQ ID NO: 17) of the J ⁇ region represented the CDR3 segment of the ⁇ chain of the OVA- specific Ts cells.
  • An unrelated peptide MIEKINVGFKAAVAAAGGVP (SEQ ID NO: 19) was synthesized and used as control peptide.
  • mice were bled 7 and 14 days later and t corresponding total Ig and IgGl Ab titers of each ser were determined by ELISA. Briefly, the plates we coated with 10 ⁇ g of either of OVA or HIgG in PB blocked with 3% of bovine serum albumin in PBS a incubated overnight with each individual mouse seru The plates were washed, treated successively wi biotinylated goat anti-mouse Ig or IgGl Ab (Zyme Mississagua, ON) and alkaline phosphatase-streptavidi conjugate (Zymed, Mississagua, ON) and finally develop with p-nitrophenyl-phosphate buffer prior to recordi their O.D. readings at 405nm. Results
  • the TCR ⁇ chain of HlgG-Ts cell possesses sequences identical to peptides #1 and #2, and that it differs from the TCR ⁇ chain of OVA-Ts cell in the composition of the CDR3 peptide (i.e. peptide #3) . Therefore, it was concluded that the difference of the effect of the mixture of the three peptides on the anti- OVA and anti-HIgG Ab responses was due to peptide #3, which represnted the TCR J ⁇ segment.
  • mice were immunized on days -5 and -3 with 25 ⁇ g each of the J ⁇ or of the control peptide followed by challenge with OVA or HIgG on day 0 and bleeding on day 14 and 21.
  • the J ⁇ peptide alone induced 90% suppression of the OVA-specific Ab response without affecting the anti-HIgG Ab response.
  • mice were pretreated with the pentapeptide corresponding to CDR3 region of the HlgG-Ts cells, and received 7 days later, a s.c. injection of OVA or HIgG and their IgG, Ab titers were determined 10 and 20 days later.
  • the CDR3 peptide of the TCR ⁇ chain of HlgG-Ts cells induced suppression in the order of 80% of IgGl Ab responses specific for HIgG, but not for OVA, as determined on days 10 and 20 after immunization.
  • This Example illustrates suppression of secondary anti-OVA Ab responses by TCR peptides of OVA-specific Ts cells 17.2.
  • TCR peptides of OVA- Ts cells Balb/c mice were treated with 25 ⁇ g each of the J ⁇ or of J/3 peptide referred to above or with a mixture of 25 ⁇ g each of them in PBS and the control mice were treated with PBS.
  • all mice were immunized with 20 ⁇ g of OVA or 20 ⁇ g of heat-aggregated HIgG as control.
  • the total Ab titers on day 35, 43 and 51 are shown in Table 6.
  • mice treated with either J ⁇ or a combination of J ⁇ and J ⁇ peptides the mice treated with J ⁇ peptide also showed suppression 60-70 of OVA-specific IgG-i titers.
  • Significant enhancement of titers was seen in mice immunized with HIgG in comparison with that of the PBS treated controls.
  • Table 7 demonstrates the OVA-specific IgG 2a titers in the same mice. The reduction of IgG 2a titers was similar to that of the IgG;-. titers; highest suppression was seen in case of J ⁇ /J/3 and J ⁇ peptide treatment and intermediate level of suppression of response was seen by treatment with J ⁇ peptide.
  • mice which were challenged with HIgG used as a control antigen showed no reduction of HIgG titers.
  • the mice injected with J ⁇ or J ⁇ /J/3 peptides exhibited suppression of HlgG- specific IgG 2a titers.
  • This Example illustrates the long lasting tolerance of OVA-specific antibody responses by pretreatment of mice with TCR peptide(s) .
  • Method Each group of six Balb/c mice were injected subcutaneously on day -5 and day -3 either PBS, or 25 ⁇ g each of ⁇ -CDR3 peptide or of /3-CDR3 peptide, or 1:1 mixture (25 ⁇ g each) of ⁇ plus /3-CDR3 peptides, in 0.2 ml PBS. On days 1, 28, 63, 93, 138 and 195 ( as is indicated by arrow) , all mice were immunized i.p. with 20 ⁇ g of aggregated OVA in PBS. Mice were bled 15 days after each immunization.
  • IgGl antibody titers for individual serum were determined by ELISA. Each point represents the mean of the titers ⁇ S ⁇ . Concordant results were obtained in a second experiment. The differences between the OVA-specific titers of mice treated with the ⁇ -peptide (or ⁇ +/3 peptides) and the corresponding titers of mice treated with PBS were statistically significant (P ⁇ 0.01). Results; The results of prior vaccination with the TCR-CDR3 peptide resulted in persistent immune tolerance at the level of production of specific antibodies is shown in Figure 7.
  • mice received (intraperitoneally) on day 0, 10 ⁇ g of aggregated OVA, and then treated subcutaneously with three courses of PBS alone or, or ⁇ -CDR3 or 1:1 mixture of ⁇ -CDR3 and /3-CDR3 in PBS -on days 15, 30 and 45. All mice were immunized i.p. with aggregated OVA or heat-treated HIgG in 0.5 ml PBS, on days 52 and 80. Mice were bled 15 days after each immunization and IgGl titers were determined for individual serum by ELISA. Each bar represents mean titer ⁇ SD. Concordant results were obtained in second experiment.
  • mice were challenged with OVA on days 52 and 80.
  • OVA specific IgGl titers were determined on day 66 (A) and day 94 (C) .
  • Differences between OVA-specific titers of mice treated with ⁇ plus /3-CDR3, and the corresponding titers with PBS were statistically significant (P ⁇ 0.01). Results;
  • mice first were immunized with OVA and treated with three courses beginning day 15 with PBS or TCR-CDR3 peptide(s) of either ⁇ chain alone or a combination ⁇ plus ⁇ chains. Mice were challenged on days 52 and 80 with either OVA or HIgG. Examination of the OVA-specific IgGl and IgE (data not shown) titers on days 66 and 94 revealed that the antibodies specific to OVA were reduced in treated group in comparison with the PBS-treated mice ( Figure 8) .
  • Ovalbumin used in this study as a model antigen, constitutes 58% of the whole egg white and plays also the role of a major allergen in individuals allergic to eggs.
  • OVA has been shown to induce antibodies of both IgG and IgE isotypes (ref. 73) .
  • Antibodies of the IgE class are elicited in response to exposure to occupational, environmental and food allergens in genetically predisposed individuals (comprising about 20% of the population) .
  • Earlier investigations had demonstrated that administration of OVA-mPEG conjugates into mice downregulated their anti-OVA IgE immune response, and that this effect was due to induction of Ts cells.
  • the effects of vaccination with TCR peptides of these Ts cells on i) the downregulation of the de novo induction, and (ii) the suppression of established IgE antibody response were investigated in this study.
  • mice were treated at day -5 and day -3 with PBS or with 25 ⁇ g each of J ⁇ , J ⁇ or a mixture of J ⁇ and J/3 in PBS. On days 1, 28, 63, 93, 133 and 195 all mice were immunized with 20 ⁇ g of OVA.
  • the IgE Ab titers of individual mice were examined on days 35, 70, 100 and 145 by passive cutaneous anaphylaxis test (47) . Results;
  • mice were vaccinated with the pentadecapeptides corresponding to the J ⁇ and/or J ⁇ regions of TCRs of the cloned OVA-Ts cell prior to initiation of immunizations with repeated injections of OVA at intervals of 4-6 weeks.
  • the IgE antibody titers were determined by the PCA assay in hooded rats.
  • the OVA-specific IgE antibody response was suppressed by treatment with J ⁇ and/or J ⁇ peptides of the TCR of cloned OVA-Ts cells.
  • treatment with a single course of injection of the TCR- CDR3 peptide induced suppression of OVA-specific IgE antibody production for more than 200 days, inspite of six injections of immunogenic OVA.
  • the magnitude of the suppression varied with different peptides. Marked reduction (>90%) in IgE antibody titers resulted upon treatment of mice with the pentadecapeptide corresponding to either J ⁇ or J ⁇ in combination with J ⁇ peptide; the J/3 peptide alone induced about 50% suppression of IgE antibody production.
  • EXAMPLE 8 This Example illustrates abrogation of an established allergen specific IgE- antibody response by TCR peptides.
  • mice received (i.p.) 10 ⁇ g of OVA on day 0, and were then treated s.c. with three courses of PBS alone or, ⁇ -CDR3 or 1:1 mixture of ⁇ -CDR3 and /3-CDR3 in PBS on days 15, 30 and 45. All mice were re-immunized (i.p.) with OVA or ha-HIgG in 0.5 ml PBS, on days 52 and 80, and were bled 15 days after each immunization; the- IgE titers of pooled sera were determined by PCA 24-hr later. Similar results were obtained in a second experimen .
  • mice were injected with OVA on days 5 (secondary) and 80 (tertiary) .
  • OVA specific IgE tite were determined on day 66 (A) and day 94 (B) .
  • mice received i.p. on day 0, 1 ⁇ g of aggregated OVA, and then treated subcutaneousl with three courses of either PBS alone, or 1:1 mixture o ⁇ -CDR3 and /3-CDR3 on days 15, 21 and 28.
  • mic were sacrificed and CD4 + T cells were separated from eac group by using polypropylene immunocolumn (collect plus Biotex Laboratories, Inc.).
  • CD T cells were separated from normal Balb/c mouse spleens OVA primed spleen cells were isolated 15 days afte immunization of mice with 50 ⁇ g OVA emulsified i complete Freund's adjuvant (subcutaneously over the hin legs) .
  • OVA primed spleen cells were cultured i quadruplate, at two different concentrations with T cells separated from mice treated with either PBS, or peptides or naive mouse, at three different concentrations, in the presence of 100 ng/ml of DNP-OVA in complete medium. Forty-eight hours later, the cells were washed and cultured in fresh medium for additional 4 days. The amount of antibody in each well, in the latter culture supernatant, was measured by ELISA using plate coated with OVA, as described above. Results; The mechanism underlying the tolerance induced by these TCR-CDR3 peptides was examined.
  • mice with TCR peptide followed by challenge with OVA-DNP resulted in the downregulation of both OVA- and DNP-specific IgGl antibody titers, suggests that the peptide treatment downregulates OVA-specific helper T cell response (data not shown) .
  • the present invention represents a unique approach to immunotherapy. While the description of - the invention contains specific embodiments thereof, it will be understood that it is capable of further modification and applications to the one skilled in the art of molecular immunology. This description is, therefore, intended to cover any variations, uses or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as would not be anticipated on the basis of known principles and and existing practice within the area to which the invention pertains.
  • CMS Update Desensitizing vaccines Brit. Med. J. 293: 948 (1986) .
  • Anti-OVA ELISA titers b Anti-HIgG ELISA titers*
  • mice received on day 0 either PBS, or a mixture A and B of the i three pentadecapeptides; consisting respectively of 25 and 50 ⁇ g of each of the three peptides.
  • PBS PBS
  • a mixture A and B of the i three pentadecapeptides consisting respectively of 25 and 50 ⁇ g of each of the three peptides.
  • One day later all mice were immunized with a mixture of 20 ⁇ g of OVA and 20 ⁇ g of heat-treated HIgG in PBS.
  • Total Ig Ab titers on day 7 and IgGl Ab titers on day 14 were determined for individual sera and the numbers in the Table represent the means ⁇ SDs. Data shown here are from one of two experiments.
  • c The differences in OVA-specific titers of groups treated with PBS or the mixture B were statistically significant (p ⁇ 0.01) .
  • Anti-OVA IgGl titers Anti-HIgG IgGl titers
  • mice received on day -5 and day -3 either PBS, or 25 ⁇ g each of Jcy or of control peptide in PBS.
  • IgGl ELISA titers were determined for individual sera on days 14 and 21. Each number represents the mean +. SD. Data shown here from one of two experiment.
  • Anti-HIgG IgG ! titers Anti-OVA IgGi titers
  • mice received on three days either PBS, or 50 ⁇ g of the J ⁇ or of the control peptide in PBS. Seven days after the last treatment all mice were immunized with 20 ⁇ g of either ha-HIgG or OVA in PBS.
  • the IgG- L ELISA titers were determined on individual sera 10 and 20 days after immunization. Each number represents the mean of the titers ⁇ SD. Similar results were obtained in a second experiment.
  • Anti-OVA ELISA titers Anti-HIgG ELISA titers
  • mice received on day -5 and day -3 either PBS, or 25 ⁇ g each of J ⁇ or of control peptide in PBS.
  • PBS normal bovine serum
  • mice received on day -5 and day -3 either PBS, or 25 ⁇ g each of J ⁇ or of control peptide in PBS.
  • IgGl ELISA titers were determined on individual sera on days 14 and 21. Each number represents the mean ⁇ SD.
  • the differences in OVA-specific titers of groups treated with PBS or the J ⁇ -peptide were significant (p ⁇ 0.01) .
  • PBS The differences in OVA-specific titers of groups treated with PBS or the J ⁇ -peptide were significant (p ⁇ 0.01) .
  • Anti-OVA ELISA titers Anti-HIgG ELISA titers
  • mice received on day -5 and day -3 either PBS, or 25 ⁇ g each of J ⁇ or of J / ⁇ peptide or mixture of 25 ⁇ g each of J ⁇ and Jj ⁇ in PBS.
  • PBS normal bovine serum
  • mice received on day 1 and day 28 either PBS, or 25 ⁇ g each of J ⁇ or of J / ⁇ peptide or mixture of 25 ⁇ g each of J ⁇ and Jj ⁇ in PBS.
  • All mice were immunized with 20 ⁇ g of OVA or 20 ⁇ g of heat-treated HIgG in PBS.
  • Total IgG ELISA titers were determined on individual sera on day 35,43 lnd 51.
  • Anti-OVA ELISA titers Anti-HIgG ELISA titers
  • mice received on day -5 and day -3 either PBS, or 25 ⁇ g each of J ⁇ or of J ⁇ peptide or mixture of 25 ⁇ g each of J ⁇ and Jj ⁇ in PBS.
  • PBS high molecular weight
  • IgG 2a ELISA titers were determined on individual sera on day 35, 43 and 51.
  • the differences in OVA-specific titers of groups treated with PBS or Jj ⁇ peptides versus J ⁇ or J ⁇ - Jj ⁇ peptides were statistically significant (p ⁇ 0.01) .
  • mice received on day -5 and day -3 either PBS, or 25 ⁇ g each of J ⁇ or of Jj ⁇ peptide, or mixture of 25 ⁇ g each of J ⁇ and Jj ⁇ in PBS.
  • PBS normal bovine serum
  • OVA-specific IgE antibodies were determined on pooled sera, one week after each immunization i.e. on days 35, 70, 100 and 145, by 24-h passive cutaneous anaphylaxis (PCA) in hooded rats.
  • PCA titer was calculated as the highest dilution giving a clearly visible skin reaction (> 5mm in diameter) .
  • NAME MOHAPATRA, SHYAM S.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

On décrit des peptides synthétiques, utilisés en immunothérapie, qui présentent une séquence d'acides aminés comprenant au moins une partie de la région CDR3 d'un récepteur de lymphocyte T et qui peuvent provoquer une réponse des lymphocytes T. En particulier, la séquence d'acide aminé peut être codée par une séquence nucléotidique comprenant le gène J de la chaîne α et/ou β du récepteur de lymphocyte T. Ces peptides se révèlent notamment utiles pour réprimer une réponse immunitaire à un antigène et notamment un antigène allergénique.
PCT/CA1994/000228 1993-04-26 1994-04-26 Moderation d'une reponse immunitaire specifique d'un allergene WO1994025489A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP94914290A EP0697024A1 (fr) 1993-04-26 1994-04-26 Moderation d'une reponse immunitaire specifique d'un allergene
AU66740/94A AU6674094A (en) 1993-04-26 1994-04-26 Downregulation of allergen-specific immune response
CA002161316A CA2161316A1 (fr) 1993-04-26 1994-04-26 Retro-regulation de la reaction immunitaire specifique aux allergenes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB939308581A GB9308581D0 (en) 1993-04-26 1993-04-26 A method for induction of antigen-specific suppression of immune-response
GB9308581.9 1993-04-26

Publications (1)

Publication Number Publication Date
WO1994025489A1 true WO1994025489A1 (fr) 1994-11-10

Family

ID=10734481

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA1994/000228 WO1994025489A1 (fr) 1993-04-26 1994-04-26 Moderation d'une reponse immunitaire specifique d'un allergene

Country Status (5)

Country Link
EP (1) EP0697024A1 (fr)
AU (1) AU6674094A (fr)
CA (1) CA2161316A1 (fr)
GB (1) GB9308581D0 (fr)
WO (1) WO1994025489A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0816496A3 (fr) * 1996-06-24 2002-01-09 Roche Diagnostics GmbH Cellules T spécifiques pour le carcinome rénal
US6905688B2 (en) 2000-04-12 2005-06-14 Human Genome Sciences, Inc. Albumin fusion proteins
US6946134B1 (en) 2000-04-12 2005-09-20 Human Genome Sciences, Inc. Albumin fusion proteins
US6972322B2 (en) 1992-01-31 2005-12-06 Aventis Behring L.L.C. Interferon and albumin fusion protein
US7045318B2 (en) 1995-12-30 2006-05-16 Delta Biotechnology Limited Recombinant fusion proteins to growth hormone and serum albumin
US7141547B2 (en) 2001-12-21 2006-11-28 Human Genome Sciences, Inc. Albumin fusion proteins comprising GLP-1 polypeptides
US7507413B2 (en) 2001-04-12 2009-03-24 Human Genome Sciences, Inc. Albumin fusion proteins
US7521424B2 (en) 2003-01-22 2009-04-21 Human Genome Sciences, Inc. Albumin fusion proteins

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992021367A1 (fr) * 1991-05-31 1992-12-10 Arthur Allen Vandenbark Peptides recepteurs de lymphocytes t utilises comme agents therapeutiques contre des maladies du systeme immunitaire
WO1993004695A1 (fr) * 1991-08-28 1993-03-18 The Wistar Institute Therapie de la polyarthrite rhumatoide basee sur le recepteur de lymphocytes t

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992021367A1 (fr) * 1991-05-31 1992-12-10 Arthur Allen Vandenbark Peptides recepteurs de lymphocytes t utilises comme agents therapeutiques contre des maladies du systeme immunitaire
WO1993004695A1 (fr) * 1991-08-28 1993-03-18 The Wistar Institute Therapie de la polyarthrite rhumatoide basee sur le recepteur de lymphocytes t

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CLAVERIE, J.-M. ET AL.: "Implcations of Fab-like structure for the T-cell receptor", IMMUNOLOGY TODAY, vol. 10, no. 1, 1989, pages 10 - 14 *
DAVIS, M.M. ET AL.: "T-cell antigen receptor genes and T-cell recognition", NATURE, vol. 344, August 1988 (1988-08-01), pages 395 - 402 *
JORGENSEN, J. L. ET AL.: "Mapping T-cell receptor peptide contacts by variants peptide immunization of single-chain transgenics", NATURE, vol. 355, 1992, pages 224 - 230 *
MOHAPATRA, S.S. ET AL.: "Analysis of T-cell receptor...", THE JOURNAL OF IMMUNOLOGY, vol. 5, 15 July 1993 (1993-07-15), pages 688 - 698 *

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7094577B2 (en) 1992-01-31 2006-08-22 Aventis Behring L.L.C. Insulin and albumin fusion protein
US7410779B2 (en) 1992-01-31 2008-08-12 Novozymes Biopharma Uk Limited Fusion polypeptides of human serum albumin and a therapeutically active polypeptide
US6972322B2 (en) 1992-01-31 2005-12-06 Aventis Behring L.L.C. Interferon and albumin fusion protein
US6987006B2 (en) 1992-01-31 2006-01-17 Aventis Behring L.L.C. Erythropoietin and albumin fusion protein, nucleic acids, and methods thereof
US6989365B2 (en) 1992-01-31 2006-01-24 Aventis Behring L.L.C. Methods of treatment with erythropoietin and albumin fusion protein
US7435410B2 (en) 1992-01-31 2008-10-14 Novozymes Biopharma Uk Limited Methods of treatment with interferson and albumin fusion protein
US7041478B2 (en) 1992-01-31 2006-05-09 Aventis Behring L.L.C. G-CSF and albumin fusion protein
US7056701B2 (en) 1992-01-31 2006-06-06 Aventis Behring L.L.C. Hormone and albumin fusion protein
US7081354B2 (en) 1992-01-31 2006-07-25 Aventis Behring L.L.C. Interleukin and albumin fusion protein
US7550432B2 (en) 1995-12-30 2009-06-23 Novozymes Biopharma Uk Limited Recombinant fusion proteins to growth hormone and serum albumin
US7045318B2 (en) 1995-12-30 2006-05-16 Delta Biotechnology Limited Recombinant fusion proteins to growth hormone and serum albumin
EP0816496A3 (fr) * 1996-06-24 2002-01-09 Roche Diagnostics GmbH Cellules T spécifiques pour le carcinome rénal
US7507414B2 (en) 2000-04-12 2009-03-24 Human Genome Sciences, Inc. Albumin fusion proteins
US6946134B1 (en) 2000-04-12 2005-09-20 Human Genome Sciences, Inc. Albumin fusion proteins
US6926898B2 (en) 2000-04-12 2005-08-09 Human Genome Sciences, Inc. Albumin fusion proteins
US6905688B2 (en) 2000-04-12 2005-06-14 Human Genome Sciences, Inc. Albumin fusion proteins
US6994857B2 (en) 2000-04-12 2006-02-07 Human Genome Sciences, Inc. Albumin fusion proteins
US7482013B2 (en) 2000-04-12 2009-01-27 Human Genome Sciences, Inc. Albumin fusion proteins
US7507413B2 (en) 2001-04-12 2009-03-24 Human Genome Sciences, Inc. Albumin fusion proteins
US7238667B2 (en) 2001-12-21 2007-07-03 Human Genome Sciences, Inc. Albumin fusion proteins
US7592010B2 (en) 2001-12-21 2009-09-22 Human Genome Sciences, Inc. Albumin fusion proteins
US7141547B2 (en) 2001-12-21 2006-11-28 Human Genome Sciences, Inc. Albumin fusion proteins comprising GLP-1 polypeptides
US8993517B2 (en) 2001-12-21 2015-03-31 Human Genome Sciences, Inc. Albumin fusion proteins
US9221896B2 (en) 2001-12-21 2015-12-29 Human Genome Sciences, Inc. Albumin fusion proteins
US9296809B2 (en) 2001-12-21 2016-03-29 Human Genome Sciences, Inc. Albumin fusion proteins
US7521424B2 (en) 2003-01-22 2009-04-21 Human Genome Sciences, Inc. Albumin fusion proteins

Also Published As

Publication number Publication date
GB9308581D0 (en) 1993-06-09
EP0697024A1 (fr) 1996-02-21
CA2161316A1 (fr) 1994-11-10
AU6674094A (en) 1994-11-21

Similar Documents

Publication Publication Date Title
AU672313B2 (en) Composition for preventing or treating a T cell mediated pathology
AU687897B2 (en) Immunoreactive peptide sequence from a 43 KD human cancer antigen
JPH05504939A (ja) 自己免疫疾患および悪性疾患の治療のためのｔ細胞リセプターペプチド
JPH11196871A (ja) 自己免疫疾患の処置としての抗−ｔ細胞レセプター決定基
US6090387A (en) Vaccination and methods against diseases resulting from pathogenic responses
CA2674888A1 (fr) Vaccins anti-ige
EP0697024A1 (fr) Moderation d'une reponse immunitaire specifique d'un allergene
US6464978B1 (en) Vaccination and methods against multiple sclerosis resulting from pathogenic responses by specific T cell populations
US6982326B1 (en) Allergenic proteins and peptides from Japanese cedar pollen
CA1327162C (fr) Methode de traitement prophylactique de personnes atteintes de maladies auto-immunes
EP0783524B1 (fr) Peptides et compositions pharmaceutiques les contenant
WO1990011293A1 (fr) Proteines allergenes tirees de l'ambrosie et utilisations de ces proteines
JP4218987B2 (ja) ペプチド免疫療法治療剤
JP4253122B2 (ja) アレルゲンの非アナフィラキシー形およびその使用
AU690234B2 (en) Peptides and methods against diabetes
US6335019B1 (en) Methods for treating sensitivity to protein allergen using peptides which include a T cell epitope recognized by a T cell receptor specific for the protein allergen
Mohapatra et al. Analysis of T-cell receptor alpha beta chains of CD8+ suppressor T cells induced by tolerogenic conjugates of antigen and monomethoxypolyethylene glycol. Involvement of TCR alpha-CDR3 domain in immunosuppression.
WO1999027957A1 (fr) Vaccination et procede de lutte contre la sclerose en plaques au moyen de peptides tcr vbeta specifiques
Schramm et al. Molecular and immunological characterization of group V allergen isoforms from velvet grass pollen (Holcus lanatus)
EP0644896A1 (fr) PEPTIDES REPRESENTANT DES EPITOPES ANTIGENIQUES DE L'IgE PRESENTS SUR LA SURFACE DES LYMPHOCYTES B MAIS NON SUR CELLE DES CELLULES BASOPHILES
WO1992016554A1 (fr) Allergenes proteiques de l'espece cynodon dactylon
McDevitt The role of MHC class II molecules in the pathogenesis and prevention of type I diabetes
US7514083B1 (en) Protein allergens of the species cynodon dactylon
WO2000014116A1 (fr) Peptides de lymphocytes t utilises comme therapie pour les maladies auto-immunes
JPH07502648A (ja) シノドン・ダクチロン（Ｃｙｎｏｄｏｎ　ｄａｃｔｙｌｏｎ）種のタンパク質のアレルゲン

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BR CA CN FI JP KR NO NZ RU UA US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2161316

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 1995 535208

Country of ref document: US

Date of ref document: 19951026

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 1994914290

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1994914290

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1994914290

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载