WO2007135684A2 - Procédé de traitement d'auto-immunité anti-cd4 - Google Patents

Procédé de traitement d'auto-immunité anti-cd4 Download PDF

Info

Publication number
WO2007135684A2
WO2007135684A2 PCT/IL2007/000627 IL2007000627W WO2007135684A2 WO 2007135684 A2 WO2007135684 A2 WO 2007135684A2 IL 2007000627 W IL2007000627 W IL 2007000627W WO 2007135684 A2 WO2007135684 A2 WO 2007135684A2
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
cells
cell
polypeptide
patient
Prior art date
Application number
PCT/IL2007/000627
Other languages
English (en)
Other versions
WO2007135684A3 (fr
Inventor
Rivka Abulafia-Lapid
Henri Atlan
Original Assignee
Hadasit Medical Research Services & Development Limited
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 Hadasit Medical Research Services & Development Limited filed Critical Hadasit Medical Research Services & Development Limited
Publication of WO2007135684A2 publication Critical patent/WO2007135684A2/fr
Publication of WO2007135684A3 publication Critical patent/WO2007135684A3/fr

Links

Classifications

    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • 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
    • C07K14/70514—CD4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2

Definitions

  • the present invention relates to immunotherapy and in particular to treatment of anti-CD4 autoimmunity, specifically in HIV-infected patients as well as to products for said treatment.
  • CD4 T-cell leukopenia persists despite a reduction in the viral load achieved by highly active antiretroviral therapy
  • HIVgpl20 activates autoreactive CD4-specific
  • T-cell responses by unveiling of hidden CD4 peptides during processing.
  • T-cell vaccination has recently evolved as a promising therapeutic modality for the treatment of autoimmune conditions. Immunization with inactivated autoreactive T-cells may induce a reaction to deplete specific subsets of autoreactive T-cells involved in the autoimmune disease. T-cell vaccination has been used clinically to induce down-regulation of the autoimmune activity, e.g. in multiple sclerosis and SLE [11-14].
  • T-cell vaccination could also have a therapeutic effect in HIV infected patients.
  • Phase I clinical trial of T-cell vaccination (TCV) in HIV-I infected patients manifesting anti-CD4 autoimmunity were performed by Abulafia-Lapid et al [17-18].
  • Vaccination of these patients with autologous anti-CD4 reactive T-cells resulted in a persistent increase in the patients' blood CD4 T-cell levels and a concomitant decrease in anti-CD4 autoimmunity.
  • a peptide or polypeptide comprising a stretch of contiguous amino acids consisting of at least 5 amino acids overlapping with 5 contiguous amino acids from a CD4 protein, wherein said stretch of contiguous amino acids is characterized by the ability to elicit a T-cell mediated autoimmune response against CD4+ T-cells in a patient with T-cell mediated autoimmunity or to impart on said peptide or polypeptide the ability to elicit a T-cell mediated autoimmune response against CD4+ T-cells in a patient with T-cell mediated autoimmunity.
  • a method of enriching a T-cell preparation comprising exposing a population of T-cells from a subject with a T-cell mediated autoimmune response to a peptide or polypeptide disclosed herein and providing conditions for selective cell proliferation of T-cells reactive with said peptide or polypeptide. Further disclosed is an enriched T-cell population obtainable by the method disclosed herein.
  • T-cell vaccine comprising the enriched T-cell population disclosed herein.
  • T-cells from a subject include cells that cause an anti-CD4+ autoimmune response, comprising contacting a sample containing T-cells of said subject with a peptide or polypeptide disclosed herein, and determining either (i) binding of said peptide or polypeptide to T-cells in said sample, or (ij) T-cell proliferation responses.
  • a conjugate comprising the peptide or polypeptide disclosed herein, conjugated to a cytotoxic moiety.
  • a method of detecting T-cell mediated autoimmunity in a patient comprising: obtaining peripheral mononuclear blood cells (PMBC) from said patient; culturing said PMBC in the presence of a peptide or polypeptide disclosed herein. providing said cells with conditions for selective cell proliferation of T-cells reactive with said peptide or polypeptide; wherein a proliferation response indicates a T-cell specific autoimmune response in said patient.
  • PMBC peripheral mononuclear blood cells
  • kits for detecting T-cell mediated autoimmune response in a subject comprising at least one peptide or polypeptide as disclosed herein.
  • TCR T-cell receptor
  • Figures 1A-1B are graphs showing proliferation of T-cells obtained from HIV-I patients detailed in Table 3 (Fig. IA) and healthy subjects (Fig. IB) in response to different CD4 peptides.
  • S.I. represents Stimulation Index.
  • the present invention is based on the finding that peptides derived from the CD4 protein evoke an autoimmune response in HIV-I infected patients.
  • the immunogenic peptides were identified by analyzing T-cell proliferation responses towards a set of synthetic CD4-derived peptides. T-cells were obtained from several HIV infected patients and healthy subjects and the proliferation responses were compared. Specifically, a set of autoimmune peptides, specific to HIV-I infected patients were identified.
  • isolated peptides or polypeptides comprising a stretch of contiguous amino acids, the stretch consisting of at least 5 amino acids overlapping with a stretch of contiguous amino acids from the CD4 protein (preferably the human CD4 protein), the peptide or polypeptide capable of eliciting a T-cell mediated autoimmune response against CD4 + T-cells in patients with anti CD4 autoimmunity.
  • isolated peptides or polypeptides comprising a stretch of contiguous amino acids consisting of at least 5 overlapping amino acids from the CD4 protein, said stretch imparting on said peptide or polypeptide the ability to elicit a T-cell mediated autoimmune response against CD4+ T-cells in a patient with T-cell mediated autoimmunity.
  • said CD4 protein is human CD4 protein.
  • T-cell mediated autoimmunity denotes a condition where a subject exhibits a T-cell mediated immune response against self antigens.
  • a non-limiting list of conditions manifesting a T-cell mediated autoimmune response include, without being limited thereto, human immunodeficiency virus (HIV) infection, multiple sclerosis (MS), diabetes (type 1 diabetes), systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA).
  • T-cell mediated autoimmunity comprises an immune response against a subjects own CD4 + T-cells.
  • said patient is an HIV-I subtype B or C infected patient, although other subtypes are also included within the scope of the present invention.
  • peptide or "polypeptide” as used herein denote any amino acid sequence comprising a stretch of contiguous amino acids consisting of at least 5 overlapping amino acids from CD4 protein (the "original" native sequence).
  • the stretch of amino acids may also include modifications of one or more amino acids from the CD4 protein sequence as long as the resulting stretch, and consequently, the resulting peptide or polypeptide exhibits a similar immunogenic character (the immunogenic character may be stronger or lower in the resulting peptide or polypeptide).
  • the CD4 protein is a human CD4 protein.
  • the term "at least S overlapping amino acids” denotes a sequence of at least 5 contiguous amino acids within said peptide or polypeptide which overlap (have per residue sequence identity) with at least 5 contiguous amino acids from the CD4 protein, preferably front the human CD4 protein.
  • immunogenic character concerns at least a capacity of the peptide or polypeptide to elicit, under suitable conditions, a T-cell proliferation response towards the peptide or polypeptide. The conditions may be in vitro conditions as well as within a mammal's (e.g. human) body.
  • substitution includes generally the replacement of one or more amino acid residues either by other naturally occurring amino acids, (conservative and non- conservative substitutions), by non-naturally occurring amino acids (conservative and non-conservative substitutions), or with organic moieties which serve either as true peptidomimetics (i.e., having the same steric and electrochemical properties as the replaced amino acid), or merely serve as spacers in lieu of an amino acid, so as to keep the spatial relations between the amino acid spanning this replaced amino acid.
  • conservative substitution in the context of the present invention refers to the replacement of an original amino acid present in the identified peptide with a naturally or non-naturally occurring amino or a peptidomimetic residue having similar steric properties.
  • side-chain of the original amino acid to be replaced is either polar or hydrophobic
  • the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the side- chain of the replaced amino acid).
  • the conservative substitution according to the definition of the invention may be with a naturally occurring amino acid, a non-naturally occurring amino acid or a peptidomimetic moiety which are charged, or with non-charged (polar, hydrophobic) amino acids that have the same steric properties as the side-chains of the replaced amino acids.
  • the purpose of such a procedure of maintaining the steric properties but decreasing the charge is to decrease the total charge of the compound, for example for improving its membrane penetrating properties.
  • substitutions are considered as conservative: replacement of arginine by cytroline; arginine by glutamine; aspartate by asparagine; glutamate by glutamine.
  • Group I includes leucine, isoleucine, valine, methionine, phenylalanine, serine, cysteine, threonine and modified amino acids having the following side chains: ethyl, n- butyl, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 OH, -CH 2 CHOHCH 3 and-CH 2 SCH 3 .
  • Group I includes leucine, isoleucine, valine and methionine.
  • Group II includes glycine, alanine, valine, serine, cysteine, threonine and a modified amino acid having an ethyl side chain.
  • Preferably Group II includes glycine and alanine.
  • Group III includes phenylalanine, phenylglycine, tyrosine, tryptophan, cyclohexylmethyl, and modified amino residues having substituted benzyl or phenyl side chains.
  • Preferred substituents include one or more of the following: halogen, methyl, ethyl, nitro, methoxy, ethoxy and -CN.
  • Group III includes phenylalanine, tyrosine and tryptophan.
  • Group IV includes glutamic acid, aspartic acid, a substituted or unsubstituted aliphatic, aromatic or benzylic ester of glutamic or aspartic acid (e.g., methyl, ethyl, n- propyl iso-propyl, cyclohexyl, benzyl or substituted benzyl), glutamine, asparagine, CO- NH-alkylated glutamine or asparagine (e.g., methyl, ethyl, n-propyl and iso-propyl) and modified amino acids having the side chain -(CH 2 ) 3 COOH, an ester thereof (substituted or unsubstituted aliphatic, aromatic or benzylic ester), an amide thereof and a substituted or unsubstituted N-alkylated amide thereof.
  • glutamic acid e.g., methyl, ethyl, n- propyl iso-prop
  • Group IV includes glutamic acid, aspartic acid, glutamine, asparagine, methyl aspartate, ethyl aspartate, benzyl aspartate and methyl glutamate, ethyl glutamate and benzyl glutamate.
  • Group V includes histidine, lysine, arginine, N-nitroarginine, ⁇ -cycloarginine, ⁇ - hydroxyarginine, N-amidinocitruline and 2-amino-4-guanidinobutanoic acid, homologs of lysine, homologs of arginine and ornithine.
  • Group V includes histidine, lysine, arginine, and ornithine.
  • a homolog of an amino acid includes from 1 to about 3 additional methylene units in the side chain.
  • Group VI includes serine, threonine, cysteine and modified amino acids having C1-C5 straight or branched alkyl side chains substituted with -OH or -SH.
  • Group VI includes serine, cysteine or threonine.
  • non-conservative substitutions concerns replacement of one or more amino acid residues present in the original molecule by another naturally or non-naturally occurring amino acid, having a different size, configuration and/or electronic properties compared with the amino acid being substituted.
  • the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the original amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted.
  • modification may also involve non-conservative substitutions, as long as the immunogenic character of the peptide is retained. Conservative substitutions are nevertheless preferable.
  • Peptidomimetic organic moiety can be substituted for amino acid residues in the peptides of the invention both as conservative and as non-conservative substitutions.
  • the peptidomimetic organic moieties often have steric, electronic or configurational properties similar to the replaced amino acid.
  • the peptidomimetics can be produced by organic synthetic techniques. Examples of suitable peptidomimetics include D amino acids of the corresponding L amino acids, tetrazol [Zabrocki et al, J. Am. Chem. Soc. 110:5875-5880 (1988)]; isosteres of amide bonds [Jones et al, Tetrahedron Lett.
  • the stretch may also include chemically modified amino acids.
  • chemically modified includes modification at the side chain of the amino acid residue, as well as modification of the peptidic bond. Accordingly, a functional group may be added to the side chain, deleted from the side chain or exchanged with another functional group. Typically, the modifications are conservative modifications resulting in conservative substitution. Examples of conservative modifications of this type include adding an amine or hydroxyl, carboxylic acid to the aliphatic side chain of valine, leucine or isoleucine, exchanging the carboxylic acid in the side chain of aspartic acid or glutamic acid with an amine or deleting the amine group in the side chain of lysine or ornithine.
  • amino acid stretch may also include modifications with respect to the original sequence at the peptide backbone, i.e. that the bond between the N- of one amino acid residue to the C- of the next has been altered to non-naturally occurring bonds.
  • modifications include, without being limited thereto, reduction (to -CH 2 -NH-), alkylation (methylation) on the nitrogen atom, or the bonds have been replaced by amidic bond, urea bonds, or sulfonamide bond, etheric bond (-CH 2 -O-), thioetheric bond (-CH 2 -S-), or to -CS-NH-;
  • the side chain of the residue may be shifted to the backbone nitrogen to obtain N-alkylated-Gly (a peptidoid).
  • the peptide or polypeptide disclosed herein may be a linear sequence as well as a cyclic peptide. Cyclization of the amino acid molecule may be performed by S-S bonds. S-S bonds may be formed via the inclusion of sulphor-containing amino acid residues, such as cysteine at each terminus of the amino acid molecule. Cyclic peptides have been shown to be more stable and with higher biological activity than the corresponding linear molecule [Tibbetts S. et al. Peptides. 21(8)1161-7 (2000)].
  • the peptide or polypeptide comprises one or more amino acid sequence comprising a stretch of at least five contiguous amino acids derived or comprise a peptide marked in the following Table 1 as Pl, P4, P21, P28 and P29 and which comprise the following respective sequences:
  • MNRGVPFRHLLLVLQLALLP SEQ ID NO:1
  • P4 KKSIQFHWKNSNQIKILGNQ (SEQ ID NO :4)
  • the peptide or polypeptide disclosed herein is that comprising a stretch of at least 5 contiguous amino acids comprises one or more of a SEQ ID NO: 1, 4, 14, 21, 28 or 29. AU these sequences were shown to elicit a proliferation response in T- cells obtained from HIV-infected patients and not in T-cells obtained from healthy (namely, HIV-negative) subjects.
  • Also disclosed herein is a method of enriching a T-cell preparation, comprising expqsing a population of peripheral mononuclear blood cells (PMBC) from a patient T- cell mediated autoimmunity to a peptide or polypeptide as disclosed herein and providing conditions allowing for selective cell proliferation of T-cells reactive with said peptide or polypeptide.
  • PMBC peripheral mononuclear blood cells
  • T-cells reactive with said peptide or polypeptide may, at times, be generally referred to as CD4 peptide-recognizing T-cells or in short, CD4 recognizing T-cells.
  • the enrichment method in accordance with one embodiment disclosed herein comprises the following steps: providing peripheral mononuclear blood cells (PBMC) from a subject; culturing said PBMC with one or more peptides or polypeptides as disclosed herein under conditions allowing selective cell proliferation of T-cells reactive with said peptide or polypeptide.
  • PBMC peripheral mononuclear blood cells
  • conditions for selective cell proliferation includes the addition to the
  • T-cell culture a media including substances required for the growth of said cells.
  • the media should preferably include at least one growth stimulating factor for stimulating the growth of said cells.
  • Growth factors for stimulating cell proliferation and cell expansion are known to those versed in the art . Such growth factors will be selected according to their ability to facilitate cell expansion essentially without inducing cell differentiation.
  • a non-limiting list of growth factors suitable for stimulating T-cell production includes phytohemmaglutinin (PHA, a mitogen known to induce non-specific T-cell proliferation), anti-CD3, and anti-CD28 (antibodies known to induce non-specific T-cell proliferation, [see, 25, incorporated herein in its entirety by reference]) IL-2 (specific for T-cell expansion) and any combinations of same as well as combinations of same with other growth factors.
  • PHA phytohemmaglutinin
  • anti-CD3 anti-CD3
  • anti-CD28 antibodies known to induce non-specific T-cell proliferation, [see, 25, incorporated herein in its entirety by reference]
  • IL-2 specific for T-cell expansion
  • the stimulating factor is phytohemmaglutinin (PHA).
  • PHA phytohemmaglutinin
  • IL-2 may be added to the T-cell culture after initial cell growth stimulation with PHA or the like, so as to facilitate expansion of the PHA-activated T-cells.
  • the conditions include the addition of irradiated CD4-pulsed antigen presenting cells (APCs) so as to "boost" the proliferation of the T- cells in the culture.
  • APCs irradiated CD4-pulsed antigen presenting cells
  • the conditions may include the addition of beads to which the peptide or polypeptide of the invention are bound, optionally in the presence of MHC molecules [23, incorporated herein by reference, in its entirety]. Such beads may replace the need to use irradiated APCs.
  • an enriched CD4-recognizing T-cell preparation obtainable by the method described above. It has already been shown that during HIV infection, CD4+ T-cells become the target of CD8+ T-cells [4,5,17,18, each incorporated herein by reference, in its entirety].
  • the enriched CD4 recognizing T-cell preparation is enriched in CD8+ T-cells and manifest high INF ⁇ and low IL-IO expression.
  • the enriched CD4-recognizing T-cell preparation may be inactivated to be used as part of a vaccine to trigger an immune response in the patient against the autoimmune T-cells
  • a method of producing an enriched, inactivated CD4- recognizing T-cell preparation comprising: - providing an enriched CD4 recognizing T-cell preparation obtainable by the method as disclosed above, and inactivating said T-cells with an inactivating agent.
  • inactivation is performed by the use of a fixation agent.
  • fixation agents include glutaraldehyde, formaldehyde or paraformaldehyde.
  • the fixating agent is then neutralized.
  • neutralization is performed in the presence of glycine.
  • the inactivated T-cell preparation is then washed at least once with a suitable washing solution, e.g. saline.
  • inactivation may be performed by irradiating the T-cells population (6,000-7,000 rads) prior to injection.
  • the thus formed enriched, inactivated, T-cell preparation may then be further treated by washing and re-suspending the cells with a washing medium, such as injection- grade saline solution.
  • the preparation may also be divided into aliquots for storage. For example, the preparation may be divided into aliquots of between 10 6 -10 8 cells for freezing in liquid nitrogen.
  • T-cell vaccine comprising an enriched, inactivated CD4-recognizing T-cell preparation obtainable by the method disclosed above, together with a pharmaceutically acceptable carrier.
  • the vaccine may be combined with other active agents, such as drugs used to treat an autoimmune disorder, anti-HIV drugs, antibiotics, vitamins etc.
  • active agents such as drugs used to treat an autoimmune disorder, anti-HIV drugs, antibiotics, vitamins etc.
  • the preparation of vaccines is well known in the art and has been described in many articles and text books see e.g. Remington's pharmaceutical sciences, Gennaro A.R. ed. Mack Publishing Co.
  • a vaccine comprising an enriched preparation of autologous inactivated CD4-recognizing T-cells obtainable by the method of the invention.
  • an immune response is generated against the T-cell recognizing CD4 or the peptide or polypeptide derived therefrom.
  • a vaccine comprising an enriched preparation of autologous inactivated CD4-recognizing T-cells obtainable by the method of the invention.
  • an immune response is generated against the T-cell recognizing CD4 or the peptide derived therefrom.
  • the vaccine is administered to the HIV infected patient by injection, preferably subcutaneous injection.
  • Each injection may comprise from about 10 6 to about 10 8 cells/ml in saline.
  • the vaccine comprises 10 7 T-cells/ml saline.
  • the immune response is triggered as immune cells recognize the injected (autologous) autoimmune cells as "foreign".
  • the patient's immune system eliminates these pathologic cells which are involved in eliciting the auto mmunity from the patients blood.
  • the HIV patient may be provided with a single dose vaccination which may be followed by one or more doses so as to increase the response to the CD4-recognizing T- cells (boosters).
  • a single dose vaccination which may be followed by one or more doses so as to increase the response to the CD4-recognizing T- cells (boosters).
  • HIV-infected patients exhibiting an auto-reactive response and treated in accordance with the invention are clinically followed, the follow-up including, for example, analysis of viral load, plasma CD4 counts, immunological follow-up including, for example, anti- clonotypic assay [20, 21, each incorporated herein by reference, in their entirety], anti- CD4 autoimmune assay, cytotoxicity assay and anti-recall antigens assay. All assays are known to those versed in the art and are inter alia described by Abulafia-Lapid R et al. [17-18, each incorporated herein by reference, in their entirety].
  • pre-clinical and clinical studies for determining therapeutic protocols of human subjects are well known to those versed in the art.
  • pre-clinical studies may involve assaying one or more (e.g. combinations) peptides as disclosed herein in an animal model.
  • developing an animal model may comprise the use of transgenic mice carrying the human CD4 coding sequences and expressing the same, such as those described by Blum MD et al. [Reconstitution of the subclass-speci ⁇ c expression ofCD4 in thymocytes and peripheral T cells of transgenic mice: identification of a human CD4 enhancer J Exp Med. 1993 ; 177(5): 1343-58].
  • the mice may be induced to develop auto-immunity to the human CD4 by injecting thereto one or more of the peptides disclosed herein.
  • Selected peptides, such as pi, p4, pl4, p28 and/or p29 may be used as a first tier of peptides in the pre-clinical evaluation.
  • the peptide or a combination of peptides, in a suitable carrier and in combination with a suitable adjuvant may be injected intravenously (or by other conventional means), with optionally a subsequent booster injection (after several days or weeks, e.g. two weeks).
  • An adjuvant may include, without being limited thereto heat killed M. tuberculosis strain H37RA (Difco) emulsified in Incomplete Freund's adjuvant (Difco).
  • anti-CD4 autoimmunity may be induced by injecting mice with one or more of the disclosed peptide, a mixture of peptides, or with rCD4 protein with an appropriate adjuvant.
  • the therapeutic effect of the T-cell vaccine disclosed herein may be evaluated.
  • the vaccine is administered in an effective amount, being an amount that is effective in achieving the desired therapeutic effect, which may be manifested by a decrease in autoimmune response, as determined by, e.g. increase CD4 cell counts in the peripheral blood.
  • the effective amount may be determined in dose-finding clinical studies in patients with T-cell mediated autoimmunity, such as HIV patients which developed the said autoimmune response, or through extrapolation from animals (such as the mice model discussed above) using one of many such extrapolation methods readily known to those of skill in the art of clinical studies.
  • the effective amount may depend on factor known per se such as weight, body surface, gender, disease history and status, concomitant medicines taken by the patient, severity of disease, frequency of administration, drug residence time in the plasma or blood, extent of binding of the drug by blood proteins and others.
  • a diagnostic method namely a method of determining whether an HIV-infected patient has an auto-reactive CD4-specific T-cell response
  • the method comprises contacting a sample of peripheral mononuclear blood cells (PBMC) containing T-cells of said patient with a peptide or polypeptide as disclosed herein and determining either (i) binding of said peptide or polypeptide to T-cells in said sample, or (ii) a T-cell proliferation response in the presence of said peptide or polypeptide.
  • PBMC peripheral mononuclear blood cells
  • a conjugate comprising a peptide or polypeptide according to the invention conjugated to a cytotoxic moiety.
  • the peptide or polypeptide will thus function as a carrier delivering the cytotoxic moiety to autoreactive T-cells.
  • the cytotoxic moiety may be a low molecular weight compound such as a radioactive label, or a high molecular weight substance such as a toxin [24, 25, 26, each incorporated herein by reference, in their entirety]. Cytotoxic compounds which may be employed in accordance with the present disclosure will be readily appreciated by those versed in the art. .
  • the peptide or polypeptide may be associated with a liposome, e.g. by conjugating (chemically or physically) to a component at the outer surface of a liposome, or by having the liposome carrying the peptide or polypeptide its intra- liposomal compartment, or by incorporating of same in the liposomal bilayer, etc. as appreciated by those versed in biochemistry, wherein the liposome also carries said cytotoxic compound.
  • the peptides and polypeptides disclosed herein may also be utilized to diagnose a T-cell mediated autoimmune response in a subject.
  • the peptides and polypeptides can be used to profile the patients' autoimmune CD4 peptide recognition pattern/profile and allow the development of a "tailor made" vaccine.
  • a method for detecting T-cell autoimmunity in a subject comprising: (a) obtaining peripheral mononuclear blood cells (PMBC) from said subject;
  • PMBC peripheral mononuclear blood cells
  • the subject is an HIV infected patient.
  • the T-cell mediated autoimmunity is anti-CD4 mediated autoimmunity.
  • a kit for profiling anti CD4 autoimmunity in a subject comprising a CD4 peptide or polypeptide as disclosed herein.
  • TCR T-cell receptor
  • Such complementary TCR peptides can be easily identified using bioinformatics tools well, known in the art and further used for therapeutic vaccination.
  • a composition comprising the complementary TCR peptides for use in therapeutic vaccination of immune deficient HIV 1 infected patients. Further, disclosed is thus a method of vaccination of such patients using the vaccine composition, optionally in combination with anti-retroviral treatment or the T-cell vaccine of the invention.
  • HIV-I subtype B infected patients (Pl to P25) with age range of 24 to 63 (median age 40 years) were recruited from the Kaplan Hospital AIDS Center (Rehovot, Israel) and the Hadassah University Hospital in Jerusalem, Israel.
  • the duration of their infection from time of diagnosis ranged from 0.3 months to 16 years (median, 7.0 years). All of the patients (but one) were treated according to the (HAART regimen, receiving a highly active anti-retroviral therapy), and the duration of treatment varied (median, 2.3 years).
  • CD4 T- cell numbers ranged from 93 to 679 cells/ml blood (mean 387, medial 376) and their plasma viral loads (Amplicor, Hoffman-LaRoche) from ⁇ 400 to 59,300 copies/ml (mean 16,307, median 14,450). Blood was also obtained from 24 HIV-sero-negative (Cl to C24) healthy donors, through the Tel Hashomer Central Blood Bank, to serve as controls.
  • Peptides were synthesized at the Biological Services Laboratory of the Weizmann Institute of Science, Rehovot, Israel and were used at a concentration of 20 ⁇ g/ml. Culturing conditions were as described previously [17, 18, each being incorporated herein by reference, in its entirety].
  • PBMC Peripheral blood mononuclear cells
  • Cells were cultured in RPMI 1640 medium (Biological Industries Ltd., Beit Haemek, Israel), supplemented with 10% fetal calf serum (FCS) (Gibco BRL, Buffalo, NY, USA), 1% sodium-pyruvate, 1% L-glutamine, 1% Penicillin/Streptomycin (10,000 U/ml/10,000 mg/ml) (Seromed, Berlin, Germany) and 2% Hepes (IM, pH7.3) (Biological Industries, Beit Haemek, Israel). Specific cell proliferation was assayed by 3 H-thymidine incorporation, as described previously [19].
  • FCS fetal calf serum
  • FCS fetal calf serum
  • 1% sodium-pyruvate 1% L-glutamine
  • Penicillin/Streptomycin 10,000 U/ml/10,000 mg/ml
  • IM Hepes
  • the antigens used were soluble recombinant human CD4 and HIV- 1, gpl20 proteins (2.5 ⁇ g/ml each), expressed in baculovirus (Intracell Corp., Cambridge, MA, USA, or other source), tetanus toxoid (5 ⁇ g/ml, Connaught Lab. Inc., PA, USA, or other source), Candida (lO ⁇ g/ml, Hollister-Stier, Toronto, Canada, or other source) (data not shown, but discussed), and PHA (0.3 ⁇ g/ml, Murex Diagnostics Ltd., Dartford, UK, or other source).
  • each of the selected CD4 derived peptides were used at a concentration of 20 ⁇ g/ml.
  • the cells were plated in 96-well round bottom microplates (Falcon, Linooln Park, NJ, USA), at a cell concentration of 2xlO 5 cells/well in medium, with or without the test antigens or mitogen.
  • the plates were incubated at 37°C in a 5% CO 2 humidified incubator for 7 days. On day 6, the cells were labeled overnight with 1 ⁇ Ci/well of 3H-thymidine and counted in a Matrix 96 -counter (Packard, Meriden, CT, USA).
  • the results of the proliferation assays are presented as stimulation indexes (S.
  • a stimulation index of above 2 was considered as a positive response. This value was chosen for two reasons: (1) the minimum concentration of CD4 was used (2 ⁇ g), and (2) the low frequency of these cells as a result of immunosuppression.
  • CD4 peptide-reactive T-cells are generated from a patient's blood sample as generally described in Materials and Methods. Briefly, PBMC are isolated from the patient's blood sample (about 30-40 ml blood is obtained from each patient), cultured with 3 ⁇ g/ml phytohemaglutinin (PHA) for 3 days, and expanded with 5-20 U/ml recombinant interleukin-2 (rIL-2) for 11 days. The cultures are re-stimulated on day 14, with irradiated CD4 peptide-pulsed antigen presenting cells (APC) [24, incorporated herein by reference, in its entirety], and are further grown for another 7 days.
  • PHA phytohemaglutinin
  • rIL-2 interleukin-2
  • the cells are inactivated for 5 minutes, at room temperature with 1.0% glutaraldehyde and prepared for injection. Aliquots of 10x10 6 CD4 peptide-reactive T- cells are frozen in liquid nitrogen, to be used later for second and third booster vaccinations, at two to six months intervals. For pure lines of anti CD4 peptide cells, several rounds of CD4 peptide re-stimulation are necessary, preferably between 4 to 8 rounds. All cultures are performed according to the procedures required for the preparation of biological products to be used in humans.
  • T-cell reactivity to recombinant CD4 (rCD4), recombinant gpl20 (rgpl20), and to the recall antigens Tetanus toxin and Candida was studied in HIV infected patients and in healthy donors. The experiments measuring the response towards the recall antigens were conducted in order to examine parameters of the immune response of the study subjects .
  • T-cell proliferation responses are summarized in Table 2A for the HIV-I infected patients (HIV subtype B) and in Table 2B, for the healthy subjects.
  • Table 2A T-cell proliferative responses in HIV infected patients
  • T-cell proliferation responses to rCD4 were significantly higher in HIV patients in comparison to the healthy controls.
  • the Stimulation Index (S.I.) in response to rCD4 had a value above 2 (mean S.I. 3.6+ 2.1), whereas in the controls, only 4 out of 24 (25%) had an S.I. above 2 (mean S.I. 1.6+0.9).
  • the major immunogenic epitopes of the CD4 molecule were then mapped by testing T-cell proliferation responses to 31 synthetic overlapping peptides obtained from the human CD4 molecule.
  • a peptide was defined as immunogenic only when at least 50% of the tested HIV- infected patients showed positive proliferation responses to this peptide (in terms of Simulation index, S.I.)- Tables 4A-4B show the mean responses of HIV patients (Table 4A) and HIV negative subjects (Table 4B) and the percentage responding to the six major immunogenic CD4 peptides.
  • Peptides pi, p28 and p29 were thus determined as major epitopes as they elicited a response in more than 50% of the patients, in the specific example, in 62.5% (Pl and P29) to 75% (P28) of the patients. Proliferation responses were observed in 4 distinct clusters (1-7, 11-15, 16-22, 25-30) with the most significant being p28 and p29.
  • the p4 and p5 peptides are located in the amino acid sequence found in CD4 domain 1, which is the HIV gpl20-binding site. Epitopes p28 and p29 are distant from the binding site. Each patient responded to at least 2 of the immunogenic peptides (except for patient P 17, see below).
  • Patient Pl 7 responded to p2, pi 2, p21 and p27. Of these 4 peptides, only p21 is considered autoimmune. Interestingly, the other 3 peptides were also responded to by the healthy subjects. This unusual response of P 17, whose disease duration is 2.6 years, may be an indication of a transition from healthy to autoimmune response.
  • the proliferation response to a mixture of 5 peptides was also tested. The mixture contained the immunogenic peptides pi, p4, pl4, p28 and p29 (p21 was excluded since its mean SI was below 2 (namely, 1.8)) at a concentration of 20 ⁇ g/ml each. The responses towards the mixture were compared to responses towards the full CD4 molecule.
  • Table 5A and 5B show responses to the peptide mixture in HIV positive (Table 5A) and HIV negative (Table 5B) donors.
  • T cells were cultured with recombinant CD4 at a concentration of 2 ⁇ g/ml.
  • SI stimulation index
  • Patients P5, P6 and P8 showed low blood CD4 counts and did not manifested a response to either the CD4 molecule or to the peptide. These patients also failed to respond to tetanus toxoid.
  • HIV gpl20 activates autoreactive CD4-specific T-cell responses by unveiling of hidden CD4 peptides during processing. J Exp Med 181:2253-2257

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Zoology (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Rheumatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Peptides ou polypeptides comprenant une suite d'acides aminés contigus qui comportent au moins 5 acides aminés chevauchant une suite correspondante de protéine CD4, et cette suite d'acides aminés est capable d'induire une réponse auto-immune à médiation assurée par lymphocytes T contre les lymphocytes T CD4+ chez un patient avec auto-immunité dont la médiation est assurée par les lymphocytes T, ou, chez un tel patient, d'amener le peptide ou polypeptide considéré à induire une telle réponse. On décrit aussi diverses applications de ces peptides et polypeptides dans la thérapie et le diagnostic.
PCT/IL2007/000627 2006-05-22 2007-05-24 Procédé de traitement d'auto-immunité anti-cd4 WO2007135684A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US80212506P 2006-05-22 2006-05-22
US60/802,125 2006-05-22

Publications (2)

Publication Number Publication Date
WO2007135684A2 true WO2007135684A2 (fr) 2007-11-29
WO2007135684A3 WO2007135684A3 (fr) 2009-04-23

Family

ID=38723699

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IL2007/000627 WO2007135684A2 (fr) 2006-05-22 2007-05-24 Procédé de traitement d'auto-immunité anti-cd4

Country Status (1)

Country Link
WO (1) WO2007135684A2 (fr)

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010092185A3 (fr) * 2009-02-16 2011-01-20 Institut National De La Sante Et De La Recherche Medicale (Inserm) Inhibiteurs de l'intération scramblase/cd4 pour le traitement d'infections à lentivirus
WO2011064407A1 (fr) * 2009-11-30 2011-06-03 Biotest Ag Agents destinés à traiter des maladies
CN103261218A (zh) * 2010-11-25 2013-08-21 Imnate责任有限公司 免疫原性肽在预防和/或治疗感染性疾病、自体免疫性疾病、针对同种异体因子、变应性疾病、肿瘤、移植排斥的免疫应答和针对基因治疗或基因疫苗接种所用病毒载体的免疫应答中的应用
US9334325B2 (en) 2008-03-13 2016-05-10 Biotest Ag Method for treating psoriasis
US9512226B2 (en) 2008-03-13 2016-12-06 Biotest Ag Agent for treating disease
US9550831B2 (en) 2008-03-13 2017-01-24 Biotest Ag Method for treating psoriasis
US10617748B2 (en) 2008-02-14 2020-04-14 Life Sciences Research Partners Vzw Immunogenic control of tumours and tumour cells
US10662232B2 (en) 2006-08-11 2020-05-26 Life Sciences Research Partners Vzw Immunogenic peptides and their use in immune disorders
US10729791B2 (en) 2015-05-18 2020-08-04 Imcyse Sa Animal models for evaluating pharmaceutical compounds
US10899795B2 (en) 2012-01-30 2021-01-26 Life Sciences Research Partners Vzw Modified epitopes for boosting CD4+ T-cell responses
US10982196B2 (en) 2008-02-14 2021-04-20 Life Sciences Research Partners Vzw Immunotherapy targeting intracellular pathogens
US11226332B2 (en) 2013-05-28 2022-01-18 Imcyse Sa Method for the detection, preparation and depletion of CD4+ t lymphocytes
US11485768B2 (en) 2016-04-19 2022-11-01 Imcyse Sa Immunogenic CD1d binding peptides
US11787849B2 (en) 2015-09-25 2023-10-17 Imcyse Sa Methods and compounds for eliminating immune responses to therapeutic agents
US12023384B2 (en) 2014-10-17 2024-07-02 Imcyse Sa Immunogenic peptides comprising an MHC class II T cell epitope and a redox motif

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ABULAFIA-LAPID ET AL.: 'T-cell vaccination against anti-CD4 autoimmunity in HIV-1 infected patients' JOURNAL OF CLINICAL VIROLOGY 2004, pages 31S, S48 - S54 *
ABULAFIA-LAPID ET AL.: 'T-cell vaccination against anti-CD4 autoimmunity in HIV-1 subtypes B and C-infected patients-An extended open trial' VACCINE vol. 23, 2005, pages 2149 - 2153 *
ATLAN ET AL.: 'Can AIDS be prevented by T-cell vaccination' IMMUNOLOGY TODAY vol. 14, no. 5, 1993, pages 200 - 202 *
ATLAN ET AL.: 'Mechanisms of autoimmunity and AIDS: prospects for therapeutic intervention' RES. IMMUNOLOGY vol. 145, 1994, pages 165 - 183 *

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11718650B2 (en) 2006-08-11 2023-08-08 Imcyse Sa Immunogenic peptides and their use in immune disorders
US10662232B2 (en) 2006-08-11 2020-05-26 Life Sciences Research Partners Vzw Immunogenic peptides and their use in immune disorders
US10982196B2 (en) 2008-02-14 2021-04-20 Life Sciences Research Partners Vzw Immunotherapy targeting intracellular pathogens
US10617748B2 (en) 2008-02-14 2020-04-14 Life Sciences Research Partners Vzw Immunogenic control of tumours and tumour cells
US9334325B2 (en) 2008-03-13 2016-05-10 Biotest Ag Method for treating psoriasis
US9512226B2 (en) 2008-03-13 2016-12-06 Biotest Ag Agent for treating disease
US9550831B2 (en) 2008-03-13 2017-01-24 Biotest Ag Method for treating psoriasis
WO2010092185A3 (fr) * 2009-02-16 2011-01-20 Institut National De La Sante Et De La Recherche Medicale (Inserm) Inhibiteurs de l'intération scramblase/cd4 pour le traitement d'infections à lentivirus
WO2011064407A1 (fr) * 2009-11-30 2011-06-03 Biotest Ag Agents destinés à traiter des maladies
JP2013511996A (ja) * 2009-11-30 2013-04-11 バイオテスト・アクチエンゲゼルシヤフト 疾患を治療するための剤
RU2598719C2 (ru) * 2009-11-30 2016-09-27 Биотест Аг Средства для лечения заболевания
US9995733B2 (en) 2009-11-30 2018-06-12 Biotest Ag Agents for treating disease
US10023847B2 (en) 2010-11-25 2018-07-17 Imnate Sarl Immunogenic peptides for use in the prevention and/or treatment of infectious diseases, autoimmune diseases, immune responses to allofactors, allergic diseases, tumors, graft rejection and immune responses against viral vectors used for gene therapy or gene vaccination
KR101902029B1 (ko) 2010-11-25 2018-09-27 임나트 에스에이알엘 감염성 질환, 자가면역 질환, 동종인자에 대한 면역 반응, 알레르기 질환, 종양, 이식편 거부 및 유전자 요법 또는 유전자 백신접종을 위해 사용되는 바이러스 벡터에 대한 면역 반응의 예방 및/또는 치료에 사용하기 위한 면역원성 펩티드
JP2018011592A (ja) * 2010-11-25 2018-01-25 イムナテ・ソシエテ・ア・レスポンサビリテ・リミテＩｍｎａｔｅ Ｓａｒｌ 感染症、自己免疫疾患、同種因子に対する免疫応答、アレルギー性疾患、腫瘍、移植片拒絶反応、および、遺伝子療法または遺伝子ワクチン接種のために使用されるウイルスベクターに対する免疫応答の予防および／または治療における使用のための免疫原性ペプチド
RU2615460C2 (ru) * 2010-11-25 2017-04-04 Имнейт Сарл Иммуногенные пептиды для применения в профилактике и/или лечении инфекционных заболеваний, аутоиммунных заболеваний, иммунных ответов на аллогенные факторы, аллергических заболеваний, опухолей, отторжения трансплантата и иммунных ответов против вирусных векторов. используемых для генной терапии или генной вакцинации
CN103261218A (zh) * 2010-11-25 2013-08-21 Imnate责任有限公司 免疫原性肽在预防和/或治疗感染性疾病、自体免疫性疾病、针对同种异体因子、变应性疾病、肿瘤、移植排斥的免疫应答和针对基因治疗或基因疫苗接种所用病毒载体的免疫应答中的应用
CN103261218B (zh) * 2010-11-25 2020-08-14 Imnate责任有限公司 免疫原性肽在预防和/或治疗疾病中的应用
EP3763729A1 (fr) * 2010-11-25 2021-01-13 Imnate Sarl Peptides immunogènes destinés à la prévention et/ou au traitement de maladies infectieuses, de maladies auto-immunes, de réponses immunitaires aux allofacteurs, de maladies allergiques, de tumeurs, du rejet de greffe, et des réponses immunitaires dirigées contre des vecteurs viraux utilisés en thérapie génique ou en vaccination génique
AU2011333749B2 (en) * 2010-11-25 2016-10-20 Imcyse Sa Immunogenic peptides for use in the prevention and/or treatment of infectious diseases, autoimmune diseases, immune responses to allofactors, allergic diseases, tumors, graft rejection and immune responses against viral vectors used for gene therapy or gene vaccination
US11193114B2 (en) 2010-11-25 2021-12-07 Imnate Sarl Immunogenic peptides for use in the prevention and/or treatment of infectious diseases, autoimmune diseases, immune responses to allofactors, allergic diseases, tumors, graft rejection and immune responses against viral vectors used for gene therapy or gene vaccination
US10899795B2 (en) 2012-01-30 2021-01-26 Life Sciences Research Partners Vzw Modified epitopes for boosting CD4+ T-cell responses
US11226332B2 (en) 2013-05-28 2022-01-18 Imcyse Sa Method for the detection, preparation and depletion of CD4+ t lymphocytes
US12023384B2 (en) 2014-10-17 2024-07-02 Imcyse Sa Immunogenic peptides comprising an MHC class II T cell epitope and a redox motif
US10729791B2 (en) 2015-05-18 2020-08-04 Imcyse Sa Animal models for evaluating pharmaceutical compounds
US11787849B2 (en) 2015-09-25 2023-10-17 Imcyse Sa Methods and compounds for eliminating immune responses to therapeutic agents
US11485768B2 (en) 2016-04-19 2022-11-01 Imcyse Sa Immunogenic CD1d binding peptides

Also Published As

Publication number Publication date
WO2007135684A3 (fr) 2009-04-23

Similar Documents

Publication Publication Date Title
WO2007135684A2 (fr) Procédé de traitement d'auto-immunité anti-cd4
Londei et al. Epithelial cells expressing aberrant MHC class II determinants can present antigen to cloned human T cells
de Smet et al. Human S-antigen determinant recognition in uveitis
US8673293B2 (en) Use of modified cells for the treatment of multiple sclerosis
Jura et al. Comprehensive insight into human aminoacyl-tRNA synthetases as autoantigens in idiopathic inflammatory myopathies
US9260692B2 (en) Use of modified cells for the treatment of multiple sclerosis
MXPA03001606A (es) Metodo de seleccion de peptidos.
Cohen Peptide therapy for Type I diabetes: the immunological homunculus and the rationale for vaccination
Sospedra et al. Antigen-specific therapies in multiple sclerosis
Johnson et al. The CD8 T cell in multiple sclerosis: suppressor cell or mediator of neuropathology?
Forrester New concepts on the role of autoimmunity in the pathogenesis of uveitis
RU2127599C1 (ru) Композиция для профилактики и лечения спида, или системной красной волчанки, или связанных с ними нарушений
Falk et al. Induction and suppression of an autoimmune disease by oligomerized T cell epitopes: enhanced in vivo potency of encephalitogenic peptides
DK2016414T3 (en) T-cell vaccine
Liu et al. Immune and metabolic effects of antigen-specific immunotherapy using multiple β-cell peptides in type 1 diabetes
CN111593022A (zh) vMIP-Ⅱ诱导CD8+ T细胞去磷酸化为Tcm及其在药物中的应用
D'Souza et al. Frontline: an in‐depth evaluation of the production of IL‐2 by antigen‐specific CD8 T cells in vivo
Massilamany et al. Identification of a second mimicry epitope from Acanthamoeba castellanii that induces CNS autoimmunity by generating cross-reactive T cells for MBP 89–101 in SJL mice
Johnson et al. Multimeric insulin desensitizes insulin-specific B cells
Holmes et al. Multiple sclerosis: MHC associations and therapeutic implications
JP6918333B2 (ja) 細胞性免疫に認識されるペプチド、及びそれを利用した医薬薬剤
Edling et al. Effect of a cyclic heptapeptide based on the human CD4 domain 1 CC′ loop region on murine experimental allergic encephalomyelitis: inhibition of both primary and secondary responses
Hellings et al. T-cell-based immunotherapy in multiple sclerosis: induction of regulatory immune networks by T-cell vaccination
Hill et al. A novel RIPK1 inhibitor attenuates GVHD
CN116589557B (zh) 一种肿瘤新生抗原特异性tcr及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07736367

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07736367

Country of ref document: EP

Kind code of ref document: A2